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Functions of a proteinase secreted by the sap-staining fungus Ophiostoma piceae Abraham, Linda Deanne 1995

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FUNCTIONS OF A PROTEINASE SECRETEDBY THE SAP-STAINING FUNGUS OPHIOSTOMA PICEAEbyLINDA DEANNE ABRAHAMB.Sc., University ofNatal, 1986M. Sc., University of Witwatersrand, 1989A THESIS SUBMITTED iN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIESDepartment ofWood ScienceWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAAugust, 1995© Linda Deanne Abraham, 1995In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department ofThe University of British ColumbiaVancouver, CanadaDate 18 €VVVDE-6 (2188)11AbstractFungal colonisation that discolours wood decreases its market value, reducing profits onCanadian lumber products. Disrupting key enzymes involved in fungal metabolism could bea way of preventing such wood-inhabiting fungi from colonising lumber. Enzyme-targetedantisapstain formulations would be expected to have a low potential for adverseenvironmental impact. The production of proteinases by sapstaining fungi may be key to theacquisition of nitrogen required for growth since protein is considered to be the major sourceof organic nitrogen in wood.Proteolytic activity detected in wood powder and culture filtrates after growth of Ophiostomapiceae was inhibited by PMSF and EDTA. The major protein detected in culture filtrates, aproteinase with a p1 of 5.6 and a molecular weight of 33 kDa, was subsequently purified byhydrophobic interaction chromatography. The proteolytic activity of the purified proteinasewas determined to be optimal at pH 7 to 9 and 40°C. The N-terminal sequence of the proteinshowed a high degree ofhomology with fungal alkaline serine proteinases classified as subtilisinclass II enzymes. Agreements in inhibition patterns, electrophoretic and catalytic propertiessuggested the secretion of the same proteinase during growth on wood. Proteinase productionwas associated with active growth, suggesting a role in primary retrieval of nitrogen from woodproteins. Preliminary attempts to selectively inactivate the proteinase by application ofchelators or serine proteinase inhibitors on wood prior to infection were inconclusive. Furtherefforts were hampered by the current lack of stable, non-toxic, specific proteinase inhibitors.iiiThe subtilisin-like serine proteinase was degraded by autoproteolysis under conditions ofheating, altered pH or partial depletion of protein-bound ions by EDTA. The proteinaseconsisted of two major hydrolytic fragments, 19 kDa and 14 kDa, which had N-terminalsequences of Ala’-Tyr2-Thr34G1n567A a8Pro9and Ser’70-Glu1Pro172Ser3174 175 176 177 178 179Val -x -Thr -Val -Gly -Ala -, respectively. Smce the former sequence was identicalto the N-terminus of the native protein, the major autoproteolytic cleavage site for a class IIsubtilase appeared to be the N-side of Ser170, consistent with a similar region identified for classI subtilases.The cleavage specificity of this subtilase was investigated on the insulin B-chain usingelectrospray ionisation mass spectrometry. Cleavage sites after hydrophobic, polar, andcharged amino acids indicated a broad specificity. Degradation of proteins extracted from thexylem tissue of poplar was observed after incubation with the proteinase. Other proteinshydrolysed by the proteinase included gelatin, collagen, albumin, edestin, globulins andcasein. This supports the conclusion that the proteinase has a broad specificity and is able todegrade physiological substrates.A thorough understanding of the nutritional requirements of staining fungi has importantimplications for preventing the growth of these fungi and other economically importantophiostomatoid fungi. The approach taken in this work - identifying key physiological enzymesas a strategy for controlling sapstaining fungi - has shown that these fungi require proteinases tobreakdown wood proteins into assimilable nitrogen. Therefore, these enzymes are vitalcomponents of fungal physiology and their selective inactivation may be the target for futurebioprotectants.ivTABLE OF CONTENTSPageAbstract iiTable of contents ivList of tables xList of figures XiiList of abbreviations XviAcknowledgement XxiChapter 1 General introduction 11.1 Problem of sapstain from an industrial perspective 11.2 Fungi causing sapstain and other wood-colonising fungi 71.3 Wood as an environment for sapstaining fungi 121.4 Nitrogen metabolism in fungi 161.5 Fungal growth and secretion of enzymes 191.6 Proteinase enzymes 211.7 Proteinase inhibition 261.8 Proteinases of wood-inhabiting fungi 281.9 Research approach and objectives 31VPageChapter 2 General methodology 342.1 Fungal strain 342.2 Growth conditions for staining fungi in culture 342.2.1 Solid media 342.2.2 Liquid media 342.3 Fungal biomass determinations 352.4 Wood material 362.4.1 Hardwood and softwood blocks 362.4.2 Inoculation of wood blocks 362.4.3 Sap pressed from wood chips 372.4.4 Milled wood powder 372.4.5 Wood protein samples 372.5 Proteinase activity assays 382.5.1 Kinetic constants 392.6 Inhibition studies 392.7 Electrophoretic analyses 402.8 N-terminal sequencing and amino acid analyses 41Chapter 3 Growth, nutrition and proteolytic activity ofOphiostomapiceae in culture and on wood 443.1 Introduction 443.2 Materials and methods 473.2.1 Culture medium 473.2.2 Temperature and pH studies 473.2.3 Nutritional studies 473.2.4 Wood analysis and inoculation 483.2.4.1 Extraction ofproteinase from wood 49viPage3.3 Results 503.3.1 Growth on solid media 503.3.2 Growth in liquid medium 503.3.3 Optimal pH and temperature for liquid cultures 523.3.4 Carbon and nitrogen requirements 543.3.5 Effect of different nitrogen sources on proteolytic activity 563.3.6 Nitrogen content of lodgepole pine and aspen sapwood 613.3.6.1 Untreated blocks 613.3.6.2 Blocks treated with solutions ofnitrogen 633.3.7 Growth and proteolytic activity on pine and aspen sapwood... 643.4 Discussion 68Chapter 4 Isolation and preliminary characterisation of a subtilisinlike serine proteinase secreted by Ophiostoma piceae 764.1 Introduction 764.2 Materials and methods 794.2.1 Chromatography of proteinase 794.2.1.1 Analytical separations 794.2.1.2 Preparative separations 804.2.2 Determination of glycosylation 804.2.3 pH and thermal stability studies 804.3 Results 824.3.1 Increasing proteinase production in liquid culture 824.3.2 Inhibition of proteinases produced in culture and in wood 82viiPage4.3.3 Purification of a serine proteinase 854.3.4 Properties of the purified proteinase 874.3.4.1 Electrophoretic properties 874.3.4.2 Catalytic properties 894.3.4.3 Stability after exposure to various pHand temperature regimes 924.3.4.4 Effect ofinhibitors 974.3.4.5 Effect ofmetal ions and buffer salts 994.3.4.6 Amino acid composition and N-terminal sequence... 1024.4 Discussion 103Chapter 5 Autolysis and substrate specificity of theproteinase purified from Ophiostomapiceae 1095.1 Introduction 1095.2 Materials and methods 1125.2.1 Proteinase preparation and purification 1125.2.2 Circular dichroic spectroscopy 1125.2.3 Generation of autolytic products 1135.2.4 Assay of activity againstp-nitrophenyl esters 1145.2.5 Insulin B-chain digestion 1155.2.5.1 HPLC conditions 1155.2.5.2 MS conditions 1165.2.6 Preparation of proteins from poplar 1165.2.7 Proteinase activity against proteins 117viiiPage5.3 Results 1185.3.1 Autolysis of the proteinase 1185.3.1.1 Autolysis 1185.3.1.2 Factors which affect autolysis 1225.3.1.3 Mechanism ofautolysis 1255.3.1.4 Identflcation ofautolytic cleavage site 1325.3.2 CD spectroscopy for structural comparisonwith proteinase K 1325.3.3 Proteinase active site specificity 1345.3.4 Cleavage specificity on insulin 1355.3.5 Substrate specificity on proteins 1415.4 Discussion 143Chapter 6 Targeted inhibition of the proteolytic enzymesproduced by sapstaining fungi 1516.1 Introduction 1516.2 Materials and methods 1546.2.1 Fungal strains and culture conditions 1546.2.2 Proteolytic activities 1546.2.3 Testing inhibitors of the proteinase in artificial media 1556.2.4 Testing inhibitors of the proteinase in wood 1566.3 Results 1576.3.1 Proteolytic activity on solid media 1576.3.2 Inhibition of proteolytic activity in culture filtrates 157ixPage6.3.3 Protein profiles 1586.3.4 Fungal growth and proteolytic activity in wood 1586.3.5 Inhibition of proteinases produced in wood 1616.3.6 Effect of inhibitors on growth of0. piceae in artificial media 1636.3.7 Effect of inhibitors on growth of0. piceae on lodgepole pine 1666.4 Discussion 167Chapter 7 Overview and future work 173Ieferences 180xList of tablesPageTable 3.1 Nitrogen content in lodgepole pine sapwood samples from variousheights and radial distances in the tree as determined by Kjeldahl analyses andexpressed as ppm N dry weight 62Table 3.2 Amino acid composition of lodgepole pine sapwood samples 63Table 3.3 Nitrogen content of lodgepole pine and aspen sapwood samplesafter soaking in nitrogen solutions 64Table 4.1 Effects ofproteinase inhibitors on the hydrolysis of azocoll bythe culture filtrate of 0. piceae strain 3 87N after growth on soybean protein 84Table 4.2 Purification of the extracellular proteinase produced by0. piceae strain 387 N 86Table 4.3 Kinetic constants for the hydrolysis of succinyl-Ala-Ala-Pro-Phe-p-nitroanilideby the proteinase purified from 0. piceae, chymotrypsin and a cuticle degradingproteinase from Metarhizium anisopliae 91Table 4.4 Half lives of the serine proteinase purified from culture filtrates of0. piceae strain 387N at different pH’s 94Table 4.5 Half life of the proteinase isolated from 0. piceae at pH 6 and 8,at temperatures ranging from 23 to 40°C 96Table 4.6 Effects of serine proteinase inhibitors on the hydrolysis of azocolland succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (sAAPF) by the proteinasepurified from 0. piceae strain 387N 98Table 4.7 Effects of different reagents on the hydrolysis of azocoll by theproteinase purified from 0. piceae strain 387N 99Table 4.8 Effect of metal ions and buffer salts on the hydrolysis of azocolland succinyl-Ala-Ala-Pro-Phe-p-nitroanilide by the proteinase purified from0. piceae strain 387N 100Table 4.9 Amino acid composition for the extracellular proteinase purifiedfrom 0. piceae strain 387N compared to those reported for serine proteinasesin the subtilisin and (chymo)trypsin families 101xiPageTable 5.1 Generation of autoproteolytic products from the proteinase isolatedfrom 0. piceae strain 387N during buffer exchange on a gel filtration column(1 ml) at room temperature 120Table 5.2 Effect of thermoprotectants on the half life of the proteinaseisolated from 0. piceae strain 387N, at pH 8, for temperatures rangingfrom4O to 50°C 124Table 5.3 P1 specificity of the 0. piceae proteinase and proteinase Kon p-nitrophenyl ester substrates 135Table 6.1 Effects of proteinase-inhibitors on the hydrolysis of azocoll bywood powders and culture filtrates that have been inoculated with differentstaining fungi 159Table 6.2 Proteolytic activity and ergosterol contents in wood infected withstaining fungi 162Table 6.3 Effect of heavy metals, chelators, serine proteinase inhibitors, anddetergents on growth of 0. piceae strain 387N on media supplementedwith organic and inorganic nitrogen 165Table 6.4 Effect of chelators, serine proteinase inhibitors and detergentson growth and pigmentation of 0. piceae strain 387N grown on lodgepole pine 166xiiList of figuresPageFigure 1.1 Canada’s exports of forest products in 1993 2Figure 1.2 Log harvests in British Columbia in 1993 3Figure 1.3 A pine board showing the radiating pattern of discolorationcharacteristic of sapstaining fungi 8Figure 1.4 Composition of wood where minor components includestarch, pectin, soluble sugars, proteins and ash 13Figure 1.5 Interrelationships of carbohydrate and amino acid metabolism 18Figure 1.6 The Schechter and Berger nomenclature for amino acid residues of asubstrate (P1 etc.) and corresponding subsites (Si etc.) of the active site of a protease 22Figure 1.7 Overall mechanism proposed for serine-proteinase catalysed reactions 25Figure 3.1 Growth of 0. piceae strain 387N at 23°C, in synthetic inorganicliquid medium at pH 6.1 51Figure 3.2 Growth response of 0. piceae strain 387N to temperature insemi-synthetic liquid medium with an initial pH of 5.1 53Figure 3.3 Growth response of 0. piceae strain 387N to pH at 23°Cin semi-synthetic liquid medium 54Figure 3.4 Growth of 0. piceae strain 387N on various carbon sourcesafter 3 days (shaded bars) and 5 days (solid bars) of incubation at 23°C,in semi-synthetic liquid medium with an initial pH of 6.1 55Figure 3.5 Growth of 0. piceae strain 387N on various nitrogen sourcesafter 2 days (darkly shaded bars), 4 days (shaded bars) and 7 days (solid bars)incubation at 23°C, in semi-synthetic medium with an initial pH of 6.1 56Figure 3.6 Azocoll assay showing proteolytic activity in culture filtratesof 0. piceae atpH 8 57Figure 3.7 Fungal biomass (open diamond) and depletion of starch(open circles) during growth of 0. piceae strain 387N at 23°C,in synthetic liquid media with an initial pH of 6.1 containing BSAas sole nitrogen source 58xliiPageFigure 3.8 Separation by PhastGel IEF 3-9 of the extracellular proteinsobtained after growth of 0. piceae 387N on BSA supplemented media 59Figure 3.9 Fungal biomass (solid circle) and depletion of ammonia(open square) during growth of 0. piceae strain 387N in mediasupplemented with equal proportions of nitrogen as anmionia and BSA 60Figure 3.10 Aspen and pine wood blocks 14 days after inoculation with 0. piceae 65Figure 3.11 Proteolytic activity in lodgepole pine (A) and aspen (B)sapwood samples after colonisation by Ophiostomapiceae 387N at 23°C 66Figure 3.12 The effect of pH on proteinase activity extracted frompine wood blocks colonised by 0. piceae for 18 days 67Figure 4.1 Fungal growth (dry weight) and extracellular proteinase productionby 0. piceae strain 387N in synthetic medium (section 2.2) containingsoybean protein as a nitrogen source 83Figure 4.2 FPLC fractionation of proteins in the culture filtrate of 0. piceaestrain 387N using a Phenyl Superose HR5/5 column 86Figure 4.3 SDS-PAGE (8-25% PhastGel) showing the purification of themajor proteinase secreted by 0. piceae strain 387N after growth on mediasupplemented with soymilk protein 87Figure 4.4 IEF (PhastGel 3-9) monitoring the purification of the extracellularproteinase from 0. piceae strain 387N 88Figure 4.5 The effect ofpH on the activity of the extracellular proteinasepurified from 0. piceae as determined at 37°C using azocoll (A) andsuccinyl-Ala-Ala-Pro-Phe-p-nitroanilide (B) as substrates 90Figure 4.6 The effect of temperature on the activity of the extracellular proteinasepurified from 0. piceae as determined at pH 8 using azocoll (A) and succinyl-AlaAla-Pro-Phe-p-nitroanilide (B) as substrates 92Figure 4.7 Effect of pH on the stability of the proteinase isolated from culturefiltrates of 0. piceae strain 387N 93Figure 4.8 Effect of temperature on the stability of the proteinase isolatedfrom culture filtrates of 0. piceae strain 3 87N 95Figure 4.9 Residual proteolytic activity after 30 mm incubation with DFP 97xivPageFigure 4.10 N-terminal sequence of the proteinase purified from 0. piceaestrain 387N and comparison with sequences reported for fungal serineproteinases that are in the subtilisin class II family 102Figure 5.1 SDS-PAGE (8-25% PhastGel) showing autolysis of theproteinase isolated from 0. piceae strain 387N during gel filtration,generating low molecular weight cleavage products 119Figure 5.2 SDS-PAGE (8-25% PhastGel) showing thermal instabilityof the proteinase isolated from 0. piceae strain 387N 121Figure 5.3 Melting curve and first derivative for the proteinase purifiedfrom culture filtrates of 0. piceae strain 387N in 20 mM Tris-Cl (pH 8) 127Figure 5.4 Melting curve and first derivative for the proteinasepurified from culture filtrates of 0. piceae strain 387N in 0.1 Mphosphate buffer (pH 7), 0.5 M KC1 128Figure 5.5 Melting curve and first derivative for the proteinasepurified from culture filtrates of 0. piceae strain 387N in20 mM Tris-Cl (pH 8) containing 27 mlvi CaCl2 129Figure 5.6 Melting curve and first derivative for the proteinasepurified from culture filtrates of 0. piceae strain 387N in 20 mMTris-Cl (pH 8) containing ammonium sulphate (1M) 130Figure 5.7 N-terminal amino acid sequence of the 14 kDa fragment fromthe autolysis of the 33 kDa proteinase isolated from 0. piceae strain 3 87N 131Figure 5.8 Far and near UV circular dichroic spectra of proteinase K (solid)and the proteinase isolated from 0. piceac (dotted) measured in 0.1 Mphosphate buffer (pH 7) containing 0.5 M KC1, in a cell with a pathlengthof 1 mm (far) and 1 cm (near) 133Figure 5.9 Mass spectra (TIC) chromatograms of proteolytic digests ofinsulin B-chain after 5 minutes (A), one hour (B) and overnight incubations (C) 136Figure 5.10 Digestion of insulin B chain (peak E) by the proteinaseisolated from 0. piceae strain 387N 138Figure 5.11 Tandem MS spectra for the parent ion mlz 1091 139Figure 5.12 Sites of cleavage of oxidised insulin B chain by various serine proteinases.... 140xvPageFigure 5.13 SDS-PAGE (8-25% PhastGel) showing degradation of caseinby the proteinase purified from 0. piceae strain 387N and proteinase K 142Figure 5.14 SDS-PAGE (8-25% PhastGel) showing degradation ofproteins extracted from poplar by the proteinase purified from 0. piceaestrain 387N and proteinase K 142Figure 6.1 Proteinase and aminopeptidase activities and ergosterol contentin lodgepole pine sapwood inoculated with 0. piceae 212375 160Figure 6.2 0. piceae strain 387N colonies (C) on media supplemented withskim milk (A) and NH4O3(B) 163xviList of AbbreviationsAEBSF aminoethylbenzene-sulphonylfluorideAla or A alanineMg arginineAsn asparagineAsp aspartic acidAsx asparagine or aspartic acidASTM American Society for Testing and MaterialsATAPNA N-acetyl-Ala-Ala-Ala-p-nitroanilideB cysteic acidBAPNA N-a-benzoyl-DL-Arg-p-nitroanilideBOC t-butyloxycarbonylBSA bovine serum albuminBTPNA N-a-benzoyl-L-Tyr-p-nitroanilideCAPS 3-(cyclohexylamino)- 1 -propanesuiphonic acidCBZ carbobenzoxy, benzyloxycarbonyleDNA copy deoxyribonucleic acidCD spectroscopy circular dichroic spectroscopyCu-8 coppper-8-hydroxyquinolinolateCys or C cysteineD aspartic acidxviiDa DaltonDCI 3,4-dichioro-isocoumarinDDBSA dodecyl benzene sulphonic acidDFP di-isopropyl-fluorophosphateDMSO dimethyl suiphoxideDTT dithiothreitolE glutamic acidE-64 L-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butaneEDTA ethylenediaminetetraacetic acidELISA enzyme-linked immunosorbent assayER endoplasmic reticulumESI MS electrospray ionisation mass spectrometryF phenylalanineFPLC fast protein liquid chromatographyGln glutamineGlu glutamic acidGix glutamic acid or glutamineGly or G glycineh hour(s)His or H histidineHPLC high performance liquid chromatographyIEF isoelectric focussinglie or I isoleucinexviiiK lysinekcat the turnover number or catalytic constant of the enzymekcat/Km specificity constantkDa kilodaltonKm the Michaelis constantLC/MS liquid chromatography mass spectrometryLeu or L leucineLys lysineMeCN acetonitrileMES 2-(N-morpholino)ethanesulphonic acidMet or M methioninemm minute(s)MS mass spectrometryMS/MS tandem mass spectrometryMW molecular weightN asparagineO.D. optical densityPAGE polyacrylamide gel electrophoresisPBS phosphate buffered salinePC paper chromatographyPhe phenylalaninep1 isoelectric pointPMSF phenylmethanesulphonyl fluoridexixPQ-8 a commercial antisapstain formulation containing DDBSA and Cu-8Pro or P prolinePVDF polyvinylidene difluorideQ glutamineR argininerDNA ribosomal deoxyribonucleic acidrpm revolutions per minuteRT room temperaturesAAPF succinyl-Ala-Ala-Pro-Phe-p-nitroanilideS.D. standard deviationSDS sodium dodecyl sulphateSer or S serineTCA trichioroacetic acidTFA trifluoroacetic acidThr or T threonineTLC thin layer chromatographyTPCK tosyl phenylalanyl chloromethylketoneTPEN N,N,N’,N’-tetrakis(2-pyridylmethyl) ethylenediamineTricine N-[ tris-(hydroxymethyl)-methyl]glycineTris tris-(hydroxymethyl)-aminoethaneTrp tryptophanTyr tyrosineVal or V valineVm maximum velocityVol volumeW tryptophanw/w weight by weightw/v weight by volumeY tyrosinexxxxiAcknowledgementsI would like to express my appreciation to Dr. Colette Breuil for her dedication in supervising thisproject. I valued her enthusiasm and interest in the work, and her willingness to generously give ofher time. I have also benefited enormously from all the members of the Chair of Forest ProductsBiotechnology group, both past and present. A special thanks to Ken Wong, Alex Yu, MikeChester and fellow graduate students who encouraged me through the “occasional” rough patchesand to my industrial mentors Paul Morris and Tony Byrne for their willingness to share their yearsof experience.I am especially grateful to people who made various scientific resources available: Dr. J. Daviesand Kevin Chow, Department of Microbiology and Immunology at UBC for use of thedensitometer, Dr. G. Mauk and Dean Hildebrand, Department of Biochemistry and MolecularBiology at UBC for access to CD spectroscopy, Forintek Canada for use of the micro-Kjeldahland testing facilities, and Dave Chow and Hamish Morrison, Biomedical Research Centre, UBCfor introducing me to the power of LC/MS. I would also like to thank Aaron Roth, ClaudiaYagodnik, Eric Yung, Dave Bradshaw, Elizabeth Molitor and Hector Gamboa for their input inthe project.Funding for this project was gratefully received through a strategic grant from Natural Sciences andEngineering Research Council of Canada, and a Graduate Research Engineering and Technologyaward from the Science Council of British Columbia. Industrial support was received fromForintek Canada Corp., Canada.I extend my thanks to my parents, family and friends for their encouragement, and to Ken, whobelieves in my ability even when I doubt it. Finally, my faith in God has sustained and supportedme through all my academic endeavours, and for that I am truly grateful.1Chapter 1.General introductionThis thesis describes the functional characterisation of a proteinase from an economicallyimportant sapstain fungus that discolours wood. The work described is part of a long-termprogramme towards developing environmentally acceptable control treatments for such fungi.While there is a considerable amount of information available in the literature on the compositionof wood, and on the physiological and biochemical features of decay fungi, there is little availableon sapstaining fungi. Effective wood protection will be impossible without a more thoroughunderstanding of the biology of sapstaining fungi (Seifert, 1993). Chapter 1 begins with adiscussion of the problem of sapstain in industry, focusing on the Canadian lumber industry. Thisis followed by a brief review of the fungi responsible for the discoloration, again emphasising thefungi isolated in Canada. The nitrogen availability in wood and nitrogen metabolism by fungi isalso summarised. Proteinase production by wood-colonising fungi and the classification ofproteinases is discussed, highlighting research on the serine proteinases. Finally the researchapproach and objectives are outlined.1.1 Problem of sapstain from an industrial perspectiveCanada’s forests make up about 11.6% of the world total and cover an area larger than all ofwestern Europe (COFI, 1994). The forests and the economic, environmental and social benefitsthey provide are part of the foundation of the Canadian way of life. In 1993, forest productscontributed $23.7 billion to the Canadian trade balance (COFI, 1994) - greater than any other2single industrial sector. Lumber constitutes more than one third of the value of forest products thatare exported (Figure 1.1).Total value of Production: $40.3 BillionTotal value of Exports: $26.6 Billionlumber 36.4%wood pulp 17.5%roundwood 1.4% panel products, 6.8%logs and chips shakes/shinglesand miscellaneousnewsprint, paper 37.9%and otherFigure 1.1 Canada’s exports of forest products in 1993 (Source: Statistics Canada)In fact, Canada is the largest exporter of softwood lumber in the world, controffing 5 5.0% of themarket in 1992 (COFI, 1994). British Columbia is the largest contributor to Canada’s softwoodlumber exports, accounting for 32.4% of world exports. Most of the lumber produced in Ontarioand Québec is used in Canada rather than being exported, whereas less than 25% of the woodproducts from B.C. were used in Canada (COFI, 1994). Spruce, lodgepole pine and true fir makeup 60% of the logs harvested in B.C., while hemlock, cedar and Douglas fir cumulatively accountfor 36% (Figure 1.2)(COFI, 1994). The major markets for forest products (lumber, plywood,shingles/shakes, millwork) from B.C. include the U.S.A., Japan, and the European community. In1993, these markets received 46.8%, 21% and 4.3% of B.C. shipments respectively. Some of theI3overseas markets require clear, unseasoned, defect-free lumber and pay higher prices for thesepremium grades.B.C. Log harvest- 1993Total: 79 239 Million cubic Coast: 25 684 Million cubic Interior: 53 555 Million cubicmetres metres metresMillion cubic metres20,000Figure 1.2 Log harvests in British Columbia in 1993(Source: Statistics Canada and B.C. Ministry of Forests)Therefore one of the challenges facing the lumber industry in B.C., as the nation’s prime exporter,is to keep wood free of discoloration during processing of logs, storage of the lumber andtransportation of logs and lumber to the market place. Although there has been a concerted effortII) D ‘-C) Oit. -g.2 a) ID U)E 0iz0.2 w1_ 0)’.-010.a)0)0-ja) a) • •-g0 C) LL.D a)-g U) 0. EO )00!a)1 )E01 U)810)00-j4to export clean wood, the industry has periodically sustained significant losses in insurance claimsdue to the discoloration caused by sapstain fungi and moulds. Actual costs to the industry aredifficult to obtain because most claims are confidential and they may not be exclusively attributedto damage by staining fungi and moulds. Upon enquiry, for the month of June in 1995, loss ofvalue in lumber exports from B.C. was about 1 million dollars in three claims alone (Tony Byrne,Forintek Canada Corp., personal communication). Over and above monetary issues, repeatbusiness with the same customer may be jeopardized (Smith, 1991). Stains on wood can beabiotic (e.g. iron stains), but for wood with a moisture content greater than 20% in the sapwoodregion, the most common stain is due to colonisation by fungi. This type of discoloration isreferred to in the industry as “blue stain” or “sapstain” or simply “stain”. In the context of thisthesis these terms will always refer to discoloration of the sapwood of logs or lumber due to thepresence of pigmented fungal hyphae.The damage to wood from sapstain is cosmetic, in contrast to structural damage produced by softrot or decay fungi (Eriksson, 1981). Sapstaining fungi have little effect on the strength propertiesof wood (Liese, 1970), generally only causing small losses in dry weight. The only majormechanical concern is the use of stained wood for applications requiring impact toughness(Scheffer, 1973; Subramanian, 1983). This property can be reduced by up to 30% in heavilystained pine (Chapman and Scheffer, 1940). However, sapstain is of significant economicimportance since the cosmetic discoloration is objectionable to buyers (Wilcox, 1973; Zabel andMorrell, 1992).5Several strategies have been used in different parts of the world to prevent losses caused bysapstain. In addition to implementation of preventative logging practices such as avoiding storageof material during high risk periods (e.g. in the warmer summer months), rapid handling of logs inwarmer climates, water storage and saturating log piles by sprinkling (Findlay, 1959; Phillips andBurdeken, 1982; Dickinson, 1988), chemical treatment and kiln-drying have been used to protecttimber during transportation and temporary storage (Byrne and Smith, 1987).In 1994, approximately 3 billion board feet of softwood lumber, with an export value exceeding$2.9 billion, was treated with antisapstain chemicals in B.C. (COFI, 1995). The market forantisapstain chemicals in Canada is currently worth about $16 to $20 million annually. The mostwidely used chemical over the past 50 years has been sodium pentachiorophenate (PCP), howeverfindings concerning the toxicity and environmental fate of the dioxin contaminants of PCP havenow severely restricted its acceptance in Europe, Pacific Rim countries and North America.Alternative chemicals have failed to satisfy an industry that had become used to the very effectivechlorinated phenols (Seifert, 1993).The ideal antisapstain product would be effective to give a 12 month shelf life to treatedlumber. To avoid toxicity to man and the environment, it should specifically target the fungicausing discoloration. Other desirable properties are that it be non-corrosive, non-leachable,water-soluble and easily handled (Zabel and Morrell, 1992). The demands on antisapstainchemicals have grown over the years. Concerns about safety with respect to the environmentand the lumber treating process have become increasingly important. However, anychemicals proposed still need to be cost effective to provide an affordable end product.6In Canada increased emphasis has been placed on formulations containing environmentallyfriendly chemicals, such as borax, soda, quaternary ammonium compounds (e.g. didecyldimethylammonium chloride), and triazoles (e.g. azaconazole and hexaconazole) (Smith,1991). There are currently seven registered active ingredients used in various formulations(Konasewich and St. Quintin, 1994), but none have all the properties of the ideal protectant asdescribed earlier.Kiln-drying has been used to prevent sapstain by reducing the moisture content of wood to levelswhere fungi are unable to grow. However, kiln-drying is only feasible in specific circumstancesbecause it can cause drying defects and it is not suitable for lumber of large dimension.Furthermore, it is expensive and the cost cannot always be recovered by increasing lumber prices(Byrne and Smith, 1987; Rayner and Boddy, 1988). The success of this treatment is alsodependent on ensuring that all the timber is adequately dried and remains so for the entire periodof transit and storage. In some countries (eg. Finland) sawn softwood is treated with antisapstainchemicals even when it is kiln-dried, as an insurance against rewetting during transportation(Dickinson, 1988).Despite a century of concern, sapstain of wood remains a serious problem for the lumber industry.Most research has concentrated on chemical protection of lumber and there is limited informationavailable on the biology and ecology of sapstaining fungi. A basic understanding of the organismsresponsible for stain and their metabolic requirements would facilitate the development of abiorational approach to preventing sapstain.71.2 Fungi causing sapstain and other wood-colonising fungiDiscoloration of lumber is caused mostly by saprobic fungi growing in and staining the sapwoodafter wood is cut (Figure 1.3). Sapstain caused by pathogenic or endophytic fungi in the livingtree may be less costly for the industry because infected wood can be discarded before or duringprocessing (Seifert, 1993). Growth of saprobic fungi is more insidious because colonisation of thewood can occur at any time after the tree is felled. Sapstain then becomes evident only when thewood is exposed to conditions favourable for fungal growth.Most staining fungi grow primarily on the nutritive substances in the parenchyma cells of thesapwood producing pigmented hyphae (Mathiesen-Käarik, 1960, Ballard et al., 1982) due tomelanin formation (Zink and Fengel, 1988). Various colours occur, although the most commonlyseen are bluish to bluish-black and sometimes brown (Zabel and Morrell, 1992). Artists in the15th century recognised the unique place for stained wood in intarsia masterpieces (Blanchette eta!., 1992), and stained wood is frequently used by Swedish wood workers for its artistic effects.Others have investigated its use for producing attractive violins (Seifert, 1993).Historically, many fungal species have been associated with stain of living trees, logs andprocessed lumber. However, if the problem is confined to the saprobic fungi then the fungi mostoften implicated are from the genera Ceratocystis and Ophiostoma and their anamorphs. Mouldsand black yeasts are also important (Seifert, 1993); especially since it is not unusual to find severaldifferent fungal species growing in close proximity on a single piece of wood. Different sapstain8species predominate in different geographical areas and although some species are limited tocertain timbers, most are found on a variety of timbers (Seifert, 1993).Figure 1.3 A pine board showing the radiating pattern of discolorationcharacteristic of sapstaining fungi (courtesy of R. Smith, Forintek Canada Corp.)In Canada, some work has been done on the taxonomy of staining fungi isolated from trees andlumber (Griffin, 1968), but published work on the prevalence and distribution of sapstaining fungiin Canadian wood is scant. Nevertheless, a survey conducted by Forintek Canada Corp. indicatedthat staining fungi were isolated from most of the economically important wood species inCanada, including those in the genera Abies, Pinus, Picea, Larix, Pseudotsuga, Populus and Tsuga9(Seifert, 1993; Seifert and Grylls, 1991). In this survey, the fungi were categorised asOphiostomatales e.g. Ophiostorna and Ceratocystis spp., black yeasts e.g. Aureobasidiumpullulans, dematiaceous moulds e.g. Cladosporium spp., or green moulds e.g. Peniciiium spp.One of the key conclusions from the survey was that Ophiostoma piceae is the most frequentsapwood inhabiting fungus on Canadian lumber, occurring on more than 50% of the samplesexamined (Seifert and Grylls, 1991).Black yeasts and dematiaceous moulds appeared to be the least important categories of sapwoodinhabiting fungi. However, they may be more important at different times of year. Black yeasts,such as Aureobasidium pullulans, are capable of causing significant sapstain, particularly of woodin service (Rayner and Boddy, 1988, Sharpe and Dickinson, 1992; Zabel and Morrell, 1992).They produce wind dispersed spores in drier environments, and slimy yeast cells that disperse inwater films or within the cells of a living tree in wet conditions (Seifert and Grylls, 1991).Discoloration by dematiaceous moulds is generally caused by masses of dark spores which can beremoved by planing, but penetration by pigmented hyphae is also common. Examples includeCladosporium and Alternaria, which produce dry masses of darkly pigmented conidia that areprobably mostly transmitted by air currents. Species of Altemaria produce a dark, penetratingstain that is similar to that produced by some members of the Ophiostomatales.The green moulds discolour wood by forming masses of pigmented asexual spores on the woodsurface (Wilcox, 1973). Because these spores can usually be brushed or planed off withoutleaving any residual discoloration, the green moulds are not usually considered sapstain fungi(Seifert, 1993). They are generally of minor economic importance in the wood industry and they10are primarily a factor in very wet wood (Zabel and Morrell, 1992). Common mould fungi includeAspergillus spp., Fusariurn spp., Gliocladium spp., Monilia spp., Penicilliurn spp., Rhizopus spp.,and Trichoderma spp.Members of the Ophiostomatales, including species of Ceratocystis and Ophiostoma, and theiranamorphs were prevalent on all woods sampled in the Forintek survey (Seifert and Grylls, 1991).Although the taxonomy of these fungi has been somewhat controversial, there appears to begeneral agreement that Ceratocystis and Ophiostoma represent discrete genera on the basis ofdifferences in cell wall composition, sensitivity to cycloheximide (Przybyl and Dc Hoog, 1989),ascospore shape, anamorph structure (De Hoog and Scheffer, 1984) and, more recently, partialrDNA sequences (Hausner et al., 1993). In this thesis, the nomenclature for species of these twogenera is consistent with that described by Seifert et a!., (1993). In particular, piceae as anaccepted species of Ophiostoma is in agreement with the body of literature currently available.The Ophiostomatales are carried by bark beetles or mites that live on bark beetles (Bridges andMoser, 1983; Seifert and Grylls, 1991). Some of the associations with insects are very specificand are restricted to single species of insect and fungus; in others, one species of fungus may beassociated with several insects or vice versa (Leach, 1940; Käarik, 1960). These fungi produceascospores in dark sexual fruiting bodies known as perithecia and conidia in asexual structuresknown as symiemata. In both cases the spores are released in a mudilagenous matrix which istransported via insect vectors (Kaarik, 1971; Dowding, 1970). These fungi invade and discolourwood during log storage and the initial stages of lumber seasoning (Zabel and Morrell, 1992).Pigmented hyphae spread primarily through the sapwood rays, although they can also spread11through the longitudinal tracheids or vessel elements (Findlay, 1959; Liese, 1970; Ballard et a!.,1984). Hyphae penetrate through pits in wood cell walls, but occasionally produce fme bore holesas well. Most experimental work suggests that the bore hole formation is by mechanical means,with only a few studies implicating enzymatic processes (Wilcox, 1973).The patterns of colonisation of untreated lumber and felled logs are quite similar in that bacteria,stain and mould fungi are isolated early on, followed by soft-rot fungi and finally decay fungi(Butcher, 1968; Clubbe, 1980; Rayner and Boddy, 1988). The establishment of decay fungi isaccompanied by a second influx of moulds which utilise cellulose or the breakdown products ofdecay fungi (Clubbe 1980). Several studies cited by Rayner and Boddy (1988) suggest thatdiscoloration is a necessary first stage before decay can develop. The early-colonising blue stainfungi may modify the wood substrate and make it susceptible to subsequent attack by rot fungi.Several explanations have been offered: (1) removal of some of the natural fungal inhibitors inwood, for example, Aureobasidiumpullulans is known to degrade phenolics (Bjurman, 1988); (2)a variety of physical and chemical changes in wood (Swift, 1976); or (3) depletion of carbonsources (Garrett, 1963).Because succession patterns are complex and variable, it is difficult to unequivocally establishexact patterns. This is due to the uncertainly of reliably isolating all the principal microorganismsinvolved (Zabel and Morrell, 1992). Despite this, three major points emerge from these studies:staining fungi cause aesthetic damage to wood, they are possibly involved in preconditioninglumber for decay, and Ophiostomatales are the most prevalent staining fungi on Canadian lumber.Therefore, an Ophiostoma species was selected as the model organism for this research.121.3 Wood as an environment for sapstaining fungiWood structure and the nutrients available in wood affect the growth and survival of fungi thatcolonise the wood. This section will begin with a description of the anatomical features in woodwhich affect fungal growth, followed by a description of the nutrients available in wood. Sapstainfungi grow in wood mainly through the natural system of passages in the wood. Natural passagesare provided by axial and radial elements that provide for the distribution of water and nutrientswithin the xylem of the living tree. Vessels and tracheids, which function in the conduction ofwater as well as providing mechanical strength, provide the major axial route for mycelialdevelopment. In angiospennous wood, known as hardwoods, the vessel elements vary in quantity,size and distribution pattern, all of which can affect fungal colonisation (Rayner and Boddy,1988). Gymnospermous wood, known as softwoods, have a more homogenous distribution oftracheid elements. In general, tracheids do not attain the dimensions possible in vessels and thusprovide a less effective pathway for axial spread than vessels. An important feature of vessels andtracheids is the occurrence of pits in the lateral walls which provide the main opportunity fortransverse passage between elements. The size, structure and distribution of pits is therefore asignificant factor affecting the accessibility ofwood to fungal hyphae.Although pits provide a limited degree of radial access, this is insignificant in comparison with theopportunities afforded by the radially elongated elements which constitute the ray system. Theseelements include parenchyma cells which may contain living protoplasts, especially in functioningsapwood. These cells act as sources of nutrients because they contain assimilable substrates, andas radial passages for fungi. Rays provide a direct route for fungal colonisation originating in bark13tissues. Sapstaining fungi which colonise the ray parenchyma produce characteristically wedge-shaped columns of stain (Figure 1.3).Wood is basically composed of the structural polymers cellulose, lignin and hemicellulose whichmake up the cell walls. Cellulose microfibrils are coated with hemicellulose and embedded inlignin to form lignocellulose (Figure 1.4). Lignocellulose is a complex polymer mixture, rich incarbon but poor in other essential nutrients for fungi (Carlile and Watkinson, 1994).cellulose 41.0%hemicellulose 26.0%minor 2.0%componentsextractives 4.0%Figure 1.4 Composition of wood where minor components include starch, pectin,soluble sugars, proteins and ash (after Fengel and Wegener, 1989)Compounds such as soluble sugars, starch, extractives, proteins and peptides occur in relativelysmall amounts (often less than 10% of the dry weight), and are found almost exclusively in livinglignin 27.0%14or recently dead sapwood parenchyma (Rayner and Boddy, 1988). Besides the structural cell wallcomponents, primary metabolites and storage compounds, wood contains a wide range ofextractive material. Extractives include waxes, fats, fatty acids and alcohols, steroids, highercarbon compounds and resins. The chemical composition, distribution and quantity of extractivesvaries between different species. Generally they are found in larger quantities in heartwood thanin the sapwood (Fengel and Wegener, 1989). They can affect the growth of fungi in three ways:as carbon sources, inhibitors or growth stimulants (Rayner and Boddy, 1988). The maincomponents of wood ash are potassium, calcium and magnesium (Fengel and Wegener, 1989).Among the nonstructural nutrients in wood, nitrogen is thought to play the most important role(Highley and Kirk, 1979).The carbon to nitrogen ratio of wood varies from about 350:1 to 1250:1 (Merrill and Cowling,1966), depending on the tree species, the individual tree, the part of the tree, the location of thetree and the time of year. At 0.01 to 0.3 % of the dry weight ofwood, little nitrogen is available tofungi colonising wood. These low nitrogen values are growth-limiting factors for fungi invadingwood (Merrill and Cowling, 1966; Levi and Cowling, 1969).Work by King et a!. (1976) and Boutelje (1990), indicated that during the drying of lumber, thesoluble nitrogen migrates to the wood surfaces, where nitrogen can accumulate to concentrationsfive times higher than in green wood. The nitrogen in wood can affect the competition betweenwood microflora and consequently the microbial activity. For example, it may block theproduction of certain enzymes and enhance the breakdown of available cellulose by decay fungi15(Fog, 1988). Theander et at., (1993) have also shown a good correlation between wood nitrogen,sugar contents and the growth of moulds at the surface of lumber.Most of the nitrogen is present in organic form, primarily as proteins (Chapin and Kedrowski,1983; Dill et a!., 1984; Wetzel et at., 1989b; Langheinrich and Tischner, 1991; Sauter andvan Cleve, 1990; Sauter et at., 1989). It is well known that trees store considerable amounts ofnitrogenous compounds such as proteins, amino acids, and nucleic acids in the parenchyma cellsof wood and bark (Laidlaw and Smith, 1965; Kramer and Kozlowski, 1979). The storageproteins are present in large quantities during winter and absent during summer (Langheinrichand Tischner, 1991). It is assumed that with budbreak, these proteins are degraded intoamino acids, which are then translocated to the growing tissues of the trees. In some woodspecies, protein bodies in the phloem and xylem ray parenchyma have been suggested to beanalogous to protein bodies of seeds (Wetzel et at., l989a). These have been shown to be thesites for the storage of specific proteins in several hardwoods and softwoods (Sauter and vanCleve, 1990; Wetzel and Greenwood, 1991). Thus overwintering protein storage in trees mayfollow sequences similar to protein deposition and subsequent catabolism during seeddevelopment and germination (Wetzet et at., 1 989a).There is not a lot of information on the amino acid composition of storage proteins found inwood, bark and leaves of trees. Most of the recent work has focused on the regulatorymechanisms controlling the production of storage proteins. However, Coleman and Chen(1993) isolated and sequenced a gene encoding a 32 kDa poplar bark storage protein, and16they showed by the derived amino acid sequence that the protein is rich in serine, leucine,phenylalanine and lysine (Coleman et at., 1992).1.4 Nitrogen metabolism in fungiNitrogen is found in nearly all complex macromolecules of all living cells and is a majorcomponent of proteins and nucleic acids. It is required to synthesise amino acids, the buildingblocks for structural proteins and enzymes. Nitrogen is an important component of the fungalprotoplasm, contributing between 2 and 5% of the dry weight, depending on the environmentalconditions and the age of the mycelium (Moore-Landecker, 1982). Thus, the nature and quantityof nitrogen available in the fungal environment will control the development of the fungus.Absence of available nitrogen may lead to autolysis or sporulation.Organic nitrogen in the form of amino acids and inorganic nitrogen in the form of ammonia can beutilised by most fungi, whereas other inorganic forms (nitrates, nitrites) may not support fungalgrowth (Jennings, 1989). Those fungi which are able to utilise nitrates take up the nitrate ion byactive transport, reduce it to the oxidation level of ammonia, and assimilate it into organiccompounds. Nitrate is not utilised by Aspergillus nidulans or Neurospora crassa unless the cellsare depleted of favoured nitrogen compounds namely ammonia, glutamate, or glutamine.Molecular biology studies have confirmed that the genes encoding the nitrate reductase enzymeare controlled at the level of transcription (Marzluf, 1993). Fungi that are unable to utilise nitrates,presumably because they cannot reduce the nitrate ion, may use nitrogen in the form of theammonium ion or in the form of organic nitrogen that has the same oxidation level as the17ammonium ion. Most of the amino acids assimilated by fungi are used directly or by initialdeamination.The principal route of amino acid catabolism is oxidative deamination, but a number of othermechanisms exist. For example, asparagine may be hydrolysed to aspartic acid and ammonium,and threonine may be cleaved to glycine and acetaldehyde. The ammonia freed by thedeamination reaction or the inorganic nitrogen enters into the amino acid biosynthetic pathway bycombining with cx-ketoglutaric acid (Moore-Landecker, 1982). Alternatively, this reaction mayfunction in reverse, in that glutamic acid may lose the amino group as ammonia, and the carbonskeleton of the amino acid is introduced into the tricarboxylic acid (TCA) cycle as cL-ketoglutaricacid, which is then oxidised (Figure 1.5). Transamination may convert amino acids into otheramino acids.18GlucoseProteinA]Pyruvic acid + NH3 •::::::::::. ProteinAmino NH + Oxaloacetic3acids .. acidfNH3 + Fumaric Amino acidsacidX—ketogIutaricacid+ L-glutamic acidNH3Figure 1.5 Interrelationships of carbohydrate and amino acid metabolism. Thoseconversions which are important in fungi are indicated by solid lines, while those inother organisms are indicated by a dashed line (from Moore-Landecker, 1982)Fungi actively decompose proteins in nature to their component amino acids, which can beassimilated. The utilisation of exogenous proteins by fungi requires the extracellular release ofenzymes and the enzymatic degradation of protein to peptides and amino acids before cellularuptake. Proteinase production in Aspergilli and N crassa is regulated by carbon, nitrogen, sulphuror phosphorus catabolite repression (Cohen, 1980; North, 1982). In Aspergillus starvation for anyone of these elements is sufficient to induce proteinase production; whereas in N crassa proteinmust also be present. Fungal growth can also be supported with small peptides generated fromprotein breakdown (Jennings, 1989), since an oligopeptide transport system capable ofTCA cycle19transporting peptides with up to five amino acid residues has been reported (Wolfinbarger, 1980).However, peptidases are widely distributed in fungi and presumably function to cleave off aminoacids from peptides and proteins either in the cell or prior to uptake (Breddam, 1986).Finally, several studies have shown that certain Basidiomycetes are able to utilise proteins as thesole source of carbon, nitrogen and sulphur (Kalisz et al., 1986, 1987). In these fungi, unlikeAspergillus and N crassa, proteinase activity was not repressed by the presence of glucose,ammonium and sulphate in the medium.1.5 Fungal growth and secretion of enzymesWith the exception of unicellular yeasts, fungi typically grow by means of hyphae that extend onlyat their apices and ramify into a mycelium (Bartnicki-Garcia, 1968). By extending at their apices,the hyphae can penetrate solid substrata such as wood, at the same time secreting the lytic enzymeswhich convert substrata polymers into products small enough to be taken up as nutrients (Wessels,1993; Wood, 1985). The efficacy of fungal hyphal spreading is enhanced by the ability of thehyphae to continue apical growth in non-nutritive substrata by translocating water and nutrientsfrom a food base (Rayner, 1991; Wessels, 1993). This ability for translocation also permits thedevelopment of aerial structures such as fruiting bodies of basidiomycetes.Various fungal genera have the dimorphic capability of changing their growth form betweenmycelial and yeast, depending on the environmental conditions. Factors such as nutrient regimes,p1-I, temperature and chemical inducers are capable of influencing the balance between yeast and20hyphal growth (Kulkanü and Nickerson, 1981; Gow, 1994). Culture methods often manipulatemany of these factors and may lead to changes in the morphological forms, particularly whenusing agitated liquid cultures. The predominance of yeast cells in liquid media is in contrast to thefilamentous growth which occurs in wood. Proteinases are likely involved in the regulation ofenzymes required for the synthesis of cell wall polymers during the mycelial form of growth. Forexample, in the case of chitin synthesis, the onset of activity and the life span of chitin synthetasemay be regulated by proteolysis (Deshpande, 1992).Filamentous fungi secrete a broad spectrum of enzymes, with the majority being hydrolyticenzymes that play an important role in nutrition, releasing carbon and nitrogen locked in insolublecompounds (Wood, 1985). Cytological studies carried out more than two decades ago providedcircumstantial evidence that secretion of proteins by filamentous fungi was probably restricted tothe tips of growing hyphae. Evidence supporting this hypothesis has been obtained byimmunocytochemical methods for the secretion of glucoamylase by Aspergillus niger and forenzymes involved in lignin degradation by Phanerochaete chrysosporium (Peberdy, 1994).The secretory pathway begins intracellularly in the lumen of the endoplasmic reticulum wherepost-translational processes are initiated. Vesicles carry molecules to the Golgi system, or itsequivalent, where the processing continues. Finally vesicles and/or vacuoles transfer the proteinsto the tip of the growing hyphae where they fuse with the plasma membrane, releasing theircontents into the periplasmic space (Wessels, 1993). Enzymes are released from the surface of theplasma membrane into the periplasmic space, where they may be incorporated into the cell wallor, in many instances, may be secreted across the cell wall into the external medium.21Before proteins are secreted, they undergo several post-translational modifications: (1) proteolyticcleavage to remove the signal sequence and a propeptide sequence, if present; (2) folding processinvolving the formation of disuiphide bonds to develop the tertiary and quaternary structures of theprotein; and (3) glycosylation (Halban and Irminger, 1994). Therefore, secreted proteins undergotwo important proteinase cleavage reactions. The first is the removal of the signal peptidesequence, possibly by an endoproteinase in the lumen of the endoplasmic reticulum. The second isthe activation of enzymes, many of which are synthesised as inactive zymogens (Peberdy, 1994).1.6 Proteinase enzymesFungi from different taxonomic classes produce extracellular and intracellular proteinases. Inaddition to a role in nutrition, proteinases have other functions such as degrading fungal proteinsfrom senescent mycelium, penetrating host tissue during pathogenesis (North, 1982), andregulating cellular functions (e.g. protein turnover, translocation, sporulation, germination)(Deshpande, 1992). The ability of proteolytic enzymes to carry out selective modification ofproteins by limited cleavage means that proteinases are well suited to a regulatory function (North,1982). Proteinases or endopeptidases are the major subject of this thesis. However, a completeunderstanding of proteolysis should also consider the action of exopeptidases (aminopeptidases,carboxypeptidases and dipeptidases). These will be covered briefly where necessary.In 1967, Schechter and Berger introduced a system of nomenclature to describe the interaction ofproteases with their substrates. In this system, the binding site for a polypeptide substrate on aprotease is envisioned as a series of subsites in the active site; each subsite interacting with one22amino acid residue of the substrate. By convention, the substrate amino acids are called P and thesubsites on the protease that interact with the substrate are called S. The amino acids residues onthe amino-terminal side of the scissile bond are numbered P1, P2, P3 as shown in Figure 1.6.reactive site of substrateor inhibitorN-terminal...-..—---i P4 i P P2 V 1P1 j P2’f_ . C-terminascissile bondactive site of enzymeFigure 1.6 The Schechter and Berger nomenclature for amino acid residuesof a substrate (P1 etc.) and corresponding subsites (Si etc.) of the active siteof a protease. The arrow indicates the peptide bond to be split by theenzyme (the scissile bond or the reactive site peptide bond).The basis for classifying proteinases is evolving. In the past proteinases were classified on thebasis of the pH range over which they are active (acid, neutral or alkaline), on the basis of theirability to hydrolyse specific proteins (keratinase, elastase, collagenase etc.), and on the basis oftheir similarity to well characterised proteinases such as pepsin, trypsin, and chymotrypsin (North,1982). Presently, the most satisfactory classification scheme is that proposed by Hartley (1960).This scheme is based on catalytic mechanisms and forms the basis for the Enzyme Commission23classification. Four different types of proteinases are recognised: aspartic, serine, cysteine andmetalloproteinases, and each type has a characteristic set of functional amino acid residuesarranged in a particular configuration to form the active site.All types of proteinases catalyse the same reaction but have different mechanisms. They can alsobe distinguished from each other on the basis of their sensitivity to various inhibitors. The serineproteinases have a reactive serine residue; the aspartic proteinases have two catalytically essentialaspartate residues; the cysteine proteinases have reactive cysteine residues and the metalloproteinases require zinc for activity. At present there are some enzymes which do not fit into oneof the four types of proteinases, e.g. the multicatalytic protease which has multiple subunits and atleast four different proteolytic activities (Powers et a!., 1993). As these new enzymes arecharacterised mechanistically, it is likely that most will be members of families within the fourtypes of proteinases and will operate by the same general mechanism of peptide bond cleavage.However, there still remains the possibility that an entirely new mechanism of peptide bondhydrolysis remains to be discovered.All four types of proteinases have been detected in fungi (Hartley, 1960; Barrett, 1977), althoughaspartic and serine proteinases predominate (North, 1982). Many fungi produce proteinases whichare active at acidic pHs, and a large proportion of these have been shown to have propertiesconsistent with aspartic proteinases. Most of these proteinases have molecular weights in therange of 30 000 to 45 000 and isoelectric points (p1 values) below 5.1. They are usually able tohydrolyse a range of native proteins but the majority have little or no activity on small synthetic24substrates. Fungal species which produce extracellular acid proteinases often acidify the media inwhich they grow, and many of the enzymes are unstable above neutral pH.The production of alkaline proteinases has been described for fungi of all major taxonomic groups.Most of those characterised were found to be serine proteinases. These proteinases generally havelow molecular weights, in the range of 18 500 to 35 000. Most have low p1 values, between 4.4and 6.2, but p1 values of 8.9 or higher have also been reported (North, 1982). The mechanism ofcatalysis has been extensively investigated and is shown in Figure 1.7. The hydroxyl group of theactive site serine residue performs a nucleophilic attack on the carbonyl carbon of the scissilepeptide bond thus forming a tetrahedral intermediate. A histidine residue in the active site servesas a general base accepting the proton from the serine residue. The acyl enzyme intermediate thusformed is broken down via a nucleophilic attack of a water molecule to complete the hydrolysis ofthe peptide bond (Neurath, 1984).Although many distinct families of serine proteinases (EC 3.4.21.-) exist, the two best studied arethe (chymo)trypsin and subtilisin (EC 3.4.21.14) families. These families are distinguished by ahighly similar arrangement of catalytic His, Asp and Ser residues in radically different 13/13(trypsin) o’J13 (subtilisin) protein scaffolds (Siezen et al., 1991). The (chymo)trypsin family isrelatively well known, with well-characterised members such as chymotrypsin, trypsin, elastase,plasmin, and factor IX. Fewer members of the subtilisin family were known until fairly recently,when a surge of interest was driven by research and industrial applications.25—CH2OH + R-Xenzyme substrateCH2O : R-&X+ R0 Michaelis complexH + ®_CH20/“xtetrahedral intermediate o®_CHO_t’ + HXR./Oacyle,zyme’R product (P1H + ®_CH2O/ OHOH : R--OHCH2O + R-&OH2enzyme product (P2)Figure 1.7 Overall mechanism proposed for serine-proteinase catalysed reactions(Murao et at., 1985)Subtilases have been further divided into two main classes (I and II) based on multiple sequencealignment of the N-terminal catalytic domains. The distinction is based on characteristic sequencepatterns and consensus residues. All subtilases contain the essential catalytic triad residues D32,H64 and S22 1. Most also contain Ni 55 that helps to stabilise the oxyanion generated in thetetrahedral transition state (Carter and Wells, 1990). The conserved secondary structure consistsof an internal core of seven parallel f3-sheet strands and two buried helices, surrounded by fiveamphipathic helices and two anti-parallel f3-sheet strands (Siezen et at., 1991). However, the26connections between conserved regions in the secondary structure are variable. They are generallyon the surface of the molecule, allowing for variation in length and amino acid sequence.Alignment of class U subtilases is fairly unambiguous due to their high degree of sequencehomology, even in the most variable regions, and the low incidence of insertions/deletions relativeto proteinase K. Therefore, as suggested previously (Siezen et at., 1991), model structures ofsubtilisin class II proteinases can be derived directly from the proteinase K structure.Proteinase K, isolated from the soil ascomycete Tritirachium album Limber, is the mostextensively characterised member of this important group of serine proteinases. Its ability todigest native proteins, including keratin (Ebeling et at., 1974), has led to its widespreadapplication in the preparation of nucleic acids. Other members of the subtilisin family havebeen extensively studied because of their practical application as additives to laundrydetergents (Betzel et at., 1990), in the preparation of proteolytic creams for medicinalpurposes, and in the food industry (Dolashka et at., 1992; Lyons, 1988).1.7 Proteinase inhibitionAny compound which decreases the measured rate of hydrolysis of a given substrate is, inprinciple, an enzyme inhibitor (Salvesen and Nagase, 1989). There are several types of proteinaseinhibitors, most of which are specific to the four types of proteases. Reversible inhibitors includesimple competitive and transition-state inhibitors. These inhibitors usually contain substrate-likefeatures and their potency depends on binding interaction with the enzyme. Irreversible inhibitorscan be active-site directed inhibitors or mechanism-based inhibitors. Active site inhibitors27resemble the substrate and contain a reactive group which can react with amino acid residues at theactive site of the proteases. Mechanism-based inhibitors, also known as suicide inhibitors, oftencontain a latent reactive group which is activated by enzyme catalysis. In this case, a normallyinnocuous reversible inhibitor is converted into a powerful irreversible inhibitor. Irreversibleinhibitors usually inactivate proteases by first forming a reversible E•I complex followed bycovalent bond formation and hence irreversible inhibition (Fersht, 1985; Powers et a!., 1993).Proteinaceous protease inhibitors form a unique category of inhibitors found almost ubiquitouslydistributed in tissues of plants and animals (Murao et a!., 1985). They have been widely used toelucidate proteinase mechanisms since the El complex exists as a stable molecular species.Inhibitors for serine proteases include simple substrate analogs, transition state analogs, alkylatingagents which react with the active site histidine, acylating agents which react with the active siteserine fornuing stable acyl enzymes, and mechanism-based inhibitors (Powers et a!., 1993). Diisopropyl-fluorophosphate (DFP) and phenylmethanesuiphonyl fluoride (PMSF) are well knownexamples of low molecular weight inhibitors which act by irreversibly modifying the active siteserine (Salvesen and Nagase, 1989). Bovine pancreatic trypsin inhibitor (BPTI) was discovered in1930 as a proteinaceous inhibitor that inactivated trypsin in the pancreas (Murao et a!., 1985).Such inhibitors do not hydrolyse under physiological conditions because the amino group that isreleased on cleavage of the peptide is constrained and cannot diffuse away from the active site ofthe enzyme (Fersht, 1985). Another example of a serine proteinase inhibitor is antipain, a peptidealdehyde which acts as a transition state intermediate by mimicking the tetrahedral intermediateformed during hydrolysis.28There is considerable interest in the design of highly specific irreversible enzyme inhibitorsbecause of their potential use as therapeutic agents, and it is likely that more suicide inhibitors,which are unreactive in the absence of the target enzyme, will be developed for future use.1.8 Proteinases of wood-inhabiting fungiFungi which grow on wood must obtain all their cellular nitrogen from wood, unless an alternativenutrient source is available. Proteinases may be essential for fungi to retrieve nitrogen from woodproteins, and may serve to recycle fungal nitrogen by autolysis (Levi et al., 1968). Whilecellulolytic and ligninolytic enzymes ofwood-inhabiting fungi have been extensively studied, theirproteolytic enzymes remain poorly characterised. The few available descriptions of proteinaseproduction in artificial media have been reported for wood-decaying Basidiomycetes,Schizophyllum commune, Postia placenta, and Trametes versicolor, for the moulds Aspergillus,Peniciiium, Trichoderma, and for the yeast Aureobasidiurn pullulans.Schizophyllum commune, a weak white rot fungus, has been used as a model system to study themechanisms of nitrogen-limited growth (Lilly et at., 1990, 1991, 1994; Sessoms and Lilly, 1986).It possesses a complex system of proteolytic enzymes (Lilly et at., 1994) which are thought to beinvolved in sustaining mycelial expansion during nitrogen-limited growth. Serine, metallo- andaspartic proteinases are produced by S. commune. Nitrogen deprivation results in a shift in thespectrum of proteinases produced: metalloproteinase activity increases and serine proteinaseactivity decreases (Lilly et at., 1994). When exponentially growing colonies are transferred tomedia low in nitrogen, radial expansion continues at nearly the same rate as in colonies transferred29to high nitrogen media. New growth is supported at the expense of existing mycelia. Proteins aredegraded in older portions of the colony and proteolytically released amino acids subsequentlyappear in the colony margins (Lilly eta!., 1991).The proteinases produced by Postia placenta, a brown rot fungus, have acidic pH optimacharacteristic of aspartic proteinases (Matsushima et a!., 1981; Micales, 1992). Proteinaseformation by different strains of the brown-rot fungus P. placenta was examined to determinewhether differences in proteolytic enzyme production could be correlated with the ability to decaywood (Micales, 1992). Although this work was not conclusive, the author suggested that theproteinases were associated with autolysis. Similarly, an association with nitrogen recycling hasbeen reported for a range of decay fungi (Santamaria and Reyes, 1988; Venables and Watkinson,1989), with the exception of T versicolor. In this fungus, the spatial distribution of the proteinasesat the margin of the agar-grown colony and the production of large quantities of proteinase beforethe onset of autolysis suggested that proteinases would be involved in primary attack of woodprotein rather than autolysis (Venables and Watkinson, 1989).Proteinases of moulds such as Aspergilli, have been extensively studied and reviewed by Cohen(1977). Much of the work examining proteinase production by Aspergiii has stemmed from theinterest in these enzymes for applications in the food industry and enzymatic depilation in thetanning industry (Malathi and Chakraborty, 1991; Tatsumi eta!,., 1989). Therefore, many studieshave used complex or undefined media. Nevertheless, all strains produced substantial levels ofextracellular neutral and alkaline proteinase activity in response to protein starvation (Cohen,1977). Mycelial extracts from Aspergillus nidulans have been shown to contain at least five30different proteinase activities (Cohen, 1973). There is an increase in proteinase activity afternitrogen starvation, and most intracellular activity is compartmentalised in vacuoles (Stevens andMcLennan, 1983). The alkaline proteinase produced by A. oryzae has been cloned and expressedinS. cerevisiae (Tatsumi eta!., 1989).Other research has investigated elastase production as a virulence determinant of Aspergillusfumigatus and Aspergillus flaws, opportunistic pathogens causing a variety of respiratorydisorders (Frosco et a!., 1992; Denning et a!., 1993; Kolattukudy et a!., 1993; Moser et a!., 1994).Production of an extracellular elastase was correlated with the ability to cause invasiveaspergillosis in immunocompromised mice. The elastase was inhibited by PMSF and EDTA, andwas classified as a subtilisin-like serine proteinase (Reichard eta!., 1990; Jaton-Ogay eta!., 1992).Closely related alkaline proteinases were secreted by other pathogenic species in contrast to non-pathogenic strains (Hanzi eta!., 1993).Trichoderma koningil, T. kzrzianum and Aureobasidium pullu!ans secreted an alkaline proteinasecompletely inhibited by PMSF (Donaghy and McKay, 1993; Geremia et a!., 1993; Manonmaniand Joseph, 1993). Peniciiium species secreted aspartic proteinases (Matsushima et a!., 1981),although serine proteinases have also been detected (Chrzanowska et a!., 1993; Yamamoto et a!.,1993). These proteinases are thought to be involved in a variety of pathogenic, nutritional ordevelopmental functions, and have been used in various industrial applications (e.g. the foodindustry, the detergent market).31To conclude, wood-inhabiting fungi have been shown to produce proteinases, and the productionof extracellular serine proteinases has been reported for decay and mould fungi. However, most ofthe research interest has resulted from the industrial or medical importance of the fungi. None ofthe work cited was conducted on wood, and none has examined proteinase production bysapstaining fungi.1.9 Research approach and objectivesThe biological control of staining fungi can be approached in many ways. Some research groupshave focused on biological control agents to outcompete the staining fungi (e.g. Benko, 1987;Stranks, 1976; Seifert et al., 1987), others have considered using colourless mutants which do notcause stain (Behrendt eta!., 1995). Some work has been conducted to quantify and detect stainingfungi on wood (Breuil et al., 1988, 1990, 1992), but little work has been conducted on thephysiology of staining fungi especially when they are grown in “solid state” as on lumber. Manyof these strategies for controffing sapstain would benefit from a better understanding of thephysiology of the organism involved.In common with all other living organisms, fungi require nitrogen in all aspects of their growthand metabolism. As discussed above, the major source of nitrogen in wood is protein, and fungiwould require extracellular proteinases to break down these proteins into more assimilable formsof nitrogen. Little information is available on the proteases of staining fungi. The work carriedout on wood-inhabiting fungi has usually involved growth on artificial media (Venables andWatkinson, 1989; Micales, 1992). Virtually no work has been done on the range of enzymes32active when fungi grow on solid wood substrates such as lumber and trees. An understanding ofproteinase production by staining fungi in wood would be essential for subsequent manipulationand disruption of the proteolytic enzyme systems. Furthermore, characterising microbial enzymeshas important practical applications. It can provide new enzymes for use in commercialapplications or supply information that permits the improvement of existing enzymes.In Canada, Ophiostoma piceae (Munch) H. & P. Sydow is the most commonly isolatedsapstaining fungus, and was therefore chosen as the model organism. Understanding more aboutthe growth and metabolism of 0. piceae is also considered important because of its possibleinvolvement with oak decline in central and eastern Europe, and its close relationship to the Dutchelm disease pathogens 0. ulmi (Buisman) Nannf. and 0. novo-uln2i Brasier (Brasier and Kirk,1993). The genera Ceratocystis and Ophiostoma also include plant pathogens causing rot of sweetpotatoes, wilt of coffee and rubber; and human pathogens causing sporotrichosis (Spatafora andBlackwell, 1994; Upadhyay, 1993). The ophiostomatoid fungi are thus of obvious economicimportance in forest products, forestry, crop plants and medical mycology.The model substrate chosen for this study was lodgepole pine, Pinus contorta var. latifolia Dougi.,since it is a high-volume lumber in B.C. that is highly susceptible to sapstain development. Aspen,Populus treniuloides Michx., was chosen as a representative hardwood species that is subject todistinct discoloration due to its intrinsic light colour.The overall goal of the work was to understand how 0. piceae retrieves nitrogen from wood. Thisinformation may be useful for controlling the growth of staining fungi in wood. Physiological33studies on wood are complicated by the intrinsic variability of wood, and the difficulty of enzymeextraction and fungal manipulation. Therefore, the approach used throughout this work was toconduct parallel studies in artificial media and in wood.The research was divided into three specific objectives:A. Characterisation of the proteinases secreted by 0. piceae• Optimise culture conditions for proteinase production• Determine the number and type of proteinases produced under various conditions inwood and artificial mediaB. Purification and characterisation of the major serine proteinase• Biochemical properties• Catalytic properties• Stability under various conditions• Sensitivity to inhibitors• Cleavage specificity on natural and synthetic substratesC. Targeted inhibition of serine proteinases on wood• Identify types of proteinases produced by staining fungi on the basis of sensitivity toinhibitors• Application of specific inhibitors to stop fungal growth on wood34Chapter 2.General methodology2.1 Fungal strainOphiostoma piceae (Munch) H. & P. Sydow strain 387N, isolated from softwood chips at theMacLaren Mill (Mason, Québec, Canada), was obtained from the culture collection of ForintekCanada Corporation (Ste. Foy, Québec, Canada).2.2 Growth conditions for staining fungi in culture2.2.1 Solid mediaSkim milk agar (1% skim milk, 1.5% agar) and malt extract agar (2% malt extract, 1.5% agar)(Difco, Detroit, USA) were used for growth of staining fungi. The former was used to determineproteinase activity by observing clearing zones during growth.2.2.2 Liquid mediaThe synthetic liquid medium contained in each litre: 0.4 g CaCI2HO; 1.0 g KH2PO4;0.8 gNa2HPO4;0.5 g MgSO47H2O;3.0 g potassium hydrogen phthalate; the micronutrients of Vogel(1956); filter-sterilised vitamins of Montenecourt and Eveleigh (1977). The carbon source was2% soluble starch unless otherwise specified. The nitrogen source was NH4O3 1.6 g/l forinorganic media and soybean proteins for organic media unless otherwise stated. Soybeanproteins were supplied as 280 mill of unsweetened soya drink (Sunrise Markets, Vancouver,35B.C., Canada) that contained 3.1% protein, 1.4% fat, and 0.9% carbohydrate. The media weresterilised by autoclaving before the vitamins were added. The final pH of the media was 5.8 ± 0.2.The inoculum (9.75 mg/i dry weight) was pre-grown for three to six days in the same medium asthat used for the corresponding experiment. These inoculum cultures were prepared from 3 mmcores of the fungus grown on 2% malt extract agar plates. Cores were stored at -80°C in 10%glycerol. Most studies were performed in 300 ml glass Erlenmeyer flasks containing 60 mlliquid medium. Unless otherwise specified, cultures were grown in the dark at 23°C on a rotaryshaker set at 250 rpm.2.3 Fungal biomass determinationsThe total fungal biomass in liquid culture was measured by filtering and washing the myceliaon pre-weighed glass fibre filters, drying the filters in a microwave (4 minutes on highsetting), cooling in a desiccator and weighing. Total fungal protein was determined using thebiuret assay (Herbert et al. 1971), and bovine serum albumin (BSA) as the calibrationstandard. Total ergosterol in extracts from freeze-dried mycelia (10-30 mg) or wood powder(1.5 g) was determined by HPLC analysis (Seitz et a!. 1977; Nilsson and Bjurman, 1990)using a Nova-Pak C18 reverse phase HPLC column on a Waters 625 HPLC system (MilliporeCorporation, Bedford, MA, USA). The method involved a one hour reflux with 60 ml of amethanol and ethanol mixture (5:1, v/v) containing 10% KOH, followed by extraction with60 ml petroleum ether. The petroleum ether was evaporated and samples redissolved inmethanol (1.5 ml) prior to HPLC analysis (Gao et al., 1993). Ergosterol (Aldrich ChemicalCo., Milwaukee, WI, USA: product number E200-0) was used as the calibration standard.362.4 Wood material2.4.1 Hardwood and softwood blocksA lodgepole pine (Pinus contorta var. latfolia) tree, approximately 45 year old, washarvested during winter in the UBC Alex Fraser Research Forest at Gavins Lake, B.C.Sapwood blocks (30 x 10 x 5 mm3) were cut with the 5 x 10 mm2 face on the transverseplane, and the 30 x 10 mm2 face on the radial longitudinal plane. Lodgepole pine sampleswere further separated and classified on the basis of distances above ground level (SectionsI-VT, 3-0 m) and radial distances from the cambium (1) to the heartwood (6). An aspen tree,approximately 70 years old, was harvested during winter in Northern Alberta. Samples werealso prepared from the sapwood as described for pine. Pine and aspen samples werepackaged, and sterilised by gamma irradiation (Gamma cell 220, Atomic Energy of Canada,Ottawa, Canada) to receive a total dose of 2.5 mRad. Sterilisation was carried out at theBiomedical Research Centre, UBC. Branches, about 3 years old, were collected from asecond lodgepole pine tree, and sterilised as described above. All the wood samples werekept at -10°C.2.4.2 Inoculation of wood blocks0. piceae was grown in liquid medium for 4 days. The biomass was harvested bycentrifugation and resuspended in 0.8% NaCl. Samples were homogenised (OmniHomogenizer model 2000, Omni International, Waterbury, USA) and centrifuged to obtain awashed pellet of cells. The cells were made up to a final concentration of 2.5 mglml dryweight. Aliquots (20 p1) were used to inoculate sterile wood blocks. Blocks were incubated37in sealed Petri dishes at 23°C for 3 to 21 days. High humidity was maintained by using watersoaked filter paper in the bottom of the Petri dishes.2.4.3 Sap pressed from wood chipsA lodgepole pine tree, approximately 40 years old, was collected from the same site at theresearch forest in B.C. It was debarked and chipped. To obtain a liquid pressate the chipswere compressed in the screw feeder of a thermo-mechanical refiner which had acompression ratio of 5:1. In this, the assistance of Dr. H. Cisneros and S. Johal of the Pulpand Paper Research Institute of Canada, Vancouver, B.C., is gratefully acknowledged.2.4.4 Milled wood powderWood blocks were milled into a powder using a micro mill (Bel-Art Products, Pequannock,NJ, USA) cooled by liquid nitrogen.2.4.5 Wood protein samplesWood powder was homogenised (Omni Homogenizer model 2000, Omni International), orground with a mortar and pestle, in a 1% sodium dodecyl sulphate (SDS) solution, andcentrifuged to remove wood fibre. Sap pressed from lodgepole pine chips was clarified bycentrifugation and then freeze dried. Material was resuspended in phosphate buffered saline(PBS), and was extracted three times with equal volumes of diethyl ether, and once withhexane. Protein was precipitated from the aqueous phase using 100% ammonium sulphate.The pellet was resuspended in PBS and desalted by ion retardation. Protein solutions were38concentrated using an ultrafiltration membrane with a molecular weight cut-off of 3000 Da(Amicon, Danvers, MA, USA).2.5 Proteinase activity assaysAzocoll (<50 mesh; Calbiochem, La Jolla, CA, USA) was used as the substrate in aspectrophotometric assay to determine proteolytic activity in filtrates from liquid culture or inwood. (Chavira et al. 1984). Assays were conducted in 1 ml reaction volumes in a water bathwith agitation of 320 rpm, using 4 mg/ml azocoll prepared in 0.1 M Tris-Cl (pH 8) or 0.1 M IVIESTris-acetate (Ellis and Morrison, 1982) and 0.09 M CAPS (Sigma, St. Louis, MO, USA) for arange of pH’s. Assays were generally conducted at 37°C, where proteolytic activity was greaterthan at room temperature. Reactions were tenninated with 50 j.tl of 50% (w/v) trichioroacetic acid(TCA). After centrifugation, supernatants were transferred into a 96 well plate and theabsorbance at 520 nm (A520) was measured using a Thermomax microplate reader (MolecularDevices Corporation, Menlo Park, CA, USA). Each well received 200 d of sample giving a pathlength of 0.7 cm. One unit (U) of proteinase activity was defmed as the amount of enzyme thatproduced a rate of increase in absorbance of 0.1 O.D. units per mm.The cleavage specificity of the proteinase was determined using the following model substratesfrom Sigma: N-c-benzoyl-DL-Arg-p-nitroani1ide (BAPNA) for trypsin-like activity; N-ctbenzoyl-L-Tyr-p-nitroaniide (BTPNA) for chymotrypsin-like activity; N-acetyl-Ala-Ala-Ala-pnitroaniide (ATAPNA) for elastase-like activity; succinyl-Ala-Ala-Pro-Leu-p-nitroanilide andsuccinyl-Ala-Ala-Pro-Phe-p-nitroanilide (sAAPF) for subtilisin-like or chymotrypsin-like activity;39and L-Leu-p-nitroanilide for amino-peptidase activity. Assays were conducted at 0.3 to 1 mMsubstrate concentrations (Sarath et al., 1989) in 20 mM Tris-Ci, pH 8 at 37°C. After completion ofthe reaction, 200 il of the reaction mixture was transferred to a 96-well plate and the absorbanceat 405 nm was measured using the microplate reader. In quantitative assays, the initial rate wasmeasured using 1 mM sAAPF (DelMar et a!., 1979) at different pH and temperature values. Therate was determined as the slope in the linear portion of a graph of absorbance against time, andwas expressed as i0 times the rate of change in O.D. per minute (mOD/mm). The assay plotswere usually linear for the first ten minutes.2.5.1 Kinetic constantsKm and Vm values were determined graphically, from the initial rates of hydrolysis ofsAAPF at five separate substrate concentrations, according to the method of Lineweaver andBurk (1934). The catalytic constant (keat) was calculated from the maximum velocity(expressed in moles per minute) using an enzyme loading of 0.6 pg and a molecular weight of33 kDa, according to the formula: kcat=(Vm)/[E].2.6 Inhibition studiesVarious compounds were examined for their effects on the proteinase enzymes in culture filtratesand on the purified enzyme, or on the growth of the fungus in wood and in artificial media.Samples were pre-incubated for 30 mm at room temperature in 0.1 mM Tris-Cl, pH 8 with thefollowing compounds: ethylenediaminetetraacetic acid (EDTA), SDS, dithiothreitol (DTT), 2-mercapto-ethanol (Bio-Rad Laboratories, Richmond, CA, USA), aminoethylbenzene40suiphonyifluoride (AEBSF) (Calbiochem), aprotinin, tartaric acid, turkey egg white inhibitor,soybean trypsin inhibitor, ethyleneglycoltetraacetic acid (EGTA), pepstatin, L-transepoxysuccinyl-leucylamide-(4-guanidino)-butane (E-64), 1,1 0-phenanthroline, PMSF,phosphoramidon, 3,4-dichioro-isocoumarin (DCI), DFP, and tosyl phenylalanylchioromethylketone (TPCK), antipain, chymostatin, N,N,N,N’-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), salicylic acid, H20ZnSO47H2O,Pb(N03)2and CuC12HO(SigmaChemical Co., St. Louis, MO, USA). PQ-8, a currently used commercial anti-sapstainfonnulation, containing the detergent dodecyl benzene suiphonic acid (DDBSA), copper andthe chelating agent 8-hydroxyquinolinoline, was obtained from ISK Biosciences, Memphis, TN,USA. Residual proteinase activity was subsequently determined using azocoll or sAAPF assubstrates.2.7 Electrophoretic analysesIsoelectric focusing (IEF pH 3-9; IEF pH 4-6.5), sodium dodecyl suiphate-polyacrylamide gelelectrophoresis (SDS-PAGE) and native PAGE (gradient 8-25%) were performed on thePhastSystem (Pharmacia, Uppsala, Sweden). Protein concentrations were measured by theBradford microassay (Bio-Rad), using BSA as the standard (Bradford, 1976). Separation bySDS-PAGE (Laemmli, 1970) and native PAGE was performed at a constant current of10.0 mA for a total 70 Vh. Separation by IEF was performed at a constant current of 2.0 mAfor a total of 431 Vh. The p1 and MW markers were from the Pharmacia kits for IEF andSDS-PAGE, respectively. For activity assays, isoelectric focusing and native gels were run induplicate: one gel was silver stained for proteins according to the manufacturer’s41recommendations (Pharmacia) and the other was used for a contact print zymogram. Proteinbands were quantified using the Discovery Series Model DNA 35 densitometer (PDI, NewYork, USA).Proteolytic bands were detected using unprocessed gelatin-coated X-ray film (Zhu et al.1990, Cheung et a!., 1991). X-Omat RP XRP-1 X-ray film (Kodak, Rochester, USA) wasincubated with its gelatin surface in contact with the gels for 4 to 15 minutes at 3 7°C. Afterincubation, the film was washed under a stream of water. Clear zones, where the gelatin hadbeen hydrolysed, indicated the presence of proteinase activity.2.8 N-terminal sequencing and amino acid analysesProtein samples were boiled for 5 minutes in SDS-PAGE sample buffer and run underconstant voltage of 110 V using a Mini-PROTEAN II cell (Bio-Rad). Separated proteinswere electroblotted onto an Immobilon-P PVDF membrane (0.45 mm pore size, Millipore) usingthe Trans-Blot electrophoretic transfer cell (Bio-Rad) for 1 h at 100 V or 3 h at 45 V. Proteins onthe membrane were stained with 0.02% (w/v) Ponceau S, and bands were cut out. The aminoacids were analysed on an Applied Biosystems Model 420 A/H (Applied Biosystems, Forster City,CA), and the N-terminal sequence was determined by automated protein sequencing on a pulsedliquid sequenator (model 473, Applied Biosystems). The sequence was compared to knownprotein sequences using the BLAST (Basic Local Alignment Search Tool) system developed bythe National Centre of Biotechnology Information at the National Library of Medicine, Bethesda,MD, USA (Altshul eta!., 1990). The BLAST system was accessed using electronic mail to carry42out the search using the BLAST heuristic algorithm for sequence alignments.For N-terminal sequencing of autolytic products, proteins were separated by discontinuousSDS-PAGE according to Schagger and von Jagow, (1987) using a 10% T, 3% C separatinggel and a 4% T, 3% C stacking gel. Tricine (0. 1M) was used as the trailing ion in the cathodebuffer. After electrophoresis, proteins were blotted onto an Immobilon-P5 PVDFsequencing membrane (Millipore) using the Trans-Blot cell (Bio-Rad) for 3 h at 45 V in10 mM pH 11 CAPS (Sigma) transfer buffer containing 20% methanol. The method ofSchagger and von Jagow (1987) was found to be useful for separating and blotting proteinssmaller than 20 kDa prior to sequencing.Amino acid composition analysis was also performed on lodgepole pine sapwood samples. Inthis case, wood powder (25 mg) was added to a rimless pyrex test tube with 3 ml of 6 M HC1.The mixture was frozen in an acetone/dry-ice mixture, the atmosphere changed to nitrogen,and the tube sealed using a flame. Samples were hydrolysed at 110°C for 24 hours. Aftercooling, samples were suction-filtered and a 10 jil aliquot of the filtrate was derivatised usingphenyl-thioisocyanate. Norleucine was added as an internal standard and the mixture wasanalysed with an amino acid analyser (model 420 H, Applied Biosystems) using a reversephase column.N-terminal sequencing and amino acid analysis was performed by Sandy KiellandiNadjaSpitzer at the Protein Microchemistry Facility at the University of Victoria, B.C. Amino acid43composition analysis was also performed by Dr. Krystyna Piotrowska at the Nucleic Acid -Protein Service at the University of British Columbia, B.C.44Chapter 3.Growth, nutrition and proteinase activity ofOphiostomapiceae in culture and on wood3.1 IntroductionWork conducted on the physiology of Ophiostomatales has been sparse, with mostcomprehensive investigations being performed on the pathogenic species of Ophiostoma - 0.ulmi. Detailed analyses of the physiology of saprophytic species was conducted by Käarik inthe 1950s and 1960s on isolates predominantly from Swedish forest products. This workdescribed the effects of growth factors on the growth and sporulation of several Ophiostomaspecies in artificial media (Kääril, 1960; Mathiesen-Kärik, 1960). Ecological studies onthese fungi have been carried out on an ad hoc basis in various research organisations, oftenpublished only as internal reports (e.g. Chung and Smith, 1986; Sutcliffe and Chan, 1992).Thus, the work reported in this chapter was necessary to establish optimum growth conditionsand proteolytic activity for 0. piceae grown in artificial media and in wood.Lodgepole pine and aspen are commercially important softwoods and hardwood speciesrespectively, and are susceptible to discoloration by staining fungi (section 1.1). For bothgroups of trees, storage of nitrogen as protein has been shown (section 1.4). However, thestorage proteins of deciduous hardwoods, especially poplar, have received more attention thanthose of evergreen conifers. Specific storage proteins with molecular weights of 32 and 36kDa have been identified in many different genera that accumulate during the late summer or45early autumn and are highly abundant throughout the winter (Langheinrich and Tiscbner,1991). However, the utilisation of these proteins by sapstaining fungi has not beendemonstrated.Regulation of proteolytic activity is a critical aspect in the physiology of an organism growingin an environment that offers little nitrogen. Little information is available on the proteinasesof staining fungi when grown in artificial media and no information is available when grownon wood. Fungi which degrade insoluble polymers outside the cell and take up breakdownproducts would require a means of regulating extracellular enzyme activity. According toCohen (1980), there are several different mechanisms by which regulation occurs. The twomost likely mechanisms for regulation of proteinases secreted by 0. piceae involve inductionor derepressed synthesis. Induction, where the substrate or an effector molecule initiatessynthesis of the enzyme, has been postulated to be involved in proteinase production byNeurospora crassa grown in the presence of BSA and other soluble proteins (Drucker, 1975).In this system, carbon, nitrogen or sulphur starvation would not induce proteinase production.For derepressed synthesis, synthesis of proteinase production is induced by nutrient limitationof carbon, nitrogen or sulphur. Derepressed synthesis is thought to be the simplest system forregulation of extracellular catabolic enzymes and has been demonstrated for the production ofneutral and alkaline proteinases ofAspergillus nidulans (Cohen, 1973).In this chapter the nutrient requirements, optimum growth conditions and proteolytic activityof the staining fungus 0. piceae were evaluated.’ Conditions under which proteinases were‘The data have been published: Abraham, L.D., Roth, A., Saddler, J.N. and Breuil, C. 1993. Growth, nutrition andproteolytic activity of the sap-staining fungus Ophiostomapiceae. Can. .J Bot. 71:1224-123046secreted during growth in culture and in wood were determined, and regulation of the activitywas considered during growth in culture and in wood. The proteolytic activity was quantifiedand the electrophoretic pattern was analysed under various conditions. The effects of alteringthe nitrogen source supplied in culture or in wood were assessed in terms of their impact onproteolytic activity.22Some of the data concerning the nitrogen in pine was presented as a conference proceedings at the InternationalResearch Group of Wood Preservation conference in Orlando, Florida: Abraham, L.D. and Breuil, C. 1993.Organic nitrogen in wood: growth substrates for a sapstain fungus. IRG Doe No. IRG/WP/10019473.2 Materials and methods3.2.1 Culture mediumThe initial work used the synthetic media (section 2.2.2) with the following modifications togive the semi-synthetic medium: for each litre, 1 ml of filter-sterilised vitamin solution (Tanand Breuil 1986) was used instead of the vitamin solution prepared as described byMontenecourt and Eveleigh (1977), NH4O3 was used at 0.8 g/l and yeast extract was addedto 0.01%.3.2.2 Temperature and pH experimentsThese studies were carried out using semi-synthetic media with initial pH set at 5.1. Fivereplicate flasks for each temperature were used in two separate experiments to determinegrowth over a broad and narrow temperature range, respectively. The pH experiments werecarried out at 23°C using semi-synthetic media adjusted to the required pH by the addition ofNaOH or HC1. The biomass was determined after 3 and 5 days of growth.3.2.3 Nutritional experimentsTo determine carbon source requirements, filter-sterilised solutions of starch, sucrose,glucose, maltose, or raffmose were added to the semi-synthetic media to give finalconcentrations of 2% (w/v). The amount of starch remaining in the culture filtrate was48determined colorimetrically (Garcia-Alvarado et a!., 1992). In nitrogen utilisationexperiments, starch was used as the carbon source and inorganic nitrogen sources (NaNO3,NH4C1, NH4O3)or organic nitrogen sources (urea, BSA, collagen, acid-hydrolysed casein,arginine, asparagine, glutamine, proline) were added to give nitrogen equivalents of 28mg/100 ml media. Nitrogen sources were also combined in the synthetic media to giveutilisable nitrogen equivalents of 28 and 65 mg/lOU ml. BSA and aninionia, in the form ofammonium nitrate, were added separately, or as mixtures at a ratio of 1:1 or 1:3. Ammoniadepletion was monitored by the phenolhypochlorite method (Solorzano 1969), and nitrate bythe method of Cawse (1967). BSA concentrations were determined by an indirect enzyme-linked immunosorbent assay (ELISA) (Kendall et al., 1983) using a monoclonal antibody toBSA obtained from Sigma (product number B-2901).3.2.4 Wood analysis and inoculationLodgepole pine and aspen wood blocks (section 2.4.1) were soaked for 2 h in sterile solutionsof BSA at 5% (w/v), ammonium nitrate at 4.6% (w/v), or water. After equilibrationovernight, wood blocks were inoculated with 0. piceae (section 2.4.2) and incubated for 3 to14 days. Inoculated and control wood blocks were milled (section 2.4.4) and samples of 40 to100 mg dry weight were individually weighed into tubes for proteolytic assay (section 2.5).Nitrogen analyses of milled wood samples were conducted by micro-Kjeldahl (Kjeldahl,1883; Pomeranz and Moore, 1975). Statistical analysis was conducted using SYSTAT 5.2 forMacintosh (Evanston, IL, USA). Results were examined by analysis of variance (ANOVA)with post-hoc comparisons using Tukey HSD tests. Ammonia analyses were also conducted49on wood powders. A known quantity of powder was homogenised in deionised water for 1mm with cooling using a Polytron Brinkman homogeniser (Brinkman, Mississauga, Ontario,Canada). The extractable ammonia was then determined in the liquid fraction using anindophenol colorimetric assay (Sheiner, 1976).3.2.4.1 Extraction ofproteinase from woodWood powder, prepared from pine sapwood colonised by 0. piceae for 18 days, was extractedin buffer overnight at 4°C. The extraction buffer was 0.1 M Tris-Ci, pH 8, containing 0.5%Triton X-l00, 5 mM sodium ascorbate, 5 mM Na2SO5,5 mM DTT, 5 mlvi MnC12, 20 mlviMgCl2, 1 mM CaC12, and 10% glycerol (Lewinsohn et al., 1991). The wood slurry wasground with silica sand in a chilled mortar, and the liquid was collected by vacuum filtration.The pellet precipitated between 50 and 90% ammonium sulphate was resuspended in PBS.503.3 Results3.3.1 Growth on solid mediaAt 23°C on malt extract agar with an initial pH of 5.0, the radial growth rates were 1.7 ± 0.4mm/day (mean ± S.D., triplicate plates). The maximum radial growth after 4 days wasobserved at 23°C. No growth occurred on plates incubated at 37°C after 8 days incubation,even after they were transferred back to 23°C for a further 14 days. At 4°C, growth couldonly be detected after 1 week. After 14 days, however, radial growth was better at 4°C(0.4 mm/day) than at 32°C (0.1 mm/day). Proteolytic activity was shown by clear zones onskim milk agar after 5 days incubation at 23°C.3.3.2 Growth in liquid medium0. piceae has two growth forms in liquid medium: hyphal and yeast-like. The hyphal formwas predominant on 2% malt extract, and the yeast-like form was predominant on semisynthetic media. Growth on malt extract resulted in 10% lower fungal biomass but strongerpigmentation.The growth curve, determined by dry weight, showed an initial 24 h lag phase in syntheticmedium, followed by a rapid increase in biomass which corresponded to nitrogen depletion(Figure 3.1). After day 3, the growth slowed and the biomass reached a maximum dry weight510JJ:(A) 2520151050(B) I0.80.6I. 0.40.2065430)2I0 •2520150IIn‘V Cl)05w00Figure 3.1 Growth of 0. piceae strain 387N at 23°C, in syntheticinorganic liquid medium at pH 6.1. Vertical bars represent 95%confidence intervals for the means: dry weight, n=4; protein, n=3;ergosterol, n=3 or 2 (the maximum coefficient of variation was 20.6%). (A) Fungal dry weight (solid circles), depletion of starch (opencircles) and depletion of ammonia (open diamonds). (B) Biomassmeasurement by protein (solid circles) and ergosterol (open circles).of 5 mg/ml at day 4. This coincided with the depletion of the nitrogen source, ammonia. Theamount of nitrogen available for growth was approximately 0.3 g/l. The carbon source was3 4Days of growthcompletely depleted after 6 days. The growth profile was also measured by determining the52protein and ergosterol contents of the cells. As for fungal dry weight, the highest amount ofprotein (0.84 mg/mi) was recorded at day 3. However, the protein expressed as a percentageof the biomass was high at 23 % on day 2 and then reached a stationary level of 15% onday 4. The ergosterol content reached a maximum of 23.8 mg/mi culture during thestationary phase of growth (Figure 3.1). All three parameters used to measure fungal growthshowed a similar pattern comprising lag, exponential and stationary growth phases. Dryweight was chosen as the parameter for monitoring growth in subsequent experiments since itproved to be the most convenient and accurate to measure.3.3.3 Optimal pH and temperature for liquid culturesAfter 2 days growth in semi-synthetic liquid medium, highest biomass values were obtainedwith cultures grown at 23°C (Figure 3.2). At low temperatures, as noted on solid medium,the organism grew slowly initially. However, after 6 days the biomass obtained at 14°C wascomparable to growth at 23°C. At 35°C growth was minimal after 2 days, but increasedrapidly when the temperature was lowered to 23°C. From light microscopic observations, thefungus seemed to be more in the yeast form at higher temperatures and more in the myceliumform at lower temperatures.An initial pH of 6.1 resulted in the greatest amount of biomass if cultures were harvested after3 to 5 days (Figure 3.3). The pH dropped by about 1.5 units over this period. If cultures wereharvested later, a higher initial pH resulted in higher amounts of biomass. Increasing acidity53appeared to enhance the ratio of yeast-like cells over hyphae. Acceptable growth wasobtained over a pH range of 3 to 9.4.1:Temperature (°C)Figure 3.2 Growth response of 0. piceae strain 387N to temperature insemi-synthetic liquid medium with an initial pH of 5.1. Dotted linesshow results from the initial experiment for day 2 (open circles) andday 6 (solid circles). Solid lines show results from a more refinedexperiment for day 2 (open circles) and day 3 (solid circles). Verticalbars represent 95% confidence intervals for each mean of 5 replicatecultures.543.2 5.1 6.1 7.1 8.3 8.9Initial pHFigure 3.3 Growth response of 0. piceae strain 387N to pH at 23°C insemi-synthetic liquid medium. Cultures were harvested after 3 days(shaded bars) and 5 days (solid bars).3.3.4 Carbon and nitrogen requirementsOn agar plates, the carbon sources resulting in the most rapid growth rates were, in decreasingorder, starch, maltose, raffinose, sucrose and then glucose. In semi-synthetic liquid media, thefungus was able to utilise monosaccharides, disaccharides, trisaccharides and polysaccharideslike starch (Figure 3.4). Starch was chosen as the carbon source for subsequent experimentsbecause it is an important source of carbon in wood.555‘LririrIriririCON MX MALT STAR SUCR GLUC RAFFCarbon sourceFigure 3.4 Growth of 0. piceae strain 387N on various carbonsources after 3 days (shaded bars) and 5 days (solid bars) of incubationat 23°C, in semi-synthetic liquid medium with an initial pH of 6.1. Thecontrol (CON) represents growth with no added carbon source. MX,2% malt extract; MALT, 2% maltose; STAR, 2% starch; SUCR, 2%sucrose; GLUC, 2% glucose; RAFF, 2% raffinose. The maximumcoefficient of variation was 4.6%.0. piceae used ammonium, but not nitrate, as an inorganic nitrogen source (Figure 3.5). Dryweight obtained after growth on sodium nitrate was comparable to the level measured whenno nitrogen source was available. The fungus was able to utilise all the organic sources ofnitrogen tested, with casein resulting in the highest fungal biomass. When supplied withidentical total levels of nitrogen, the same amount of fungal biomass was obtained withammonium and urea. Growth was approximately the same with all the different amino acidssupplied. Except for asparagine, the pH of the medium decreased with fungal growth for allnitrogen sources. The greatest decrease in pH was recorded for media supplemented withammonium and urea.56iIiI.i idi1i111 1CON Ni N2 N3 UREA ASN GLN ARG PRO CAS BSANitrogen sourceFigure 3.5 Growth of 0. piceae strain 387N on various nitrogensources after 2 days (darkly shaded bars), 4 days (shaded bars) and 7days (solid bars) incubation at 23°C, in semi-synthetic medium with aninitial pH of 6.1. The control (CON) represents activity in cultures withno added nitrogen. Ni, NaNO3; N2, NH4O3;N3, NH4C1; ASN,asparagine; GLN, glutamine; ARG, arginine; PRO, proline; CAS,casamino acids; BSA, bovine serum albumin. All nitrogen sourceswere added to give nitrogen equivalents of 28 mg/i 00 ml. The averageof duplicate cultures are shown (maximum coefficient of variation was12.7%).3.3.5 Effect of different nitrogen sources on the proteolytic activityIn liquid media supplemented with various nitrogen sources, proteolytic activity in thefiltrates was assayed at pH 5 and pH 8. Proteinase activity was minimal in mediasupplemented with easily assimilable nitrogen (e.g. ammonium, urea and single amino acids)57(Figure 3.6). Small amounts of activity were detected at day 4, when ammonia was depleted.Similarly, activity was measured in filtrates of cultures grown in media devoid of nitrogen(CON) or supplemented with a non-assimilable source of nitrogen such as NaNO3(Figure 3.6).3.0.2.0.1.0.c0)’-CON BSA Ni N2 UREA ASN GLN ARGNitrogen sourceFigure 3.6 Azocoll assay showing proteolytic activity in culture filtrates of 0. piceae atpH 8. Cultures were supplemented with various nitrogen sources and grown for 2 days(darkly shaded bars), 4 days (shaded bars), or 7 days (solid bars) at 23°C, in semi-synthetic liquid medium with an initial pH of 6.1. The control (CON) representsactivity in cultures with no added nitrogen. BSA, bovine serum albumin; Ni, NH4O3;N2, NH4C1; ASN, asparagine; GLN, glutamine; ARG, arginine. All nitrogen sourceswere added to give nitrogen equivalents of 28 mg!100 ml. Proteinase activity units aredefined as the amount of enzyme which produced an A520 of 0.1 units! (ml mm) at37°C and 320 rpm.As expected, the best activity was recorded when protein was used as the source of organicnitrogen (e.g. BSA, collagen). This activity was approximately 300 fold greater than incontrol cultures lacking a nitrogen source.58In all filtrates, proteinase activity measured at pH 8 was higher than at pH 5. The optimal pHfor activity was confirmed in a more detailed study, where assays were conducted at a rangeof pH values, for cultures grown with BSA as the nitrogen source. The optimal pH foractivity was 8 for each day of growth. For all pH’s, the relationship between activity and daysof growth followed a trend similar to that shown for pH 8 (Figure 3.7).30T/25 4‘0___Days of growthFigure 3.7 Fungal biomass (open diamond) and depletion of starch (opencircles) during growth of 0. piceae strain 387N at 23°C, in synthetic liquidmedia with an initial pH of 6.1 containing BSA as sole nitrogen source.Vertical bars represent 95% confidence intervals for each mean of 4 replicatecultures. Proteinase activity units (solid circles) are defined as the amount ofenzyme which produced an A520 of 0.1 units/(ml . mm) at 37°C and 320 rpm(maximum coefficient of variation was 19.9%).59The total proteolytic activity in the culture filtrate increased from day 1 to day 9. However,when the activity was expressed as a function of the biomass present, the maximum activitywas observed at days 2 and 3. BSA was utilised by the cultures during the first 5 days ofgrowth, whereas starch was not depleted (Figure 3.7). Using ELISA to measure BSA, wefound that the initial concentration of 0.2% BSA was reduced to 0.0 15% by day 7. Theseresults were in agreement with the depletion of BSA (p1 4.8) as seen in electrophoretic gels(Figure 3.8).1 2345 6p1 --::j5.85—-a - —6.55—47.35—i8.15—8.45—8.65— —9.30— -A -Figure 3.8 Separation by PhastGel IEF 3-9 of the extracellular proteinsobtained after growth of 0. piceae 387N on BSA supplemented media.Lanes 1, 6: p1 markers; lane 2: day 1; lane 3: day 3; lane 4: day 6; lane5: day 9. (A) silver-stained gel showing location of proteins. (B) X-rayfilm overlay of the gel after 4 minutes incubation at 37°C, showingproteolytic activity.1 2345 6In order to investigate the regulation of the proteinase, nitrogen was supplied in the form ofboth aminonium and protein. An increase in proteinase activity was detected which coincidedwith ammonia depletion after four days of growth (Figure 3.9).60Electrophoresis of culture filtrates by IEF showed a major protein band which focused at a p1value of 5.6 and corresponded to the site of clearing on the zymogram. The amount of proteinand the size of the clearing zone increased with growth (Figure 3.8). Similarly, a single majorband of clearing was obtained for native-PAGE gel overlays. Other proteinase bands at p1 5.2and 7.6 were detected when the incubation time for the IEF gels in contact with the overlaywas increased from 15 to 30 minutes.110IT II00 1 2 3 4 5Days of growthFigure 3.9 Fungal biomass (solid circle) and depletion of ammonia(open square) during growth of 0. piceae strain 387N in mediasupplemented with equal proportions of nitrogen as ammonia and BSA.Proteinase activity units (open circles) are defined as the amount ofenzyme which produced an A520 of 0.1 units/(ml mm) at 37°C and 320rpm.6 761When higher amounts of nitrogen were added to media containing both ammonia and BSA,such that the ammonium exceeded the nitrogen required for ten days of growth, proteinaseactivity was not detected at all during ten days of growth.3.3.6 Nitrogen content of lodgepole pine and aspen sapwood3.3.6.1 Untreated blocksThe moisture content of the wood expressed as a percentage of dry weight was 111 ± 11.5 and125 ± 6.8 % for aspen and pine wood blocks, respectively. Nitrogen contents of lodgepolepine sapwood samples taken from different positions within the tree were very similar,although there seemed to be a slight decrease in nitrogen content of about 30 ppm withincreasing height (from VI 1 to I 1). However only the value from the lowest sample (VI 1)was statistically greater than some of the samples at higher heights (III 1 to I 1). Samplestaken from the eambium to the heartwood showed that the part closest to the cambium (VI 1)and that closest to the heartwood/sapwood boundary (VI 6) had slightly higher nitrogencontents than the samples between these regions.Analyses of the ammonia content of pine and aspen showed that it accounted for less than 5%and 2% respectively, of the total nitrogen present (Table 3.2). Although pine contained lesstotal nitrogen than aspen, it contained almost double the amount of extractable ammonia.62Table 3.1 Nitrogen content1 in lodgepole pine sapwood samples from variousheights and radial distances in the tree as determined by Kjeldahl analyses andexpressed as ppm N dry weightVI I VI 2 VI 3 VI 4 VI 5 VI 6VI 1 483 ± 14cc 447 ± 9a 458 ± 16ab 452 ± 4a 463 ± 5abc ± 8bcVi 46l±lvi 465±18’’1111 452±2wII! 4S5±7YIi‘mean ± S.D., n =4; ANOVA of the radial and vertical samples were carried out separately, P<O.005;Tukey test, P<O.05; values with the same letter are not statistically differentOrganic nitrogen levels were difficult to quantif’. SDS-PAGE of extracts from mature pinesapwood, sapwood from branches and sap pressed from pine wood chips (section 2.4.3)showed protein bands in the molecular range of 14 to 21 kDa, with minor bands between 31and 45 kDa (gel not shown). Amino acid composition analysis of acid-hydrolysed pinesapwood samples indicated relatively high levels of Asx, Gix, Ser, Gly and Ala (Table 3.2).Asx and Glx are frequent in vegetative storage proteins (Wetzel and Greenwood, 1991). Suchproteins would have been hydrolysed during acid digestion prior to amino acid analyses. Thehighest yield obtained was about 0.986 p.g amino acids per mg wood. This corresponded toabout 170 ppm nitrogen per mg wood.63Table 3.2 Amino acid composition of lodgepole pine sapwood samples. Results areexpressed as mass percentage of amino acids in each sample.Amino acid VI 3 II 1 II 1ASX 10.5 8.3 10.1GLX 12.4 11.0 13.2SER 9.5 14.5 11.8GLY 5.8 16.8 6.4HIS 1.0 1.7 1.4ARG 4.5 3.3 5.0THR 6.8 5.9 6.4ALA 9.4 8.5 8.7PRO 5.0 4.1 4.8TYR 1.8 2.8 1.1VAL 6.3 6.0 7.2MET 0.8 0 0ILE 11.8 2.4 5.1LEU 5.1 11.6 5.3PHE 3.1 7.4 5.7LYS 6.1 5.8 7.9CYS ND’ ND NDTRP ND ND NDTotal ammo acids(ng) 197.9 121.5 116.2Sample size (mg) 24.1 25.4 27.8‘not determined3.3.6.2 Blocks treated with solutions ofnitrogenIn experiments with addition of nitrogen to the wood, the nitrogen content of pine and aspenblocks increased significantly after 2 h soaking in solutions ofN11403and BSA (Table 3.3).The sapwood of both wood species took up two times more nitrogen when soaked inammonium nitrate than when soaked in BSA.64Table 3.3 Nitrogen content of lodgepole pine and aspen sapwood samples after soakingin nitrogen solutions.Sample description TKN’ Extractable ammonia(ppm dry weight) (ppm N dry weight)Pine unsoaked control 549 25soaked in dH2O 537 19soaked in 4.6% NH4O3 4495 ND2soaked in 5.0% BSA 2242 NDAspen unsoaked control 807 12soaked in dH2O 638 7soaked in 4.6% NH4O3 5900 NDsoaked in 5.0% BSA 2587 ND‘Total Kjeldahl nitrogen2not determinedFor the ammonium nitrate the nitrogen content was more than seven times that in the originalwood. For BSA it was about four times greater. Control blocks soaked in water lost 12 and169 ppm TKN from pine and aspen blocks, respectively. This probably represents the loss ofsoluble nitrogen.3.3.7 Growth and proteolytic activity on pine and aspen sapwood0. piceae grew actively on aspen and lodgepole pine. Visual differences in growth wereobserved between growth on wood blocks soaked in organic and inorganic nitrogen solutions.BSA appeared to accelerate growth and pigmentation on pine and aspen. Asexual reproductivestructures, synnemata, were observed on the surface of wood after three days. Discoloration ofpine and aspen was noticeable after 3 days, and intensified over two weeks.65ElI pineHj41‘IA.. aspenLi LA BFigure 3.10 Aspen and pine wood blocks 14 days after inoculation with 0. piceae.From left to right, the blocks were uninoculated controls (A), soaked for 2 h in sterilesolutions of water (B), 4.6% NH4O3(C) and 5% BSA (D)Findings were similar for blocks soaked in water, although growth did not appear to be as rapidas on blocks soaked in BSA. In contrast, growth of 0. piceae on wood soaked in inorganicnitrogen was white and filamentous on the surface of the blocks with little pigmentation, evenafter two weeks of fungal growth (Figure 3.10).Initial assays indicated that proteolytic activity in wood was higher at pH 8 than at pH valuesless than 7. Therefore, subsequent assays were conducted at pH 8. Proteolytic activity ininoculated wood increased with time and reached its maximum after six or nine days. Thelevel of proteolytic activity in pine (Figure 3.11 A) was approximately ten times higher in the66BSA-soaked wood than in the water-soaked wood, whereas the activity in NH4O3-soakedwood was less than in water-soaked wood.(A)3. 2D>‘o-(B) 10I:15Days of incubationFigure 3.11 Proteolytic activity in lodgepole pine (A) andaspen (B) sapwood samples after colonisation by Ophiostomapiceae 387N at 23°C. Proteinase activity units are defined asthe amount of enzyme which produced an A520 of 0.1 units/gdry wood/mm at 37°C, pH 8 and 320 rpm. Activity in woodsoaked for 2 h in 5% BSA (solid squares), in water (solidcircles) and in 4.6 % NH4O3 (open triangles). The pointsplotted are the mean values of 4 determinations with standarddeviations shown by error bars.In aspen the pattern was similar (Figure 3.11 B), although the levels of activity were higher inall cases. In untreated or water-soaked wood, proteolytic activity was up to ten times higherI067in aspen than in pine. This was not accounted for by a ten times increase in biomass levelswhich were measured by ergosterol. Ergosterol contents in pine and aspen were 38 and65 ig/g wood respectively, after 14 days growth of 0. piceae.The presence of the proteinase in the wood was confirmed by IEF analyses of protein extractsfrom infected pine. A protein band that corresponded to the site of clearing on thezymogram, focused at a p1 value of 5.6 which was comparable to the proteinase produced inliquid medium. Maximum proteolytic activity in wood extracts was detected at pH 8 (Figure3.12), which again correlated with the optima pH for the proteinase produced in liquid medium.21.5I0.50pHFigure 3.12 The effect of pH on proteinase activity extractedfrom pine wood blocks colonised by 0. piceae for 18 days.Proteinase activity units (U) are defined as the amount ofenzyme which produced an A520 of 0.1 units/mm per ml extractat 37°C and 320 rpm.2 3 4 5 6 7 8 9683.4 DiscussionOur results showed that 0. pieeae can grow on a defined synthetic media supplemented withvitamins. Various carbon sources were assimilated equally well, including carbon polymerssuch as starch. Starch is generally recognised to be an important storage carbohydrate intrees, but soluble sugars can also constitute a major proportion of the total non-structuralcarbohydrates present (Cranswick et a?. 1987). Further work in our laboratory (Gao et a?.,1994) has examined the ability of 0. piceae to utilise wood lipids as carbon sources.Triglycerides and fatty acids were identified as nutrients for this fungus. Although 0. piceaecould degrade xylan and carboxymethyl cellulose when present in artificial media (data notshown), it was unable to utilise avicel, solka floc and wood cellulose and lignin. Generally,staining fungi cause minimal structural damage to wood (section 1.1) because they are unableto degrade the structural polysaccharides and lignin.Like most Ophiostoma species (Käärik 1960), 0. piceae utilised ammonia or organicnitrogen, but not nitrate, as a nitrogen source. Similarly, synthetic medium supplementedwith nitrate did not support the growth of 41 wood-rotting basidiomycetes (Highley and Kirk,1979). Growth and amnionium utilisation when ammonium nitrate was used as the solenitrogen source provided a measure of the nitrogen requirement for 0. piceae. Approximately0.3 mg ammonia-N was assimilated to give 5 mg/ml biomass. Therefore the N-requirementof 0. piceae was about 0.06 mg N/mg dry weight, which is consistent with known values forthe nitrogen composition of fungi and in agreement with values of 0.05 and 0.08 mg N/mg dryweight determined for basidiomycete fungi (Kalisz et a?., 1986).69Apparently proteins (e.g. BSA) may be metabolised both as carbon and nitrogen sources,since starch was not utilised for the first 5 days when both BSA and starch were supplied inthe medium (Figure 3.7). 0. piceae is an ascomycete, but found in the same habitat as thedecay fungi which are basidiomycetes. Therefore, it is interesting that basidiomycete fungihave also been shown to utilise protein as sole source of carbon, nitrogen and sulphur (Kaliszet al., 1986). These nutritional requirements of 0. piceae are similar to those found inprevious investigations on sapstaining fungi (Käärik 1960, 1974).Ambient temperature, moisture content and pH are other parameters that influence the rate ofdevelopment of sapstain on wood. Ophiostoma species are mainly found in temperateclimates. 0. piceae is a mesophilic fungus that grows well at moderate temperatures between14 and 32°C. The organism did not grow at 35°C and did not survive at this temperature formore than one week. After prolonged growth at 6°C, its biomass was similar to the amountdetected at higher temperatures (Figure 3.2). This would explain why serious stainingproblems can occur when the wood is stored at 3 to 8 °C, or when the trees are cut in the earlyspring (personal communications from saw mill staff).Apart from the effect of environmental factors, the form and concentration of nitrogen inwood was found to influence the wood discoloration which is caused mainly by the melaninin hyphal cell walls. When wood was soaked in an inorganic nitrogen solution, there wasvery little discoloration compared to wood in which protein was the major nitrogen source.The effect of the nitrogen source on pigmentation has also been demonstrated in liquid culture(A. Brisson, UBC, personal communication) where the nutrients available for growth can be70more readily defined and manipulated. Although the reasons for this observation are not fullyunderstood, it is possible that the catabolism of proteins may result in the formation ofproducts which are needed for melanin biosynthesis. These precursors may not be formedfrom the utilisation of inorganic nitrogen.Fungal proteinases play a major role in nutrition, development and pathogenesis, and areproduced by fungi of all major taxonomic groups (North 1982). When cultures of 0. piceaewere supplemented with protein, e.g. BSA, proteinase activity per unit of fungal biomassincreased during the active growth of the organism, reaching a maximum between days twoand three, and dropped sharply after day three. The low amount of proteinase activitydetected on day one (Figure 3.7) could be due to the binding of the enzyme to the substratepresent in the medium, or to the fungal cell wall, or to an extracellular sheath attached to thecell wall. These mechanisms have been reported in other fungi (Kalisz eta!., 1987), and mayconfer a competitive advantage to an organism growing in a solid substrate such as wood.The presence of a hyphal sheath in liquid cultures of 0. piceae has been observed (Hoffert,1995). In addition, production of extracellular membranous structures, which may resemble asheath, were observed during growth of 0. piceae in wood (Luck et a!., 1990).The role or function of a proteinase must be directly related to its location in situ. Enzymesand their products are unlikely to diffuse away from their hyphae when they are growing inwood, and a fungal sheath is probably “instrumental” in retaining enzymes and products ofdigestion close to the hyphae (Venables and Watkinson, 1989). A polysaccharide hyphalsheath is a morphological feature of many staining and decay fungi, occurring around the71growing points and along the length of hyphae (Eriksson et al., 1990). It appears to beinvolved in the support and transportation of fungal enzymes (Palmer eta!., 1983), and in thecreation of a micro-environment for optimal enzyme activity (Eriksson et a!., 1990).Naturally immobilised enzymes would offer advantages to organisms growing on solidsubstrates in ensuring efficient utilisation of substrate macromolecules such as proteins(Kalisz et a!., 1987). It may also provide a form of protection against dehydration andenvironmental injury (Green et a!., 1992), and assist in the attachment of the hyphae to thewood cell wall (Ruel and Joseleau, 1991)Traces of proteinase activity were detected in liquid cultures of 0. piceae supplemented witheasily assimilable organic nitrogen sources like amino acids and urea. Similarly, very littleactivity was recorded when inorganic nitrogen was added to the media. In contrast, when nonitrogen source was present in the media, proteolytic activity was detected; however, theactivity was about 300 fold lower than with organic nitrogen. When combined inorganic andorganic nitrogen sources were supplied in culture, proteinase production was only observedafter the ammonium had been depleted. Proteinase production under conditions of nitrogenstarvation suggests that the maj or regulatory mechanism for control of proteolytic activity wasderepression. Results in wood appeared to support this form of regulation.Proteinase activity in wood soaked in ammonium nitrate was consistently lower than in woodsoaked in water, suggesting a preferential use of inorganic nitrogen in the presence of protein.However, in wood the results were more difficult to interpret since the distribution of addednitrogen was unknown, and the form of nitrogen prior to the addition of nitrogen was not72completely defined. After pine sapwood had been soaked in inorganic nitrogen and infectedby 0. piceae, very little proteinase activity was detected. However, when the wood wassoaked in protein solutions, three fold differences in proteinase levels were detected.Interestingly, proteolytic activity in untreated aspen was about 10 fold higher than in pine.Yet the total nitrogen content of aspen was not quite double that of pine, and the total biomassproduced on aspen was only twice that on pine. Therefore, it appears that factors other thanthe nitrogen content may influence the growth and proteolytic activity of 0. piceae. Thepresence of natural endogenous proteinase inhibitors associated with proteinases has oftenbeen reported (DeMartino, 1989) and may explain some of the differences observed in wood.Fungal inhibitors were previously shown to be present in pine sapwood (Bjurman, 1986), andextremely potent, specific proteinase inhibitors have been isolated in the past from such sourcesas the seeds of leguminous plants, grains and potatoes (Murao et aL, 1985). Other factors suchas differences in the pH, extractive content or type of protein substrate may also explain someof the differences in activity on pine and aspen.Qualitative and quantitative data on the nitrogenous compounds in lodgepole pine haveconfirmed the presence of proteins and amino acids. The nitrogen content of pine and aspendetermined by the Kjeldahl method agreed with previous data (Merrill and Cowling 1966),falling in the expected range of less than 0.1% of the dry weight of wood. Ammoniumnitrogen represented less than 5% of the total nitrogen in pine and less than 2% of the totalnitrogen in aspen. Wood samples were not analysed for nitrate because it was not a potentialnitrogen source in these experiments with 0. piceae, however, previous studies have shownthat it is usually present at 1 to 2 percent of the total nitrogen (KäArik, 1960). Proteins were73shown to be present in mature pine wood, branch wood, and sap pressed from green chips oflodgepole pine. The proteins extracted from wood were mainly low molecular weightproteins which may be more amenable to extraction than higher molecular weight,hydrophobic proteins. Proteins are probably the most important nitrogen reserves in trees (vanden Driessche, 1984), comprising 75 to 80% of the nitrogen in bark and 50 to 60% of thenitrogen in the wood of young apple trees. However, woody tissues are heavily lignified andcontain relatively few living cells per unit mass. Furthermore, woody tissues generally containhigh levels of phenolic compounds which often create difficulties in isolating and quantifyingproteins from wood. Phenolics interfere with many of the protein assay procedures. In addition,the presence of wood extractives appears to further complicate extraction protocols. However,all evidence suggests that the protein in wood was likely responsible for proteinase productionby 0. piceae.Proteinases secreted by 0. piceae during growth on wood or in liquid culture were alsoevaluated after electrophoresis. The use of X-ray film following native PAGE or 1EF was avery quick, and effective way to localise the proteolytic band after non-denaturingelectrophoretic separation. Compared to the traditional zymogram procedure (Brown et a!.,1982) using a second substrate gel containing skim milk, the use of X-ray film was considerablycheaper and more convenient. Furthermore, the results were immediately visible after rinsingthe film whereas the traditional overlay requires staining before zones of clearing are distinct.In the case where protein was the sole source of nitrogen, this work showed that an alkalineproteinase with a p1 of 5.6 was produced. This proteinase appeared to function in the primary74retrieval of nitrogen during growth on wood and in artificial media. The proteolytic activitymeasured spectrophotometrically in culture filtrates was in agreement with the results fromnative and isoelectrofocusing gels followed by overlays. A major proteinase band with a p1 of5.6 and optimum activity at pH 8 was detected after 24 hours of growth, and increasedthroughout the growth. In general the pH of wood varies from 4 to 6, but there can beconsiderable heterogeneity between different tissues. Wood pH is measured on wood powder(sawdust) in a suspension. It is difficult to know how closely such measured pHs correspond toactual microenvironmental conditions (Rayner and Boddy, 1988). For example, the pH of theparenchyma cells may be quite different from the other woody cells. Similarly, the pH in themicroniche of the hyphal sheath may also be different than in the rest of the wood in order tocreate optimal conditions for enzyme reactions.Late in the stationary growth phase, two other minor proteinase bands were detected in culturefiltrates, at p1 7.6 and p1 5.2. Similar results were found in cultures grown on other sources ofprotein such as casein, gelatin, collagen and soybean. The proteinase activity profile of 0.piceae was linked to growth, in contrast to reports on the proteinase activity of Penicilliumroqueforti, which produces acid proteinases independently of biomass (Petrovic et a!., 1991).In general, in wood, as in synthetic media, two types of proteinases may be produced -- one tobreak down wood protein into easily assimilable compounds, and the other to recycle fungalprotein when the external source of nitrogen is depleted (Micales, 1992). Recycling andtranslocation of nitrogenous compounds within the fungal cell may provide a nitrogen pooi.Lilly and co-workers (1991) suggested that under nutrient limitation, fungi may notnecessarily sporulate, but may divert all energy resources to continued hyphal extension in75order to increase the chance of reaching a fresh nutrient supply. Then, autolytic breakdown ofproteins in older mycelia is the major source of translocatable nitrogen (Fenn and Kirk, 1981).In summary, in this chapter the growth characteristics of 0. piceae on its natural substrate,wood and in artificial media were described. The production of extracellular proteolyticenzymes during growth under various conditions was examined. Proteinases appeared to besecreted under conditions of starvation, and in the presence of protein when no other simplenitrogen sources were available. Proteinase production was associated with active growth,suggesting an essential physiological role in primary retrieval of nitrogen. Production ofproteinases during growth on wood confirmed that protein was the major nitrogen source inwood, and analyses confirmed the minimal content of ammonia in pine and aspen.Electrophoretic separation of secreted proteins suggested that one major proteinase wasproduced with an acidic p1 and an alkaline pH optimum.76Chapter 4.Isolation and preliminary characterisation of a subtilisin-likeserine proteinase secreted by Ophiostomapiceae4.1 IntroductionThe introduction to this thesis outlined the classification of proteinases according to theircatalytic mechanism (section 1.6) and the use of inhibitors to distinguish the four types ofproteinases (section 1.7). One of the useful steps in the classification of a newly discoveredproteinase is the exposure of the enzyme to a limited number of “standard” inhibitors. For eachtype of proteinases specific inhibitors acting on the crucial amino acid or metal ion at the activesite are known. For example, sensitivity to PMSF in addition to DFP permits the identificationof serine proteinases, E-64 can be used to assay for cysteine proteinases and pepstatin foraspartic proteinases. Similarly, chelating agents such as EDTA and 1,10 phenanthroline identifymetalloproteinases (Dunn, 1989). Once the initial testing has established which types ofproteinases are present, the response to a broader range of inhibitors can provide more data onthe mechanism of action.While inhibitors are often useful in determining the type of proteinase in crude extracts, fordefmitive testing purified enzymes should be used. In general, the purification of proteolyticenzymes presents the normal challenges associated with the purification of all proteins. Inaddition there are specific problems, such as autolysis, inherent in dealing with the purificationof this class of enzymes (DeMartino, 1989). Many proteinases can be purified with a77combination of conventional steps. The proposed purification steps can be selected on the basisof their resolving power, sequence compatibility, capacity for amount of sample (in terms ofvolume and protein concentration), cost, protein yield and the necessity of preserving theactivity of the proteinase (Harris, 1989). In the early stages of a purification strategy, capacityand low cost are important while at the later stages high resolution is important.For the purification of an extracellular protein, the first stage is generally clarification (e.g.centrifugation), usually followed by a concentration step (e.g. ultrafiltration or precipitation).Chromatography techniques succeeding the primary separation techniques can includehydrophobic interaction, ion exchange or affinity chromatography. Chromatofocusing and gelfiltration are usually only considered towards the end of the purification strategy when thesamples are smaller (DeMartino, 1989). Assays for yield and degree of purification will indicatewhether the yield is acceptable and whether one technique is more effective than another.Analysis by gel electrophoresis will also indicate the purity of the protein and how manycontaminants are present (Harris, 1989).Initial work, described in Chapter 3, showed the production of proteinases during growth of 0.piceae 3 87N in protein-supplemented liquid culture and in blocks of lodgepole pine and aspen.The results showed that the major proteinase produced had an alkaline pH optimum, and thissuggested that the enzyme was a serine proteinase. In this chapter inhibitors were used toconfirm the secretion of a serine proteinase during active fungal growth in wood in liquidculture, and to determine whether any other types of proteinases were produced. Results on thepurification and characterisation of the major proteinase in terms of electrophoretic properties,78catalytic properties, amino acid content and N-terminal sequence are presented. The stability ofthe proteinase was characterised under various pH/temperature regimes and the resultscompared with data for other proteinases. Some of the characteristics which may influenceboth the function and practical application of the purified proteinase are detailed.’‘These data were submitted for publication: Abraham, L.D., and Breuil, C. 1995. Isolation and characterizationof a subtilisin-like serine proteinase secreted by the sap-staining fungus Ophiostoma piceae. Enzyme andMicrobial Technology. Accepted.794.2 Materials and methods4.2.1 Chromatography of proteinase4.2.1.1 Analytical separationsFungal cultures grown in the synthetic medium containing 3% starch and 0.6% soybean protein(section 2.2.2) were harvested after 3 to 6 days growth. Culture filtrates were partiallyconcentrated by ultrafiltration using the Minitan system equipped with four PLCC IVlinitan plateswith a molecular weight cut-off of 5 kDa (Millipore), or the Amicon system fitted with a DiafloYM3 membrane with a 3 kDa cut-off (Amicon). Proteins were then precipitated using ammoniumsulphate at 90% saturation, and the pellets resuspended in Tris-Ci (0.1 Iv!, pH 8) and stored at-20°C. Various chromatographic separations were tested using the FPLC (fast protein liquidchromatography) system (Pharmacia).Anion exchange chromatography was conducted using a MonoQ HR5/5 column (Pharmacia)equilibrated with 20 mlvi Tris-Ci, pH 8. Samples were first desalted using a Bio-gel P6 column(Bio-Rad) equilibrated with the equilibration buffer. Elution from the anion exchange column wascarried out by applying a linear salt gradient from 0 to 0.5 M NaC1 in the buffer. Gel filtration wasconducted using a Superose 12 HR 10/30 column (Pharmacia) equilibrated with 20 mM Tris-Cl,pH 8. Chromatofocusing was conducted using a MonoP HR 5/20 column (Pharmacia)equilibrated with 25 mM piperazine-Cl, pH 6.3. Elution was carried out using 1/10 dilutedPolybuffer 74-Cl (Pharmacia) at pH 4.5. Hydrophobic interaction chromatography was performedon a Phenyl-Superose HR 5/5 column (Pharmacia) equilibrated with 100 mlvi Tris-Ci,801.7 M ammonium sulphate, at pH 8. Proteins were eluted by applying a decreasing linear saltgradient from 1.7 toO M ammonium sulphate in the buffer.4.2.1.2 Preparative separationsLarger scale purifications were carried out by preparative hydrophobic interactionchromatography. The culture filtrate after 5 days of growth (approximately 5.8 1) was collected byfiltration and ammonium sulphate was added immediately to give 2 M salt. The sample wasloaded onto a 4.8 x 17 cm phenyl-Sepharose column (Pharmacia) equilibrated with 100 mMTris-Ci, 1.7 M ammonium sulphate, at pH 8. Elution was carried out by applying a decreasinglinear salt gradient from buffer containing 1.7 to 0.5 M salt. The fractions containing the peak ofproteinase activity were pooled, and precipitated using ammonium sulphate at 90% saturation.4.2.2 Determination of glycosylationGlycosylation in the purified proteinase was determined using the digoxigenin (DIG) glycandetection kit (Boehringer-Marmheim, Mannheim, Germany). Transferrin and creatinase served aspositive and negative controls for glycoproteins, respectively.4.2.3 pH and thermal stability studiesThe proteinase was mixed with 0.1 M MES-Tris-Acetate broad range buffer (Ellis andMorrison, 1982) at a ratio of 1:10 by volume. After incubation at set pH’s or temperatures,81residual proteinase activity was assayed at 37°C, in 0.1 M Mes-Tris-Acetate (pH 8) usingsAAPF as substrate (section 2.5). Half lives were determined by interpolating the leastsquares linear fit of a plot of the ln of remaining activity versus time.824.3 Results4.3.1 Increasing proteinase production in liquid cultureWhen BSA (0.2%) was used as the nitrogen source in liquid media, proteinase activity in theculture filtrate reached 2.5 U/mi after 9 days of growth (section 3.3.5). To increase yields ofproteinase, the medium composition was modified to support more fungal biomass. Higherconcentrations of protein (0.6% soyprotein) and starch (3%) were used to increase fungal growthby four times. This increase in biomass to 20 mg/mi was accompanied by elevated levels ofproteinase activity which reached 14 U/ml. A time course study showed that proteinases wereproduced during active fungal growth (Figure 4.1). When the proteinase activity was expressedrelative to the amount of protein measured in the culture filtrate, the specific enzyme activityremained constant during the stationary phase of growth (Figure 4.1).4.3.2 Inhibition ofproteinases produced in culture and in woodThe proteinases produced by 0. piceae were classified according to the effects that inhibitorshad on proteolytic activity (Table 4.1). Total inhibition of activity in culture filtrates wasobtained with PMSF, a known inhibitor of serine proteinases. An inhibitor specific forchymotrypsin-like serine proteinases, TPCK, and classical metalloproteinase inhibitors (1,10-phenanthroline and phosphoramidon) enhanced rather than inhibited proteinase activity.However, all other serine proteinase inhibitors caused some degree of inhibition (23 to 47%) atthe concentrations used. Significant inhibition (88%) was also obtained with the chelating agentxEC)EU)U)CaE0.0C)C,CC’)CCDCD .-‘CD.CDC •U)CO CDU)CD .—83EDTA. Aspartic and cysteine proteinase inhibitors had no inhibitory effect on activity, evenwith assays performed at acidic pH values.20000 1 2 3 4 5 6 7 8 9 10Days of incubation2520151050Figure 4.1 Fungal growth (dry weight) and extracellular proteinaseproduction by 0. piceae strain 387N in synthetic medium (section 2.2)containing soybean protein as a nitrogen source. Proteinase activity units(U) are defined as the amount of enzyme that produced a rate of increasein A520 of 0.1 units/mm at 37°C and 320 rpm. A similar trend wasobserved in duplicate experiments.Proteinases produced by 0. piceae strain 387N on wood (section 3.3.7) showed a similarinhibition pattern. There was no inhibition by 1,10-phenanthroline, pepstatin or E-64. Howeversignificant inhibition (greater than 50%) was observed after incubation with PMSF and EDTAThese results suggested that extracellular proteinases secreted by 0. piceae when the organismgrew both in wood containing protein as the nitrogen source and in liquid culture weremechanistically similar.84Table 4.1 Effects of proteinase inhibitors on the hydrolysis of azocoll by theculture filtrate of 0. piceae strain 387N after growth on soybean proteinType of Inhibitor A520, % InhibitionproteinaseNo inhibitor With inhibitoraspartic pepstatin2 0.80 0.80 0.0cysteine E-643 0.75 0.83 -10.7metallo EDTA4 0.75 0.09 88.01, 10-phenanthroline5 0.75 0.86 -14.7phosphoramidon6 0.75 0.80 -6.7serine PMSF7* 0.57 0.00 100.0AEBSF8 0.57 0.30 47.4DCI-1009 0.57 0.43 24.6DFP1° 0.53 0.41 22.6TPCKII* 0.50 0.70 -40.41% Inhibition was calculated as the percentage difference between the absorbances of the reactionswith and without inhibitors. The values for no inhibitor controls represent the activity where only theinhibitor was absent.2pepstatin (1 tM in 1% DMSO), 3E-64 (10 jiM),4EDTA (10 mM), i, 10-phenanthroline (10 mMwith 1% methanol) and6phosphoramidon (10 jiM),7PMSF (2 mM in 1% DMSO),8AEBSF (2 mM),9DC1 (100 jiM in 1% DMSO), ‘°DFP (100 jiM in 1% 2-propanol), “TPCK (100 jiM in 1% ethanol)TPCK and PMSF are also known to affect cysteine proteinases854.3.3 Purification of a serine proteinaseIn order to further characterise the major proteinase produced by 0. piceae, variouschromatographic methods were tested for purifying the proteinase from culture filtrates. Theseparation of a proteolytic peak from other proteins was obtained by hydrophobic interactionchromatography on the FPLC system (Figure 4.4). Recovery of the proteinase activity in thepeak was between 86 and 100% in duplicate experiments. This was in contrast to separations byanion exchange (MonoQ HR5/5), chromatofocusing (MonoP HR5/20) and gel filtration(Superose 12 HR1O/30) where recoveries of proteinase activity were low, about 20 to 50% ofapplied activity. This was possibly due to autolysis of the proteinase, a phenomenon that hasbeen examined further in Chapter 5.To purify large quantities of the proteinases for subsequent characterisation, preparativehydrophobic interaction chromatography was used as a first step. In preparation forhydrophobic interaction chromatography, ammonium sulphate was added to culture filtratesimmediately after harvesting by filtration. This was also found to stabilise the proteolyticactivity in the crude culture filtrate. After chromatographic separation at 4°C, the single peakcontaining the proteolytic activity was concentrated for storage by precipitation with ammoniumsulphate (Table 4.2). This procedure resulted in a 2.2 fold purification, which was low becausethe protein of interest was the major protein from the outset. Additional hydrophobic interactionchromatography on FPLC resulted in an increase of specific activity from 243 to 280 U/mgprotein.86CCco4-cU)C.)CCD-e0C’).0Ca)Sample Vol (ml) Total Total Specific Purifica- YieldActivity (U) Protein Activity tion (activity) %(mg) (U/mg) (fold)Culturefiltrate 5100 56100 510 110 1.0 100Preparative 845 33 000 150 220 2.0 59hydrophobicinteractionchromatographyAmmonium 10.3 25750 106 243 2.2 46sulphateprecipitation2.01.61.20.80.40 50 100 150Elution time (mm)330C3Cl)Ca)CDFigure 4.2 FPLC fractionation of proteins in the culture filtrate of 0.piceae strain 387N using a Phenyl Superose HR5/5 column. Theproteins (1.7 mg) were loaded in 1.7 M ammonium sulphate and elutedwith 75 ml of a decreasing salt gradient (dotted line) from 1.7 to 0 Mammonium sulphate which was applied at a flow rate of 0.5 mi/mm.Elution of the proteins was monitored at 280 nm. The arrow indicates theonly peak showing proteinase activity using azocoll as substrate.Table 4.2 Purification of the extracellular proteinase produced by 0. piceae strain 387 N874.3.4 Properties of the purified proteinase4.3.4.1 ElectrophoreticpropertiesElectrophoretic analyses of the culture filtrate showed one major extracellular protein in theculture filtrate of 0. piceae when grown in the modified medium (section 4.3.1). A 33 kDaprotein was observed by SDS-PAGE (Figure 4.3, lane 2), and a p1 5.6 protein was shown byIEF (Figure 4.4A, lane 2). The major band after IEF corresponded to a proteinase that is able todegrade gelatin coating the X-ray film used as an overlay (Figure 4.4B, lane 2). A secondproteinase band at p1 5.2 was detected when the incubation time for the overlay was increasedfrom 3 to 10 minutes.Figure 4.3 SDS-PAGE (8-25% PhastGel) showing the purification ofthe major proteinase secreted by 0. piceae strain 387N after growth onmedia supplemented with soymilk protein. Lane 1, low molecular weight(MW) standards (Pharmacia) with MW expressed in kDa; lane 2, culturefiltrate after 6 days of growth; lane 3, protein of 33 kDa obtained fromculture filtrate after purification using hydrophobic interactionchromatography. Proteins were loaded at 300 ng/lane and stained usingsilver as recommended by Pharmacia.2HMW94674330—i20.1-i14.4883Figure 4.4 IEF (PhastGel 3-9) monitoring the purification of theextracellular proteinase from 0. piceae strain 387N. Lane 1, wide rangep1 standards (Pharmacia); lane 2, culture filtrate after 6 days of growth;lane 3, protein band of 33 kDa obtained from culture filtrate afterpurification using hydrophobic interaction chromatography. Proteinswere loaded at 500-600 ng/lane. (A) Silver-stained gel showing locationofproteins and proteinase (arrow), (B) X-ray film overlay of the gel after3 mm incubation at 37°C showing the proteolytic activity (arrow).To determine the p1 and molecular weight of the purified proteinase, protein preparationsobtained after the second round of hydrophobic interaction chromatography were analysed bySDS-PAGE and IEF. On SDS-PAGE a major band of 33 kDa (Figure 4.3, lane 3) and minorbands of lower MWs were observed. These minor bands could be degradation products of theproteinase with similar hydrophobic properties, or protein fragments of the proteinase formedafter the purification. A major protein band at a p1 value of 5.6 was observed on IEF gel with aminor band at apI of 5.2 (Figure 4.4, lane 3). These values were confirmed using IEF gels withA B89a narrow ampholyte range of pH 4.5 to 6. Most of the proteolytic activity shown by the X-rayfilm overlay was associated with the band at a p1 of 5.6. The purified enzyme was notglycosylated as shown by the lack of reaction with the colorimetric substrate (NBT-BCIP) usingthe digoxigenin (DIG) glycan detection kit.The MW and p1 values for the purified proteinase were the same as those for the major protein inculture filtrates after growth on soyprotein (Figure 4.3, lanes 2 and 3; Figure 4.4, lanes 2 and 3).Furthermore, the electrophoretic pattern on SDS-PAGE and IEF was the same as that found forthe major proteinase produced on BSA-supplemented medium (section 3.3.5). This confirmedthat the same major alkaline serine proteinase was produced when protein was present as anitrogen source.4.3.4.2 CatalyticpropertiesThe optimal activity of the purified proteinase was found to be between pH 7 and 9 when assayedon azocoll at 37°C (Figure 4.5A), and to be at 40°C when assayed at pH 8 (Figure 4.6A). Therange of pHs for the optimal activity indicated that the proteinase belonged to the alkalineproteinase family. Proteinase activities dropped off rapidly at pH values greater than 10. The pHwas determined at the start of the reaction and remained constant over the course of the reaction.90(A) 70D50m 40U)C20100(B) 2520EQ 1511:2 3 4 5 6 7 8 9 10 11 12pHFigure 4.5 The effect of pH on the activity of the extracellular proteinasepurified from 0. piceae as determined at 37°C using azocoll (A) and succinylAla-Ala-Pro-Phe-p-nitroanilide (B) as substrates. Mes-Tris-Acetate (0.1 M)was used from pH 3 - 9 and CAPS (0.09 M) was used from pH 8.3-pH 10.5for activity using azocoll. The proteinase activity units (U) determined inCAPS buffer were adjusted by a factor of 1.38 to normalise the activity at pH8.3 to that observed in Mes-Tris-Acetate buffer. Reaction rates on thesubstrate succinyl-Ala-Ala-Pro-Phe-p-nitroanilide was expressed as 1 0 timesthe rate of change in O.D. per mm (mOD/mm). Points plotted are the meanvalues of 2 to 4 determinations.91To investigate the cleavage specificity, model substrate peptides were used (Dunn, 1989). Theproteinase had no activity on the substrates BAPNA, BTPNA, and ATAPNA used to detecttrypsin-, chymotrypsin- and elastase-like activities, respectively. The aminopeptidase substrateL-Leu-p-nitroanilide was not hydrolysed. However, activity was observed using the subtilisinsubstrates succinyl-(Ala)2-Pro-Phe-p-nitroanilide (sAAPF) and succinyl-(Ala)2-Pro-Leu-p-nitroanilide. Phenylalanine was more readily hydrolysed than leucine in the P1 position on thesubstrate. The kinetic constants for hydrolysis of sAAPF were calculated from LineweaverBurk plots (Table 4.3). The catalytic constant was calculated from the maximum velocity asdescribed in section 2.5.1. For comparison the table also includes values for another fungalproteinase and a mammalian proteinase. The substrate sAAPF is a better substrate forchymotrypsin and the proteinase from Metarhizium anisopliae both in terms of Km and kcat.Table 4.3 Kinetic constants for the hydrolysis of succinyl-Ala-Ala-Pro-Phe-p-nitroanilide bythe proteinase purified from 0. piceae, chymotrypsin and a cuticle degrading proteinase fromMetarhizium anisopliaeProteinase Substrate concentration Km k k/Km—1 1 —1range(mM) (mlvi) (s ) (Ms )0. piceae protemase 0.06 - 1 3 2.3 7.7 x 102chymotrypsin’ 0.02-2 0.11 39.4 3.6 xPri proteinase ofM anisopliae’ 0.02 -2 0.27 28.1 1.0 x‘St. Leger et al., 1987Temperature and pH activity profiles on sAAPF (Figures 4.5B, 6B) were similar to thoseobtained on azocoll.92(A) 70- 60• 5020°- 100(B) 3025020 30 40 50 60Temperature (°C)Figure 4.6 The effect of temperature on the activity of the extracellularproteinase purified from 0. piceae as determined at pH 8 using azocoll(A) and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (B) as substrates.Reaction rates on the substrate succinyl-Ala-Ala-Pro-Phe-p-nitroanilidewas expressed as 1 0 times the rate of change in O.D. per minute(mOD/mm). Points plotted are the mean values of 2 to 4 determinations4.3.4.3 Stability after exposure to variouspH and temperature regimesThe proteinase isolated from 0. piceae was exposed to pH’s from 3 to 11 before being assayedfor residual activity at pH 8, 37°C. Greater than 80% of the initial activity was recovered aftera 4 h exposure to pH’s 6 to 11 at room temperature, whereas 73% of the initial activity wasrecovered after 4 h at pH 5 and no activity was detected after incubation at pH 3. In fact, after093just 5 mm exposure to pH 3 at room temperature, only 40% of activity was recovered. At3 7°C, the proteinase was even less stable across the p11 range. Less than 30% of the originalactivity remained after 1 h incubation for all pH’s tested (Figure 4.7). At 3 7°C, the proteinasewas most stable at pH 6, at which it had a half life of 30.4 mm (Table 4.4).100- • I I I>•. ,.4-,>Cu60-Cu40-20COa)• I I I I I0 2 4 6 8 10 12pHFigure 4.7 Effect of pH on the stability of the proteinase isolated fromculture filtrates of 0. piceae strain 3 87N. Protein concentration during theincubation was at 0.04 mg/ml with 0.1 M ammonium sulphate.Incubations of 60 mm duration were carried out at a range of pH’s inMES-Tris-Acetate (0.1 M), at room temperature (square symbols) or at37°C (circles). Samples were then assayed at 37°C, pH 8 using succinylAla-Ala-Pro-Phe-p-nitroanilide as substrate. The points plotted are themean values of 3 determinations, with standard deviations shown by errorbars.94The thermostability of the proteinase isolated from 0. piceae was evaluated at pH 6and 8.5. The results showed that the proteinase was relatively thermolabile (Table4.5). At room temperature, the half life of the proteinase at pH 8 was 6.7 days.However, as the temperature increased, the half life decreased rapidly (Figure 4.8).At temperatures higher than 40°C the half life was always less than 5 minutes.Table 4.4 Half lives of the serine proteinase purified from culture filtrates of 0.piceae strain 387N at different pH’s. Enzyme preparations were exposed to a rangeof pHs in MES-Tris-Acetate buffers (0.1 M) at 37°C for 0 to 60 mm before beingassayed at 3 7°C, pH 8 using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. Proteinconcentration was at 0.04 mg/mi with 0.1 M ammonium sulphate during the preincubation.Conditions t112 (mm) Correlationcoefficient’pH3.4 less than 5pH 5.1 15.1 0.99pH 6.1 30.4 1.00pH 7.0 27.3 1.00pH 8.4 20.3 1.00pH 8.9 20.4 1.00pH 11.5 17.7 0.99‘Correlation coefficients for the best-fit lines to the In residual activity versus time.95>13U)U)CU)0IU)ci)Temperature (°C)Figure 4.8. Effect of temperature on the stability of the proteinaseisolated from culture filtrates of 0. piceae strain 387N. Proteinconcentration during the incubation was at 0.04 mg/mi with 0.1 Mammonium sulphate. Enzyme preparations were incubated at 23 to50°C for 5 mm at pH 6 (squares) or at pH 8.5 (circles) before beingassayed at 37°C, pH 8 using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide assubstrate. The points plotted are the mean values of 3 determinations,with standard deviations shown by error bars.20 25 30 35 40 45 50 5596Table 4.5 Half life of the proteinase isolated from 0. piceae at pH 6 and 8, attemperatures ranging from 23 to 40°C. Protein concentration during theincubation was at 0.04 mg/ml with 0.1 M arnmonium sulphate. Residual activitywas assayed at 3 7°C, pH 8 using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide assubstrate. A plot of ln residual activity (%) versus incubation time was used tointerpolate half lives.Condition t,,2 CorrelationpH Temperature (°C) (h) coefficient’8 23 160.8 0.9730 8.1 1.0035 0.6 1.0040 0.1 1.006 23 76.8 0.9830 7.7 0.9935 0.9 1.0040 0.2 1.00‘Correlation coefficient for the best-fit lines to the In residual activity versus time974.3.4.4 Effect ofinhibitorsThe effects of selected serine proteinase inhibitors and chelating agents on the purifiedproteinase were examined. DFP, specific for the active site of serine proteinases, inhibitedproteolytic activity in a dose-dependent manner (Figure 4.9), confirming that this proteinasewas a serine-type proteinase.60>Coci)U)CoCci00.Coz-020U,ci)Figure 4.9 Residual proteolytic activity after 30 mmincubation with DFP. The activity was measured on azocoll(open circles) and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide.80Concentration DFP (mM).498Other classic serine proteinase inhibitors similarly caused a reduction in activity on protein(azocoll) and peptide substrates (Table 4.6). However, two high molecular weight inhibitorswhich are known to inhibit some serine proteinases, aprotinin and soybean trypsin inhibitor,had no effect on the purified proteinase. Activity was reduced significantly by chelatingagents, such as EDTA and salicylic acid (Table 4.7). This was in contrast to other classicalmetallo-proteinase inhibitors (1,1 0-phenanthroline) which enhanced, rather than inhibited,proteinase activity. Proteolytic activity was also inhibited by SDS, while reducing andoxidising agents had little effect (Table 4.7).Table 4.6 Effects of serine proteinase inhibitors on the hydrolysis ofazocoll and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide by the proteinasepurified from 0. piceae strain 387NInhibitor Concentration % inhibition’(mM) azocoll sAAPFPMSF 0.2 ND2 572 100 100AEBSF 2 33 204 40 39antipain 0.01 ND 190.1 ND 98chymostatin 0.01 ND 930.1 ND 100aprotinin equimolar3 0 0soybean trypsin inhibitor equimolar 0 0turkey egg white inhibitor equimolar 78 01the difference in absorbance between inhibited and uninhibited enzyme, expressed as apercentage of the uninhibited enzyme2not determined3equimolar with proteinase99Table 4.7 Effects of different reagents on the hydrolysis of azocoll by theproteinase purified from 0. piceae strain 387NType Compound Concentration % inhibition’(mM)chelators EDTA 1 8910 89EGTA 1 8710 88TPEN 1 13salicylic acid 10 1850 100tartaric acid 10 2350 100detergents SDS 0.35 123.5 100reducing agents DTT 1 12-mercaptoethanol 1 7oxidising agents H20 8.8 01the difference in absorbance between inhibited and uninhibited enzyme, expressed as apercentage of the uninhibited enzyme4.3.4.5 Effect ofmetal ions and bufftr saltsMercury, cobalt, nickel, copper, zinc and lead resulted in a decrease in proteolytic activity onazocoll (Table 4.8). However, only mercury caused a significant reduction in activity whenmeasured on a short peptide substrate. Similarly, sodium citrate, sodium carbonate andsodium phosphate appeared to affect activity on azocoll but not on sAAPF. In contrast,divalent ions such as calcium, manganese and magnesium increased activity on bothsubstrates. A commercial antisapstain formulation, PQ-8, was included in these experimentssince it contains oxine copper as its prime active ingredient (section 2.6). It resulted in 96 %inhibition on azocoll when the concentrate (5.4% Cu-8, 5 5-65% DDBSA) was used at a 1000times dilution.100The assay using the sAAPF was performed at a higher ammonium sulphate concentration thanthe azocoll assay. This is known to stabilise the proteinase. Therefore, inhibition of activitymeasured by the sAAPF assay was more likely to be at the active site rather than an effect onstability of the enzyme. Ammonium sulphate increased the rate of hydrolysis of sAAPF evenat concentration of 1 M. However, at the same concentrations it caused a decrease in thehydrolysis of azocoll.Table 4.8 Effect of metal ions and buffer salts on the hydrolysis of azocolland succinyl-Ala-Ala-Pro-Phe-p-nitroanilide by the proteinase purified from0. piceae strain 387NInhibitor Concentration (mM) % inhibition’azocoll sAAPFCaCI2 10 -15.9 -16.5CuCI2 10 23.4 -1.9CoC12 10 61.4 0.9MgC12 10 -20.7 -3.9MnC12 10 -14.4 -40.8HgCI2 10 100 80.0NiC1 10 13.6 -17.5KCI 10 -10.6 ND2CaSO4 10 -8.6 NDZnSO4 10 100 -6.8Ca(N03)2 10 -21.1 NDPb(N0 10 59.7 NDAgNO3 10 -10.2 NDBa(OH)2 10 -1.5 NDNa•acetate 100 3.5 -9.7Na•carbonate 100 12.3 0.0Na•H carbonate 100 -3.6 -9.7Na•citrate 100 64.3 -5.8Na•phosphate 100 14.8 -1.9‘inhibition was calculated as the percentage difference between the absorbances of thereactions with and without inhibitors. A negative sign indicates the reaction wasenhanced not inhibited2ND not determined101Table 4.9 Amino acid composition of the extracellular proteinase purified from0. piceae strain 387N compared to those reported for serine proteinases in thesubtilisin and (chymo)trypsin familiesAmino acid Mole %0. piceae proteinase K subtilisin BPN’3 bovine trypsin4proteinase1 (T album)2ASX 10.4±2.0 11.3 10.2 9.7GLX 7.3 ± 1.0 4.4 5.5 6.2SER 10.9± 1.9 13.5 13.5 14.6GLY 16.3±0.8 12.4 12.0 11.1HIS 1.2 ± 0.2 1.5 2.2 1.3ARG 1.2 ± 0.1 4.4 0.7 0.8TRR 6.8 ± 1.0 8.0 4.7 4.4ALA 17.2±2.9 12.0 13.5 6.2PRO 2.5±0.3 3.3 5.1 4.0TYR 2.8±0.5 5.5 3.6 4.0VAL 6.8± 1.3 6.2 10.9 5.3MET 0.5±0.0 1.8 1.8 0.9ILE 4.4± 1.1 3.3 4.7 7.5LEU 6.3 ± 1.1 4.7 5.5 4.4PHE 1.3 ±0.5 2.2 1.1 6.2LYS 4.0±0.9 2.9 4.0 1.81/2CYS ND5 1.8 0.0 5.3TRP ND 0.7 1.1 1.81mean of 4 separate determinations ± S.D.2Jany and Mayer, 19853Tsuchiya et al., 19924MilIer et al., 19745not detemuned1024.3.4.6 Amino acid composition and N-terminal sequenceThe proteinase was rich in glycine, alanine, serine and aspartic acid/asparagine but low inhistidine, arginine, tyrosine, proline, methionine, isoleucine, phenylalanine and lysine(Table 4.9).The N-terminal sequencing of the 33 kDa protein yielded a sequence of 18 amino acids, A Y TT Q T G A P W G I S R L L H K (Figure 4.10). Using the BLAST system, we found thefollowing proteins with similar sequences: a serine proteinase from Schizosaccharomyces, analkaline proteinase from Aspergillus fumigatus, an alkaline proteinase from Trichodermaharzianum, an elastase from Metarhizium anisopliae, a serine proteinase from Acremoniumchrysogenum. These enzymes are from the subtilisin family of serine proteinases sometimesreferred to as subtilases. Based on its NH2-terminal sequence, the proteinase purified from 0.piceae can be classified as a subtilisin class II serine proteinase.A Y T T Q T G A P W G I S R L L H K OphiostomapiceaeA A Q T N A P W G L A R I S S Tritirachium albumA L V T Q N G A P W G L G T I S H R AcremoniumchtysogenumG L T T Q K S A P W G L G S I S H K Aspergillus o?yzaeA L T T Q K G A P W G L G S I S H K AspergillusfumigatusA L T T Q 5 G A P W G L G T V S H R Trichoderma harzianumG I T E Q S G A P W G L G R I S H R Metarhizium anisopliaeFigure 4.10 N-terminal sequence of the proteinase purified from 0. piceae strain387N and comparison with sequences reported for fungal serine proteinases that arein the subtilisin class II family. Black boxes show residues that are conserved inthese enzymes.1034.4 DiscussionInitial experiments reported in this chapter were conducted on crude culture filtrate and woodpowders after growth of 0. piceae. They showed that the proteinases produced in protein-supplemented liquid media and on wood were sensitive to PMSF and EDTA. Inhibitors forother types of proteinases did not cause a reduction in proteolytic activity. We concluded thatthe proteinase(s) produced by 0. piceae in liquid media and wood were serine-type proteinaseswith a requirement for metal ions. Many proteinases classified as serine proteinases have beenshown to be sensitive to EDTA. They include fungal proteinases from Aspergillusfumigatus, A.flavus (Frosco et a!., 1992; Kolattukudy et a!., 1993), A. niger (Barthomeuf et al., 1989),Aureobasidium pullulans (Donaghy and McKay, 1993), and Trichoderma koningii (Manonmaniand Joseph, 1993), and a bacterial proteinase from Xanthomonas maltophilia (Debette, 1991).The basis for the classification of metalloproteinases is the presence of a metal ion (usuallyzinc) which participates in catalysis. Therefore 1,10-phenanthroline is usually preferred as adiagnostic indicator of metallo-proteinases because it has a much higher stability constant forzinc than for calcium. In contrast, chelating agents such as EDTA can inactivate both the zinc-dependent metalloproteinases and some calcium-stabilised proteinases from other classes(Salvesen and Nagase, 1989).Studies investigating the conformational integrity of subtilisin have shown that calciumchelating agents lead to autolytic digestion (Wells and Estell, 1988). It is thought that theremoval of enzyme-bound calcium by chelation increases the flexibility of the protein, and104thereby its rate of autolysis (Siezen et a!., 1991). This may explain the sensitivity of theproteinase to inhibition by EDTA and will be explored further in the following chapter.Subsequent experiments to purify the proteinase from 0. piceae used liquid media. Initialattempts to purify the proteinase were frustrated by repeated losses in enzyme activity. It wasfound that good activity recoveries were possible after separation by hydrophobic interactionchromatography, where the proteinase eluted at about 1 M animonium sulphate. Other studieshave reported the use of ammonium sulphate to prevent self digestion (Kolattukudy et a!., 1993).The conformation of the protein in high salt concentrations may not be favourable for autolyticdigestion. Our experiments showed that the purified proteinase was stable in 1 M ammoniumsulphate for several days at room temperature. However, autolysis of the proteinase may haveoccurred during separations when animonium sulphate was not present, leading to poor activityrecoveries after gel filtration, anion exchange and chromatofocusing. Interestingly, the rate ofhydrolysis of a peptide substrate was enhanced in the presence of ammonium sulphate.However, at the same concentration of ammonium sulphate, activity could not be detected onthe protein substrate azocoll.Ammonium sulphate appeared to affect the substrate-enzyme interaction in ways which weredependent on the nature of the substrate. Previous research has shown that the lack ofinhibition of subtilisin by bovine pancreatic trypsin inhibitor is most likely caused by a sterichindrance (Mitsui, 1985). Steric collision of the inhibitor and the proteinase occurs at sites farfrom the reactive site of the inhibitor (and thus the active site of the enzyme) preventingbinding. Similarly, ammonium sulphate could affect the conformation of the proteinase and105the substrate such that steric hindrances of this kind affected digestion of larger proteinsubstrates but not small peptide substrates. Interestingly, a high molecular weight proteinaseinhibitor, turkey egg white inhibitor, caused significant reduction in activity on azocoll, but noinhibition on the peptide substrate. This suggests that the peptide was hydrolysed as asubstrate in preference to the inhibitor or pseudo-substrate.The purified proteinase preparation was analysed by SDS-PAGE and IEF. Although a singleband at 33 kDa was observed on SDS-PAGE gels, a major band at p1 5.6 and a minor band at p15.2 were observed on IEF gels. A single polypeptide giving 2 bands on IEF may be an artifactor due to microheterogeneity of the protein (Gianazza and Righetti, 1980). Artefact formationduring IEF has been largely attributed to protein-ampholyte interactions. Microheterogeneityamong proteins is now recognised to be widespread, even though its underlying causes are notfully understood. Microheterogeneity can, for example, represent alternative stableconformations of the same polypeptide. These may arise from post-translational modificationssuch as phosphorylation and glycosylation. Glycosylation was not detected in the purifiedproteinase, and the presence of carbohydrate in purified fungal serine proteinases has beenreported in only a few instances (North, 1982; Tunlid et a!., 1994). Interestingly, serineproteinases from A. fumigatus, A. flavus and A. oryzae have been shown to contain a putativeglycosylation site in their protein sequences, but glycosylation has not been shown (Ramesh eta!., 1994).106It is probable that the structure and properties of this proteinase have evolved to suit proteindigestion in wood, which proceeds at ambient temperatures and at pH values between 4 and 7.Proteolytic activity of the purified proteinase dropped off at pHs greater than 10 as describedfor alkaline proteinases from Aspergillus flavus, Aureobasidium pullulans, Verticilliumsuch?asporium (Malathi and Chakraborty, 1991; Donaghy and McKay, 1993; Lopez-Llorca,1990). However, the proteinase appeared to be stable over a wide pH range in vitro, beinginactivated at pH values less than 3. Inactivation at lower pH values was irreversible. Theinactivation may be confonnational, involving misfolding, aggregation and adsorption; orchemical, involving deamidation of AsnJGln residues, f3-elimination of Cys residues andhydrolysis of peptide bonds at Asp residues (Tatsumi eta?., 1994). It is unlikely that autolysiswas involved, since the enzyme did not have any detectable activity at pH 3. Similar pHstability results have been found for proteinase K, where changes were observed in thesecondary structure below pH 3 (Dolashka eta?., 1992).The proteinase was extremely unstable at temperatures above 23°C. Since fungal growth isoptimal at this temperature (section 3.3.3), the physiological function of the enzyme in vivo isunlikely to be affected by its thermolability. In contrast, other fungal subtilases, includingproteinase K, are remarkably heat stable (Betzel et a?., 1988). The thermostability ofproteinase K has been partially attributed to the presence of two disulphide bonds (Betzel eta?., 1990). While there seems to be agreement that disuiphide bridges contribute to the overallstability of a protein, attempts to introduce new S-S bonds to enhance thermostability ofsubtilases have generally met with little success (Siezen et a?., 1991). Thermostability of107proteins is determined by factors such as structural stability or resistance to chemicaldegradation processes. However, in the case of proteinases, resistance to autolysis plays amajor role (van den Burg et al., 1990).Amino acid composition analysis indicated that subtilisin BPN’ and proteinase K from thesubtilisin family of serine proteinases have amino acid compositions similar to that of theproteinase from 0. piceae. In contrast, serine proteinases such as bovine trypsin from the(chymo)trypsin family appear to have a significantly lower content of alanine and a highercontent of phenylalanine.High homology at the N-terminus is sufficient to distinguish two definite classes of subtilases(Siezen et al., 1991). Class I subtilases include proteinases from Gram positive and Gramnegative bacteria, as well as mammalian, insect and yeast proteinases. Class II subtilasesinclude many fungal proteinases including proteinases from Tritirachium album, Aspergillusoryzae, and Acremonium chrysogenum. In addition to having homology with the N-terminus ofthe class II subtilases, the proteinase purified from culture filtrates of 0. piceae was shown todisplay similar properties to those reported for this class of serine proteinases. These includedthe inhibition pattern and cleavage specificity. Furthermore, polyclonal antibodies against thesubtilisin class II enzyme, proteinase K, cross-reacted with the proteinase purified from 0.piceae. These results support the classification of the purified enzyme as a subtilisin class IIserine proteinase.108In summary, for the first time, an enzyme from the sap-staining fungus Ophiostoma piceae wasdescribed and purified. The subtilisin-like serine proteinase isolated was produced as the majorprotein in the extracellular filtrate during growth on protein-supplemented medium. It appearsto be actively secreted during growth on wood where similar inhibition patterns and biochemicalprofiles were obtained. The properties of the proteinase, particularly the autolysis and substratespecificity will be explored further in Chapter 5.109Chapter 5.Autolysis and substrate specificity of aproteinase purified from Ophiostoma piceae5.1 IntroductionDuring the purification of the p1 5.6 proteinase from 0. piceae, the proteinase was found to beunstable under certain conditions. Autoproteolytic cleavage was considered a likely explanationfor losses of activity. This observation prompted a detailed examination of this phenomenon inattempts to identify conditions which favoured stability or degradation of the isolatedproteinase.’ It was also necessary to understand autolysis in order to carry out substratespecificity experiments. Therefore the two objectives for this section were the evaluation of theautolysis and substrate specificity as part of the functional characterisation of the proteinasepurified from 0. piceae.Autolysis or autoproteolysis is influenced by several factors, including the cleavage specificity ofthe proteinase. Autolysis has been observed in different types of proteinases. Recent studieshave characterised autoproteolysis in aspartyl proteinases of retroviruses (Rose et a!., 1993),neutral proteinases (van den Burg et a?., 1990), and class I subtilases (Kim et a?., 1990; Braxtonand Wells, 1992; Jang et a?., 1993). The identification of the cleavage sites of autoproteolysis iscritical for designing mutations which can slow the rate of autolysis. A considerable research‘These data were submitted for publication: Abraham, L.D., and Breuil, C. 1995. Factors affecting autolysis of asubtilisin-like serine proteinase secreted by Ophiostoma piceae and identification of the cleavage site. Biochimicaet Biophysica Acta. In press.110effort has focused on improving the stability of the subtilases in order to enhance theirapplication, particularly in the detergent industry. However, autoproteolytic cleavage sites havenot been previously identified for class II subtilases. In this chapter, the role of autolysis instability was examined and factors which affect autolysis evaluated. By characterising theenzyme fragments produced by autolysis, the position of autoproteolytic cleavage for subtilaseclass II enzymes was determined.The second experimental objective was to determine the relative activity of the purifiedproteinase towards various specific substrates in order to define the specificity, and compare itwith that of proteinase K. To achieve this purpose, simple synthetic substrates in which there isonly one bond susceptible to enzymatic hydrolysis can be used as described by Walsh andWilcox (1970) and Sarath et al. (1989). Amino acid esters and amides blocked at the N-terminalwere used in spectrophotometric assays. The mechanism of action was also evaluated using theoxidised B-chain of insulin which has served for over 30 years as the substrate of choice for theinitial screening of the specificity of a newly discovered proteinase (Dunn, 1989).The insulin sequence contains a selection of peptide bonds in an unfolded structure so that all thepeptide bonds should be freely accessible (Dunn, 1989). Much of the previous published work todetermine points of cleavage in oxidised insulin B-chain has used HPLC, paper chromatographyor TLC separation of peptides after digestion, followed by amino acid analysis and/or N-terminalsequencing of the separated peptides. These procedures are both time-consuming and laborious.In this work, peptides generated by digestion of insulin with the proteinase from 0. piceae wereseparated by HPLC coupled to an on-line to electrospray ionisation mass spectrometer (ESI MS).111The cleavage sites were determined by matching the peptide masses with the theoretical cleavageof insulin, and by sequencing the peptides using tandem MS.2Finally the functional interaction of the purified proteinase was studied with native proteins,including extracts from wood. Protein extracts were incubated with enzyme preparations andanalysed electrophoretically after various incubation times. The final part of this study wasdesigned to demonstrate that the function of a proteinase purified from a staining fungus is tobreakdown wood proteins and use these proteins as a source of nitrogen.2These data were submitted for publication: Abraham, L.D., Chow, D. and Breuil, C. 1995. Characterization of thecleavage specificity of a subtilisin-like serine proteinase by liquid chromatography/mass spectrometry and tandemMS. FEBS Letters. Submitted.1125.2 Materials and methods5.2.1 Proteinase preparation and purificationThe proteinase used for this study was purified to homogeneity, according to SDS-PAGE, byhydrophobic interaction chromatography (section 4.3.3). The purified proteinase was storedat -20°C in 0.1 M Tris-Ci (pH 8) buffer containing 1 M ammonium sulphate.5.2.2 Circular dicbroic spectroscopyCircular dichroic (CD) measurements were made on a Jasco J-720 spectropolarimeter (JapanSpectroscopic Co. Ltd., Tokyo, Japan). Spectra were recorded between 200 and 350 nm usingcuvettes of 10 mm or 1 mm pathiength. Protein concentrations for near (250 - 350 nm) and far(200 - 250 nm) UV spectra were 0.4 - 0.8 mg/mi and 0.09 mg/mi, respectively. CD data wereexpressed in terms of eliipticity measured in degrees (1 mdeg = 0.001 deg). Proteinase K(Boehringer Mannheim) and the proteinase isolated from 0. piceae were prepared for CDspectroscopy as described previously (Genov and Shopova, 1978). Briefly, they were inactivatedwith PMSF and the intact proteinases were separated from autoiytic products by gel filtrationusing a Superose 12 column equilibrated with 0.1 M phosphate buffer (pH 7) containing 0.5 MKC1 on the FPLC (Pharmacia).The melting temperatures (Tm), defined as the midpoint in the thermally induced transition fromthe folded to the unfolded state, were determined in 0.1 M phosphate buffer (pH 7) containing1130.5 M KCI for the PMSF-inactivated proteinases. Spectral changes were recorded at 221 nm asthe temperature was increased using the JTC-340 temperature control programme (JapanSpectroscopic Co. Ltd., Japan) and the Neslab RTE-1 10 temperature controller (NeslabInstruments Inc., Newington, NH, USA). The effects of calcium (27 mM) and ammoniumsulphate (1 M) on the Tm of the proteinase from 0. piceae were also determined in 20 mMTris-Cl buffer (pH 8). Re-folding of the proteinase in 20 mM Tris-Cl (pH 8) with 1 Mammonium sulphate was assessed by recording spectra at 221 nm while decreasing thetemperature.5.2.3 Generation of autolytic productsAutolytic degradation products were generated by desalting samples (83 tg - 2.6 mg) at roomtemperature on a Bio-gel P6 polyacrylamide gel column (1 ml)(Bio-Rad) equilibrated with thedesired buffer. Alternatively, they were generated by adding active proteinase to proteinaseinactivated by incubation with 2 mM PMSF for 30 mm at room temperature, or by boiling inbuffer containing 2.5% SDS and 5% -mercaptoethanol for 5 min. Before use, these inactivatedsubstrates were transferred to 0.1 M Tris-Cl (pH 8) or 25 mM piperazine-Cl (pH 6.3) containing10 mM EDTA by ultrafiltration using the Microcentricon 10 (Amicon). Inactivated, washedproteinase substrates (86 jig) were digested with 0.5 ig active proteinase at room temperature.The digestion was stopped by the addition of equal volumes of SDS-PAGE sample buffer andboiling for 5 minutes.Glycerol, B SA, DTT, MgCl2 CaC12, and ammonium sulphate were tested as protectants againstautolysis during heating. The proteinase was mixed with 0.1 M IvWS-Tris-Acetate buffer, pH 8114containing the protectants at a ratio of 1:10 by volume. After incubation at set temperatures,proteins remaining were examined by SDS-PAGE (section 2.7), and residual proteinase activitywas assayed at 37°C, in 0.1 M Mes-Tris-Acetate (pH 8) using sAAPF as substrate (section 2.5).Half lives were determined by interpolating the least-squares linear fit of a plot of the in ofremaining activity versus time.5.2.4 Assay of activity againstp-nitrophenyl estersVarious phenyl ester substrates were purchased from Sigma and prepared at 2.4 mM in dioxane.They were always prepared fresh on the day of use. A methanolic buffer (30 mlvi Tris-Cl, 20%methanol, 0.1 M CaC12, pH 7.5) was added to each well in a 96 well plate. Enzyme dilutionswere prepared for the purified 0. piceae proteinase and proteinase K, in 20 mM Tris-Cl, 0.1 MCaC12,pH 7.5 immediately prior to use. Dilutions were adjusted to be in the linear range of theassay. The reaction was initiated by the addition of the substrate, to give a fmal concentration of79 p.M. Four replicate wells were used for each of three enzyme concentrations. The progress ofeach reaction was followed, at 405 nm at room temperature for 10 mm with shaking, using aThermomax microplate reader (Molecular Devices Corporation). Each well received 200 p.1 ofsample giving a path length of 0.7 cm. The initial reaction rate was determined as the slope in thelinear portion of a graph plotting absorbance against time, and was expressed as 1 0 times the rateof change in O.D. per minute (mOD/mm). The assay plots were usually linear for the first fiveminutes.1155.2.5 Insulin-B chain digestionAn aliquot (100 j.tl) of bovine insulin B-chain (Sigma) prepared at 1 mg/mi in 0.1 M Tris-Ci,pH 8 containing 2 mM CaC12, and 2 jig 0. piceae proteinase were incubated at roomtemperature. Substrate cleavage sites were determined after stopping the reaction at 2 mm,5 miii, 1 h and 24 h.5.2.5.1 HPLC conditionsAt the specified times, a 5 jil aliquot was removed from the reaction mixture and diluted 10times with HPLC solvent A (pH 2.2) to stop the reaction. Peptides in an aliquot (20ji1) wereseparated on an Ultrafast Microprotein Analyser (Michrom BioResources Inc., Auburn, CA,USA) by reverse phase HPLC on a 1 x 150-mm Reliasil C-18 column (5 jim, 300-A).Chromatography solvents were 2% acetonitrile (MeCN), 0.05% trifluoroacetic acid (TFA)(solvent A) and 80% MeCN, 0.045% TFA (solvent B). The column was developed with alinear gradient from 5% to 50% solvent B in 30 miii, followed by a gradient from 50 to 80%solvent B in 2 minutes. The flow rate was SOjil/min, and the UV absorbance was measured at214 nm in a 300-ni flow cell with a pathlength of 2 mm. A post column flow split diverted 85%of the column eluate for fraction collection, and 15% to an ion spray mass spectrometer (Hess etal., 1993).For analysis by tandem MS (MS/MS), column fractions containing the peak of interest wereeach injected onto a concentrator ITPLC column (0.5 x 150-mm Reliasil Cl8 column) at a flowrate of 20 jil/min. A 10 to 50% gradient of solvent B was applied over 12 miii, followed by agradient from 50% to 90% solvent B in 1 minute, with all the eluate fed into the MS.1165.2.5.2 MS conditionsMass spectra were recorded on a PE-Sciex API III triple quadrupole MS (PE-Sciex, Thornhill,Ontario, Canada) equipped with a Ion Spray ion source. The ion spray voltage wasapproximately 5000 V and the nebulizer gas pressure was 40 psi. All LC/MS experiments weredone in a single quadrupole operating mode using quadropole 3 of the mass analyser. The massrange from 175 to 2200 Da was scanned with a step size of 0.5 Da and a dwell time of 1 ms perstep.For LC/MS/MS the spectrum was obtained by selectively introducing an ion of a single mass-tocharge ratio from the first quadrupole (Qi) into the collision cell (Q2) and observing thedaughter ions in the third quadrupole (Q3). Thus, Qi was locked on a particular mlz ratio, andthe Q3 scan range was adjusted to cover the range from 50 to higher than the mlz ratio selected.Conditions: collision gas thickness = 4.3 x i0’ molecules/cm2(COT = 430), collision gas wasN2/Ar mix in 10/90 proportion, step size = 0.5 or 1, orifice energy = 80, dwell time = lms.5.2.6 Preparation of proteins from poplarA three year old branch was removed in April from an eight year old Populusdeltoides/trichocarpa (hybrid DT 49-177) grown in Agassiz, B.C. The bark and cambium layerswere peeled back and the differentiating xylem was removed by scraping. The wood was groundwith silica sand in a mortar and pestle with liquid nitrogen. Chilled buffer (50 mM Tris-Ci, pH 8containing 5 mM MgC12) was added, mixed gently, and the solution clarified by repeatedcentrifugation at 10000 g. The supernatant was dried under vacuum (Speedvac, Savant,117Farmingdale, NY, USA) and resuspended in 20 mM Tris-Ci containing 27 mM CaC12immediately prior to proteolytic digestion.5.2.7 Proteinase activity against proteinsProtein substrates including BSA, casein, globulins from pumpkin seeds (Cucurbita pepo) andedestin from hemp seeds (Sigma) were prepared at 4 mg/mi in 20 mM Tris-Cl, pH 8. Thepurified 0. piceae proteinase and proteinase K were added at 1:200 ratio (w/w) and incubatedwith the substrate at room temperature for 5 to 150 mm. Proteins extracted from poplar xylemwere digested for 2 to 5 h at an enzyme to substrate ratio of 1:50. Reactions were terminated byboiling in SDS-PAGE sample buffer. Proteins in the digested samples were separated by SDSPAGE.1185.3 Results5.3.1 Autolysis of the proteinase5.3.1.1 AutolysisPreliminary work suggested that the proteinase may be unstable. Proteinase activity losses werefound after desalting into various buffers for FPLC techniques. To determine if low activityrecovery was due to protein degradation, proteinase preparations were desalted into various buffers,and the protein bands observed on SDS-PAGE gels (Figure 5.1). By SDS-PAGE, a major proteinband at 33 kDa was observed when the purified proteinase was inactivated by 2 mM PMSF anddesalted into buffer (25 mM piperazine-CI, pH 6.3 + 10 mM EDTA) (Figure 5.1, lane 4).However, when PMSF was not added, two lower molecular weight protein bands of about 14 and19 kDa were observed in addition to the 33 kDa band (Figure 5.1, lane 3). This distinct change inthe protein banding pattern was accompanied by a proteolytic activity loss of about 80% (Table5.1). PMSF inhibited the formation of smaller molecular weight proteins, which suggested thatthese were proteolytic degradation products from the major proteinase. The fragments wereconsidered to be the major degradation products with the sum of the individual molecularweights being the molecular weight of the native protein.119MW!1 3 4 5 6 7 843—30——20.1—144—Figure 5.1 SDS-PAGE (8-25% PhastGel) showing autolysis of theproteinase isolated from 0. piceae strain 387N during gel filtration,generating low molecular weight cleavage products. Lane 1, lowmolecular weight (MW) standards (Pharmacia) with MW expressed inkDa; lane 2; proteinase purified by hydrophobic interactionchromatography prior to gel filtration; lanes 3 to 8, proteins bandsobtained after desalting into piperazine-Ci, pH 6.3 + 10 mM EDTA (lane3); into piperazine-Ci, pH 6.3 + 10 mM EDTA with prior inactivation ofthe proteinase with 2 mM PMSF (lane 4); into piperazine-Cl, pH 6.3 + 10mM EDTA + 0.15 M ammonium sulphate (lane 5); into piperazine-Cl, pH6.3 + 10 mM EDTA + 10 mM CaC12 (lane 6); into piperazine-Ci, pH 6.3(lane 7); into Mes-Tris-Acetate, pH 2.4 (lane 8). Proteins were desalted at5 mg/mi and loaded onto gels at 100 ng/lane. Staining was carried outusing silver as recommended by Pharmacia.Table5.1Generationofautoproteolyticproductsfromtheproteinaseisolatedfrom0.piceaestrain387Nduringbufferexchangeonagelfiltrationcolumn(1ml)atroomtemperature.Proteinasepreparationswereappliedatthespecifiedconcentrations,andelutedwiththeequilibrationbuffer.Proteinbandsobservedaftersilverstainingofthegelwerequantifiedbydensitometry.0EquilibrationbufferProteinActivityQuantityofprotein(%)concentration,recoveredinmajorbandsobserved(mg/ml)±SD1)afterSDS-PAGE(kDa)SaltConcentrationpHAdditive331914(mM)Piperazine-Ci256.3None5.019.8±0.5572914Piperazine-CI256.310mMEDTA5.020.1±1.5512821Piperazine-CI256.310mMEDTA5.0106.4±1.39910mMCaC12Piperazine-Ci256.310mMEDTA5.099.4±2.3990.15M(NHJ2SO4Piperazine-CI256.310mMEDTA5.03.4±0.51002mMPMSF2Piperazine-Ci209.5None8.0ND99MES-Tris-Acetate1002.4None5.03.2±0.895Na-acetate1005.0None5.025.5±2.5243632Tris-Ci1008.0None8.088.8±4.198S.D.indicatesthestandarddeviation2sampleswerepre-treatedwithPMSFpriortoloadingonthecolumn121In order to ascertain whether the loss of activity after heating was due to irreversibleinactivation or autolysis of the purified proteinase, protein preparations were analysed bySDS-PAGE after incubation for various times at elevated temperatures. The native proteinband at 33 kDa decreased with increased exposure time, but no protein breakdown productswere detected (Figure 5.2).1 4:—Figure 5.2 SDS-PAGE (8-25% PhastGel) showing thermal instabilityof the proteinase isolated from 0. piceae strain 3 87N. Lane 1, lowmolecular weight (MW) standards (Pharmacia) with MW expressed inkDa; lanes 2 to 4, proteinase preparations incubated at 55°C for 5 mmwith no additive, with 0.9 M (NH4)2S0 and with 29 mM CaC12 (lanes2 to 4, respectively); proteinase preparations incubated at 40°C with 29mM CaC12 for 0, 10 and 30 minutes (lanes 5 to 7, respectively); lane 8,proteinase preparation incubated at 40°C for 30 minutes without anyadditive. Proteins were incubated at 0.4 mg/ml and 0.5 ml were loadedon each lane. Staining was carried out using silver as recommended byPharmacia.MW9467435 6 7 83O—I20.1,—14.4.122After incubation of the purified proteinase at 55°C for 5 mm or at 40°C for 30 mm, the proteinband at 33 kDa was not observed (Figure 5.2, lanes 2 and 8). This observation was consistentwith studies on autolysis of other proteinases (Braxton and Wells, 1992; van den Burg et a!.,1990) that reported difficulties in isolating breakdown products under similar conditions.These results suggested that loss of proteinase activity after incubation at higher temperatureswas not due to irreversible inactivation but that elevated temperatures resulted in loss ofnative protein as a result of autolysis. Autolysis of the proteinase occurred at temperatures upto 80°C as indicated by the disappearance of the 33 kDa band on SDS-PAGE gels (gel notpresented).5.3.1.2 Factors which affect autolysisGlycerol (1.95 M), BSA (18.4 mM), DTT (0.9 mM), MgCl2(9 mM), CaC12 (9 to 27 mM), andanimonium sulphate (0.76 M), were all tested as protectants against thermal inactivation.MgCl2,BSA and DTT had little effect as thermoprotectants at 40°C (Table 5.2). However,glycerol, CaCl2 and ammonium sulphate significantly extended the half life of the proteinase.The half life of the proteinase without additives was about 7.7 mm at 40°C, whereas an halflife of 2.3 h was found in the presence CaC12. At 45°C the half life increased from 1.9minutes for the unprotected enzyme to 9.4 mm, 40.4 mm and greater than 2 h when incubatedwith glycerol, CaC12 (27 mM) and ammonium sulphate, respectively.The observed effect of “thermoprotectant& on the half life of the proteinase was furtheranalysed by examining the protein bands by SDS-PAGE. In the presence of CaCl2 at 40°C123there was no significant observable decrease in the native protein band at 33 kDa over 30minutes (Figure 5.2, lanes 5 to 7) whereas the native protein band had disappeared after 30minutes at 40°C for the unprotected enzyme. At 55°C, anunonium sulphate provided themost protection against autolysis (Figure 5.2, lane 3), followed by CaCl2 (Figure 5.2, lane 4),and then glycerol.Ammonium sulphate and CaC12 were similarly shown to prevent autolysis of the purifiedproteinase during buffer exchange at room temperature. The appearance of lower molecularweight bands (Figure 5.1, lanes 3 and 7) was inhibited when ammonium sulphate and CaC12were present in the equilibration buffer (Figure 5.1, lanes 5 and 6 respectively), and most ofthe applied activity was recovered after gel filtration (Table 5.1).124Table 5.2 Effect of thermoprotectants on the half life of the proteinaseisolated from 0. piceae strain 387N, at pH 8, for temperatures ranging from40 to 50°C. Protein concentration during incubation was at 0.04 mg/miwith 0.1 M ammonium sulphate. Residual activity was assayed at 3 7°C, pH8, using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as substrate. A plot of inresidual activity (%) versus incubation time was used to interpolate halflives.Temperature Additive Concentration t112 (mm) Correlation(°C) (mM) coefficient40 None NA 7.7 1.00BSA 18.4 8.8 0.95MgCI2 9.0 9.3 1.00DTT 0.9 6.5 1.00Glycerol 1950.0 46.5 0.98CaC12 9.0 140.0 0.97CaC12 27.0 170.0 0.94(NH4)2S0 760.0 65.3 0.9845 None NA 1.9 0.98Glycerol 1950.0 9.4 1.00CaCI2 27.0 40.4 0.99(NH4)2S0 760.0 123.0 0.9750 None NA <1.0 NAGlycerol 1950.0 1.7 0.99CaCI2 27.0 5.2 1.00(NT{4)2S0 760.0 13.7 0.98NA not applicable* correlation coefficients for the best-fit lines to the ln residual activity versus time.1255.3.1.3 Mechanism ofautolysisExposure to elevated temperatures causes a shift from the native, catalytically active enzymeto the unfolded, catalytically inactive enzyme. The Tm values thus provide an estimate ofrelative thermodynamic stability. The transition temperature for the proteinase isolated from0. piceae was determined by following the changes in absorbance induced by temperature.The Tm for the proteinase from 0. piceae was 37.1°C in 20 mM Tris-Ci (pH 8) (Figure 5.3)and 40.3°C in 0.1 M phosphate buffer (pH 7) with 0.5 M KC1 (Figure 5.4). These transitiontemperatures were much lower than the 66.3°C determined for proteinase K in phosphatebuffer. The addition of CaC12 (27 mM) increased the Tm for the proteinase isolated from0. piceae from 37.1°C to 49.4°C (Figure 5.5), and that of ammonium sulphate (1 M) shifted itfurther to 5 0.7°C (Figure 5.6). The thermal unfolding was not reversible. Although theproteinase was inactivated by PMSF, the possibility of autolysis during the melting processcannot be ruled out at this point.It has been speculated that autolysis could proceed by an intramolecular mechanism, in whicheach molecule of enzyme digests itself, or by an intermolecular mechanism, in whichmolecules of proteinase digest other molecules of proteinase. If each molecule of proteinasedigested itself, the rate of autolysis should be independent of the applied proteinconcentration. During gel filtration experiments, the amount of autocatalytic degradation wasfound to depend on the protein concentration applied. At lower protein concentrations(0.25 mglml), degradation products were not observed as major protein bands, and greaterthan 85% of the applied proteinase activity was recovered (Table 5.1). Higher proteinconcentrations (5 mglml) desalted under identical conditions, resulted in the recovery of less126than 20% of the applied activity as well as the identification of the aforementioned 19 and 14kDa breakdown products on SDS-PAGE gels (Figure 5.1, lane 7). These bands accounted for29% and 14%, respectively, of the total protein in the preparation. These data suggested that,under the experimental conditions used, autolysis was an intermolecular rather than anintramolecular event.To further investigate the ability of the proteinase to act intermolecularly, proteinase sampleswere inactivated with PMSF prior to digestion with small amounts of active proteinase.When the PMSF-inactivated substrate was transferred into 0.1 M Tris-Cl (pH 8), and digestedwith active proteinase, no degradation was observed, even up to 48 h. However, when thePMSF-inactivated substrate was transferred into 25 mM piperazine-Ci (pH 6.3) containing10 mM EDTA, and digested with active proteinase, the 19 and 14 kDa degradation productswere observed on SDS-PAGE gels after 10 mm digestion at room temperature. These studiesindicated that, at pH 6.3 in the presence of a calcium chelator, the enzyme’s conformation wassusceptible to proteolytic attack by active proteinase. When active proteinase was used todigest proteinase which had been inactivated by boiling with SDS, four breakdown productswere observed with approximate molecular weights of 18, 17, 13 and 12 kDa. This indicatedthat the sites of proteolytic cleavage in a linearised substrate were different from those foundwith the same substrate in a folded state, and suggested that folding rendered these sitesinaccessible to enzymatic attack.ci)EEC4CN0.4-wC-)00)I—127—1•1.5-2-2.5-3.03.02.01025 30 35 40 45 50Temperature ‘C)Figure 5.3 Melting curve and first derivative for the proteinasepurified from culture filtrates of 0. piceae strain 387N in 20 mMTris-Cl (pH 8). Spectra were determined at a protein concentration of0.1 mg/mI in a 1 mm cell. Spectra are the average of three scans.a)EEc’J>%.1-’C)4-wC)00)a)0I—•0128-2-3-4-5-6-7.06.04.02025 30 35 40 45 50Temperature C)Figure 5.4 Melting curve and first derivative for the proteinasepurified from culture filtrates of 0. piceae strain 387N in 0.1 Mphosphate buffer (pH 7), 0.5 M KC1. Spectra were determined at aprotein concentration of 0.1 mg/mi in a 1 mm cell. Spectra are theaverage of three scans.C)U)•CEEc’.Jc’J>‘4-C)4-w129-.5—1-1.5-2-2.5-3-3.5-4Temperature C)Figure 5.5 Melting curve and first derivative for the proteinasepurified from culture filtrates of 0. piceae strain 387N in 20 mMTris-CI (pH 8) containing 27 mM CaC12. Spectra were determined at aprotein concentration of 0.1 mg/ml in a 1 mm cell. Spectra are theaverage of three scans.0)U)•0EECc.Ic’.J>%04-LU1302-4-645 50 55.15.1040 60Temperature &C)Figure 5.6 Melting curve and first derivative for the proteinasepurified from culture filtrates of 0. piceae strain 387N in 20 mM TrisCl (pH 8) containing ammonium sulphate (1M). Spectra weredetermined at a protein concentration of 0.1 mg/mi in a 1 mm cell.Spectra are the average of three scans.160170180190XXXXXXXXXXSEPSVXTVGATDSDDLXAEYSNFN0.piceaeDARNYSPASEPSVCTVGASDRYDRRSSFSNYGproteinaseKDASSSSPASEESACTVGATDKTDTLAEYSNFGproteinaseTtGSSSTVGYPGKYPSVIAVGAVDSSNQRASFSSVGsubtil±sinBPN’GNTNTIGYPAKYDSVIAVGAVDSNSNRASFSSVGsubtilisinCarlsbergtFigure5.7N-terminalaminoacidsequenceofthe14kDafragment fromtheautolysisofthe33kDaproteinaseisolatedfrom0.piceaestrain387N.Thesequenceiscomparedtocorrespondingsequences(Siezenetal.,1991) oftwoclassIIsubtilases:proteinasesTandproteinaseKfromTritirachiumalbum,andtwoclassIsubtilases:subtilisinBPN’andCarlsbergfromBacillussubtilis.Xrepresentsunidentifiedresiduesandthearrowshowsthenewlyidentifiedcleavagesitesfor subtilaseclassIIenzymesbycomparisonwiththatreportedforclassIenzymes(Kimetal.,1990;BraxtonandWells,1992).1325.3.1.4 Identflcation ofautolytic cleavage siteTo determine the site of autocatalytic degradation, the N-termini of the 14 kDa and 19 kDaautolysis products were sequenced. The N-terminus of the 19 kDa fragment was A Y T T Q TG A P W G I S R L L H K, which was identical to the N-terminus of the native protein. Thefirst 24 residues of the second fragment were defined with the exception of positions 6 and 17(Figure 5.7). There was a high degree of similarity with corresponding regions in proteinaseK and proteinase T, with 13 and 16 amino acids identical to those in proteinase K andproteinase T, respectively. Proteinase K and proteinase T are both produced by T album, andare classified as class II subtilases which display a high degree of similarity at the N-terminusto the proteinase isolated from 0. piceae. The position for autolytic cleavage was found toexist at the N-side of Ser’70, when abiding by the numbering system pre-established forproteinase K. Although the entire amino acid sequence of the proteinase from 0. piceae isunknown at this point, this site is likely to be near residue 170 based on the sizes of thecleavage products.5.3.2 CD spectroscopy for structural comparison with proteinase KThe structures of the proteinase from 0. piceae and proteinase K were assessed by near andfar UV CD (Figure 5.8). Proteinase K and the proteinase from 0. piceae display a similarpattern in the far UV region, dominated by a large negative band at 221 rim, arising mainlyfrom c-he1ices (Dolashka et al., 1992). The similar spectra in this region suggest theseproteinases have comparable secondary structural contents in solution. According to the Xray model (Betzel et al., 1988), proteinase K contains 32.4% cz-helix and 32% 3-sheet. Since133the secondary structure of the proteinase from 0. piceae is similar to that of proteinase K,many of the properties of 0. piceae’s proteinase can be interpreted without detailedknowledge of its crystal structure.The near UV spectra for the two proteinases are quite different, indicating differences in theiraromatic amino acid contents. Proteinase K has several peaks in the aromatic region of thenear UV with positive peaks at 292 and 304 nm, and a negative peak centred at 274 nm. TheCD spectrum of the proteinase from 0. piceae in the near UV spectral range has positivebands at 281 and 291 nm, and a shoulder at 260 nm. According to analyses by Kolvenbach etaL, 1990, all three types of aromatic residues, tryptophan, tyrosine and phenylalanine,contribute to the CD spectra in this region. They also speculated that the two disulphidebonds present in proteinase K contribute to the observed signals in the near UV spectralregion.+5—‘ 0C)E -5: -100.-15-20220 240 260 280 300Wavelength (nm)Figure 5.8 Far and near UV circular dichroic spectra of proteinase K(solid) and the proteinase isolated from 0. piceae (dotted) measured in0.1 M phosphate buffer (pH 7) containing 0.5 M KC1, in a cell with apathlength of 1 mm (far) and 1 cm (near). Spectra are the average ofthree scans.1345.3.3 Proteinase active site specificityAs described in Chapter 4 (section 4.3.4.2), no activity was detected on the model substratesBAPNA, BTPNA, and ATAPNA used to detect trypsin-, chymotrypsin- and elastase-likeactivities respectively. However, activity was observed using the subtilisin substrates succinyl(Ala)2-Pro-Phe-p-nitroanilide and succinyl-(Ala)2-Pro-Leu-p-nitroani ide. Phenylalarilne wasmore readily hydrolysed than leucine in the P1 position using the nomenclature of Schechter andBerger (1967) as described in section 1.6.The active site specificity on model substrates was also assessed from the relative rates ofhydrolysis ofp-nitrophenyl ester substrates (Table 5.3). The 0. piceae proteinase appeared topreferentially hydrolyse aromatic (Tyr, Leu, Phe) or apolar (Gly, Ala) residues at the P1 site.The best in a series of substrates was CBZ-Gly-p-nitrophenyl ester. Ala, Tyr, Leu and Phewere hydrolysed in decreasing order. Val and Pro were poor substrates. The results alsoillustrated the importance of the N-acyl substituent of the amino acid esters. Replacing abenzyl group (CBZ) with an aliphatic group (BOC) decreased the rate of hydrolysis ofotherwise similar substrates by up to 10 times. Presumably the CBZ group provides a usefularomatic binding site for the enzymes. The only case where there was little difference in theCBZ and BOC substrates was for phenylalanine. Proteinase K displayed a similar specificityto the proteinase from 0. piceae on these substrates (Table 5.3).135Table 5.3 P1 specificity of the 0. piceae proteinase and proteinaseK onp-nitrophenyl ester substrates.Substrate Proteinase activity (U’)0. piceaeprotemase proteinase KCBZ-G1yNP 57.4 57.5CBZ-A1aNP 19.9 38.1CBZ-TyrNP 17.1 11.1CBZ-LeuNP 11.9 14.8CBZ-Phe NP 3.2 6.3CBZ-Val NP 0.4 2.0CBZ-Pro NP 0.3 0.8BOC-Gly NP 5.2 4.7BOC-Phe NP 3.0 3.3BOC-Ala NP 2.3 4.9BOC-Leu NP 2.3 1.61Units represent the initial reaction rate expressed as i0 times the rate ofchange in O.D. per minute (mOD/mm)5.3.4 Cleavage specificity on insulinThe digestion of insulin by the 0. piceae proteinase was analysed by LC/MS after incubationtimes ranging from 2 mm to 24 h. After 2 mm of digestion (data not shown) major peakswere C, D and E, with the last corresponding to the molecular weight of undigested insulin.MS chromatograms (TIC) of 5 mm, lh, and 24 h digestion times are shown in Figure 5.9 asA, B, and C, respectively. Only peaks containing peptide masses that matched variousfragments of insulin B-chain, as determined by LC/MS or tandem MS, are labelled. After 5mm of digestion, the major fragments identified as peaks C and D corresponded to theprimary cleavage of insulin (peak E) between Leu15 and Tyr’6. The remaining peaks labelledin Figure 5.9 A correspond to peptides resulting from cleavage at secondary sites of fragmentsC and D. Upon increasing digestion times, the observation made in Figure 5.9 A was136confirmed in Figure 5.9 B and C which show the disappearance of peaks C, D and F withtime and the relative increase of smaller fragments. The proposed fragments of insulin areshown in Figure 5.10.27.120.413.6A17B:: —_______________________________________________.1:11.E28.4 B0 I 1721.3 o(I) IC II_142 1 A7.1a,>0.0 —‘1715213 C1511.4 1197.66 101138 2 A0.0 - .0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0Time (mm)Figure 5.9 Mass chromatograms (TIC) of proteolytic digests of insulin B-chain after 5minutes (A), one hour (B) and overnight incubations (C). All labelled peaks were identifiedby LCIMS or tandem MS.137The location of the cleavage sites giving rise to the observed peaks was determined bymatching the masses of the peptides with masses of various fragments of insulin. Whereambiguities existed, or where more than one insulin fragment had the same mass as theobserved peptide mass, comfirmation of the peptide sequence was resolved by sequencing bytandem MS as shown in Figure 5.11 for the analysis of peak 19 in Figure 5.9. The daughterions obtained were mainly type b ions, where the charge is retained on the amino terminus ofthe ion to form an acylium ion; or type-y ions where the charge is retained on the carboxyterminus (Siuzdak, 1994). Amino acid composition analyses of selected proteolytic fragmentswere in agreement with the sequences determined by tandem MS (data not shown).The results indicated that fragments C and D resulted from the initial cleavage between Leu15and Tyr’6, which happens to be the observed primary cleavage site for proteinase K andsubtilisins Carlsberg and Novo (Kraus et al., 1976). Some of the other cleavage sites (Figure5.10) have previously been identified for other subtilisin-like serine proteinases, includingsubtilisin (Morihara and Tsuzuki, 1969) and proteinase K (Kraus et a!., 1976), but thereappears to be unique differences between the digestion patterns (Figure 5.12). For example,no evidence was obtained in this study for cleavage after Tyr’6or Tyr26, which were commoncleavage sites for several other serine proteinases. Cleavage after His’°, Val’8 and Arg22 wasunusual when compared to the other digestion patterns, and may be unique target sites for the0. piceae senile proteinase.Figure5.10DigestionofinsulinBchain(peakE)bytheproteinaseisolatedfrom0.piceaestrain387N.AllpeptidesshownwereseparatedbyHPLCandtheirpositionintheinsulinB-chaindeterminedbyMSortandemMSortandemMS/aminoacidanalysis.AminoacidresiduesattheP1positionareunderlinedandinbold.Dottedandsolidarrowsrepresent thepartialorcompletebreakdownoffragments,respectively.Theminorpeaksobservedandidentifiedhavenotbeenshownonthisdiagram.Bintheaminoacidsequencesinthediagramrepresentscysteicacid(R=CH2SO3H).VpeakC151015FVNHLBGSHLVEALINSULINpeakES1015202530FVNQHLBGSHLVEALVLVBGERGFFYTPKANpeakD16202530VLVBGERGEEYTPpeak414FVNQpeakB51015HLBGHLVEALpeak151015HLVALVpeak61013HLVEpeak159HLBGSçji DopeakF16292529VLVBGERGFFYTPKpeak13162022VLVBGERpeak17162024VLVBGERGFpeak13162022YLVBGERpeakA2530FVTPKA‘Ipeak132529FYTPKpeak19162025VLVBGERGFFVpeak13162022VLVBGERpeak52630YTPIAVpeak22629YTPK16427737652750 38Cl) U) C ci:. > c’ a:.25 12 0Figure5.11TandemMSspectrafortheparentionmlz1091.Thedaughterionsgeneratedbythefragmentationprocessresultedindifferentclassesof fragmentions.Fragmentionsofthetype-b(toprow)andtype-y(bottomrow)aregeneratedifthechargeisretainedontheNterminusandCterminusof thepeptide,respectively.w10919288157165655083792231663793431091716100960070080090010001100100200300400m/z151015202530F-V-N-Q-H-L-B-G-S-H-L-V-E-A-L-Y-L-V-B-G-E-R-G-F-F-Y-T-P-K-A0.piceaeproteinase:***INITIALCLEAVAGE************COMPLETEDIGEST*****SUBTILISINBPN’********A.oryzae****chyrnotrypsin****************proteinaseK0Figure5.12SitesofcleavageofoxidisedinsulinBchainbyvariousserineproteinases.Initialcleavagebythe0.piceaeproteinaserepresentsthesitesdeterminedafter5minutedigests.Completedigestsindicatethesitescleavedafterextendeddigestsovernight.Binthediagramrepresentscysteicacid(RCH2SO3H).Splittingsitesfor theremainingproteinasesweretakenfromMoriharaandTsuzuki,1969; Kraus€1at,1976.1415.3.5 Substrate specificity on proteinsResults detailed in Chapters 3 (section 3.3.5) and 4 (sections 4.3.4.1 and 4.3.4.2) indicated thatthe purified proteinase was able to degrade azocoll and gelatin coating X-ray films. Caseinpresent in skim milk was also degraded when it was used as a overlay following non-denaturingelectrophoresis. Analysis by SDS-PAGE also showed the disappearance of proteins whencasein, BSA and plant proteins, such as globulins and edestin, were digested by the 0. piceaeproteinase. Most of the bands in the preparation of casein had been digested to peptides oramino acids that were not detected on the gels after only 5 mm incubations (Figure 5.13). Thehydrolysis of casein by proteinase K, at equal enzyme loadings, was more complete than that ofthe proteinase from 0. piceae. Similar differences in the extent of hydrolysis were observed forproteins extracted from wood (Figure 5.14). Both proteinases appeared to be most active on thehigher molecular weight components, with the gels showing an accumulation of lowermolecular weight products as digested proceeded. Interestingly, there was little change in the2 h and 5 h digestion patterns for both the 0. piceae proteinase and proteinase K, suggesting thedigest had reached completion or conditions were no longer suitable for prolonged enzymeactivity.94—-67—•43— i43cr-2O.1- •14.4——Figure 5.13 SDS-PAGE (8-25% PhastGel) showing degradation of casein by theproteinase purified from 0. piceae strain 387N and proteinase K. Lane 1, lowmolecular weight (MW) standards (Pharmacia) with MW expressed in kDa; lane 2,casein undigested control; digestion with 0. piceae proteinase for 5 mm (lane 4) and 10mm (lane 3); digestion with proteinase K for 5 mm (lane 6) and 10 mill (lane 5).5-94— .67— .443— . S —Sll1422 3_ 4 5 61MW_i .2_Z4.. .2_ 8Figure 5.14 SDS-PAGE (8-25% PhastGel) showing degradation of proteins extractedfrom poplar by the proteinase purified from 0. piceae strain 387N and proteinase K.Lanes 1 and 8, low molecular weight (MW) standards (Pharmacia) with MW expressedin kDa; crude protein extracts prior to digestion (lanes 2, 7); protein extracts afterdigestion by 0. piceae proteinase for 2 h (lane 3) and 5 h (lane 4); protefti extracts afterdigestion by proteinase K for 2 h (lane 5) and 5 h (lane 6).1435.4 DiscussionAs discussed in the preceding chapters, the subtilisin-like serine proteinase secreted by 0.piceae was the major protein secreted under conditions where protein is supplied as thenitrogen source, and is likely a key enzyme in the physiology of this fungus. Therefore thefactors which influence autolysis and substrate specificity of the proteinase may be critical tothe growth and survival of this fungus.The results for the 0. piceae proteinase suggested that under conditions of heating, altered pHor partial depletion of protein-bound ions by EDTA, the structure of the proteinase wasperturbed or relaxed, becoming a more suitable substrate for proteolysis. Many proteins canbe partially unfolded by relatively minor environmental changes, such as a modest elevationin temperature or a shift in pH (Geisow, 1991). In the case of proteinases, this unfolding maybe sufficient for it to become a substrate for autoproteolysis. Our results indicated thatautolysis proceeded by an intermolecular mechanism. This mechanism might be accentuatedat high temperatures where either populations of both active folded and inactive unfoldedproteins exist in concert, or all enzymes are active but partially unfolded.Autolysis has been reported for many of the subtilisin-like serine proteinases, often leading tomultiple protein bands in purified samples (Frosco et a!., 1992; Reichard et a!., 1990) ordiscrepancies in size of proteins determined by SDS-PAGE and gel filtration (Bajorath et al.,1988; Kolattukudy et a!., 1993). Calcium ions and ammonium sulphate contributedsignificantly to the stability of the functional enzyme folded conformation as indicated by the144higher melting temperatures when they were present. The use of ammonium sulphatesolutions for storage of proteinases to prevent autolysis has been reported previously(Kolattukudy et al., 1993), but it has not been the subject of extensive investigations, incontrast to calcium. The mechanism of stabilisation by calcium is thought to be through acontribution to the overall stability of the surface regions of the enzyme, and a reduction in theflexibility of the protein, both of which reduce its susceptibility to partial unfolding andsubsequent autolysis (Betzel et al., 1990). Recent work with subtilisin BPN’ (Braxton andWells, 1992) demonstrates that molecules do not need to unfold fully for autolysis, but thatthe rate-limiting process involves local unfolding.Calcium ions are required by some proteinases for folding of the polypeptide chain and hencefor activity (e.g. proteinase K and thermitase). It has been suggested that this may haveoriginated as a protective action by the cells against intracellular proteolysis (Betzel et a!.,1990). Intracellular calcium levels are too low to produce folded, active enzyme, whereasextracellular levels are high enough to activate secreted proteinases. Significantly, calcium ispresent in wood at levels greater than that typical in fimgal cells, with 550 ppm calciumdetected in lodgepole pine.The sites of autoproteolysis in subtilisin BPN’ were shown to be a region of high mobility asestimated from crystallographic B-factors (Braxton and Wells, 1992). Fontana and coworkers (1986) showed that cleavage sites did not occur within the segments of regularsecondary structure (such as helices), but instead at loops or turns characterised by the highestdegree of flexibility. The ioop containing the identified proteolysis site has also been145identified as being actively involved in the binding of Ca2 ions in many subtilases (Siezen eta!., 1991). Therefore, it seems likely that during the gel filtration experiments reported in thisstudy, protein-calcium interactions were disrupted, causing some unfolding and subsequentproteolysis.The position for autolytic cleavage of the proteinase isolated from 0. piceae was identified asthe N-side of Ser17° according to the numbering system of proteinase K. This cleavage siteoccurs in a variable region located in a ioop on the surface of the molecule connectingconserved elements of the f3-pleated sheet structure (Siezen et a!., 1991). Previous studies onsubtilisin BPN’ (Braxton and Wells, 1992), a subtilisin class I enzyme, identified anautoproteolysis site at the N-side of Thr164, in a similarly accessible exposed loop structurenear the active site. Since 0. piceae’ s proteinase has been characterised as a class II subtilase,these results suggested that the autoproteolysis sites in class I and II subtilases are similar.In most other subtilase class II enzymes, Ser’7° follows after a conserved alanine residue atposition 169, suggesting a P1 cleavage specificity for autoproteolysis. Interestingly, subtilisinBPN’ rapidly cleaves Ala48-Ser9 and Ser163-Thr’M(Braxton and Wells, 1992), indicating thatone autolysis site in subtilisin BPN’ also contains alanine in the P1 position. As discussed byBraxton and Wells (1992), these sites are not necessarily cleaved on the basis of primaryspecificity. Factors influencing the globular nature of proteins, such as folding, flexibility,and stereochemistry may be the critical determinants of stability, and cleavage specificityalone may not determine the suitability as a proteinase substrate.146Proteolysis is generally directed and limited to the cleavage of specific peptide bonds in thetarget protein. One of the keys to this selectivity is the accessibility of the scissile peptide bondto the processing proteinase. Compact protein domains are usually resistant to proteolysis, incontrast to more flexible surface loops and interdomain regions that can be exposed to the activesite of proteinases (North, 1982). In addition, many proteinases are specific for the amino acidside chain at the P1 position, using the nomenclature of Schechter and Berger, 1967 (section1.6). For example, trypsin cleaves after positively charged amino acids, namely lysine andarginine. Chymotrypsin cleaves after large hydrophobic residues such as tryptophan, tyrosineand phenylalanine, and elastase cleaves after alanine and serine amino acids with small sidechains. In some cases, the specificity may be determined by amino acids further removed fromthe scissile bond.The advantage of using simple model substrates to investigate specificity is that the observedkinetics are usually relevant to a single cleavage event. The use of small peptide substrates alsoeliminates problems associated with the structural conformation of proteins becauseoligopeptides usually do not have stable well-defined structure, and exist as random, extendedchains. However, several studies have indicated the role of secondary enzyme-substrateinteractions. These include for example, the effects of elongation of the peptide substrateresulting in a dramatic changes in binding and catalytic efficiency (Dunn, 1989). Therefore,in assaying the cleavage specificity, factors which influence either the binding or catalysiswill affect the results. For this reason the synthetic substrates have a major drawback in thatthey are generally soluble only in organic solvents which may have a significant influence oncatalysis. Homopolymers of single amino acids were soluble in different solvents, making147true comparisons impossible. The phenyl ester substrates had an additional drawback thatspontaneous hydrolysis occurred in the substrate. This was mentioned by Walsh and Wilcox,1970 who recommended simply subtracting the rate of the background reaction from the assayrate. The spontaneous hydrolysis was affected significantly by temperature. Therefore it wasbetter to perform the assay at 23°C rather than 25°C or higher. It was also necessary toperform the experiment with appropriate controls. An additional problem was thatreproducibility depended greatly on precise technique. There was always quite a bit ofvariation between replicate samples, and linear reaction rates were often not observed.Despite these problems, the results from this assay suggested that the 0. piceae proteinase hada specificity similar to that of proteinase K. Aromatic or apolar residues at the P1 site werepreferred. Leucine at the P1 site was a better ester substrate than phenylalanine at the P1 site.However, this was in contrast to the results using the model substrates Suc-(Ala)2-Pro Phe-p-nitroanilide and Suc-(Ala)2-Pro-Leu p-nitroanilide (section 4.3.4.2). When using insulin asthe substrate, leucine at the P1 site was also the first bond to be cleaved.The cleavage specificity of the proteinase on insulin indicated a broad specificity leading todoubts concerning the homogeneity of the proteinase preparation. Therefore, LC/MS wasused to conclusively demonstrate the purity of the enzyme used for digestion. Only oneprotein species was present upon injection of the proteinase preparation. The only otherspecies detected in the TIC were peptides that are probably due to autolytic degradation of theproteinase. Therefore the observed broad specificity could be attributed to a single proteinase.Aromatic residues were hydrolysed preferentially at the P1 site. Although alanine was themost likely residue at the P1 site for autoproteolysis, cleavage after alanine was not observed.148Interestingly, the proteinase was also able to cleave after polar, positively charged ornegatively charged residues. Although the first bond to be cleaved had Leu at the P1 site,cleavage was not observed after Leu6, Leu”, or Leu’7. The lack of cleavage may beinfluenced by the P4 residue, which was hydrophobic for several of the cleavage sitesobserved. Furthermore, cleavage after Phe24 and Phe25 was observed, but there was noevidence of cleavage after Phe’. This suggests the need for more than one amino acid forbinding and subsequent cleavage. In fact, work with proteinase K has shown that the smallestpeptide hydrolysable by this subtilisin class II enzyme is a tetrapeptide (Kraus et al., 1976).In general, the binding site of subtilases can be described as a surface channel or creviceaccommodating at least six amino acid residues (P4 to P2’) of a polypeptide substrate.Substrate binding is predominantly determined by the binding of the P1 and P4 residues intwo pockets on either side of the backbone strand 125 to 128. In many subtilisins, includingproteinase K, both pockets are large and hydrophobic, giving rise to a broad specificity with apreference for aromatic or large nonpolar P1 and P4 substrate residues (Siezen et al., 1991).In this study, ESI MS offered a rapid, simple and accurate means of obtaining molecularweight information, which was sensitive in the low picomole range. Analysis by tandem MSprovided sequence information allowing a direct correlation to sites of cleavage. Using thistechnique it was possible to follow the digestion of insulin over time and identify the primaryand secondary cleavage points in a relatively short time frame. With the development of moresensitive instruments and instruments capable of auto MS/MS, cleavage specificities of newproteinases could be determined with even greater rapidity. This method could be further149developed as a general strategy for characterising sites of cleavage using other enzymes andsubstrates.That 0. piceae ‘S proteinase had broad specificity was consistent with the enzyme’s functionwhen produced by the fungus during growth in wood where a variety of protein substrates areavailable. Substrate specificity may indicate the range of proteolytic events in which aproteinase might be able to participate, as discussed by North (1982). However, it cannotalways be related directly to activity on the physiological substrate. Therefore, it wasconsidered important to investigate activity on proteins extracted from wood. Degradation ofproteins extracted from the xylem tissue of poplar was observed after incubation with theenzyme for two hours. Disappearance of protein bands on SDS-PAGE gels indicated mostproteins in the extract were susceptible to the action of the proteinase. Similar results havebeen recently reported for xylem proteins from American Elm (Ulmus americana L.) aftercolonisation by the dutch elm disease fungus Ophiostoma ulmi (Eshita et al., 1994). Otherproteins hydrolysed by the proteinase include gelatin, collagen, albumin, edestin, globulinsand casein. These data support the conclusion that the proteinase has a broad specificity andis able to degrade a variety of substrates, including physiological substrates.In summary, this chapter addressed two key factors in the action of the isolated proteinase: itsautolysis and substrate specificity. Under conditions of heating, altered pH or partialdepletion of protein-bound ions, the structure of the proteinase was susceptible toautoproteolysis. Glycerol, calcium ions and animonium sulphate increased the thermalstability. The major autoproteolytic cleavage site was identified and by comparison with150other subtilases, located in an outer exposed ioop. The secondary structure was similar to thatof proteinase K, and aspects of the substrate specificity were also comparable to proteinase K.The proteinase had a broad specificity with a slight preference for hydrophobic or aromaticamino acids at the P1 site. It was able to hydrolyse esters and a number of different proteins,including proteins isolated from the xylem of poplar.151Chapter 6.Targeted inhibition of proteolytic enzymesproduced by sapstaining fungi6.1 IntroductionAs discussed in section 1.2, sapstain of lumber is caused mainly by Ophiostoma andCeratocystis species. For this reason, 0. piceae, a frequently isolated sapstaining fungus, hasbeen used for most of the work described in this thesis so far. However, black yeasts such asAureobasidium, and dark moulds such as Alternaria can also cause discoloration of sapwood(section 1.2). In addition, the green moulds, Trichoderma and Penicillium species, whichsporulate on the surface of lumber, also contribute to the decrease in wood value. These othergroups of sapstain and mould fungi would become increasingly important if members of theOphiostomatales were prevented from colonising lumber. The ecology of these fungi hasbeen covered to some extent (Seifert and Grylls, 1991; Seifert, 1993), but there is littleinformation on the physiology and the biochemical characteristics of these organisms,especially proteolytic enzyme production in solid wood. Therefore, before attempting toinhibit the proteinase enzymes as an anti-sapstain strategy, it was necessary to evaluate thenumber and type of proteinases produced by other staining and mould fungi. 1‘Some of these data have been published or presented at conferences:Breuil, C., Abraham, L. and Yagodnik, C. 1995. Staining fungi growing in softwood produce proteinases andaminopeptidases. Mat. u Org. 29:15-25;Abraham, L.D., Banerjee, S., Yagodnik, C. and Brown, D. 1994. Proteinase and animopeptidase activity in sap-staining fungi. Poster presented at the Fifth International Mycological Congress (August 14-21, 1994;Vancouver, B.C., Canada)152Previous studies have shown that a range of staining fungi produce proteinases andaminopeptidases when growing in protein-supplemented artificial media (Breuil and Huang,1994; Banerjee et al., 1995a). Carboxypeptidase activity has also been detected in 0. piceaegrowing in wood and liquid media (Banerjee et a!., 1 995b). Aminopeptidases andcarboxypeptidases catalyse the hydrolysis of amino acid residues from amino and carboxytermini of protein and peptide substrates respectively. They are found in organelles, in thecytoplasm and in secreted forms in the media (Taylor, 1993). As with proteinases, peptidasescan be classified according to susceptibility to inhibitors, location, and optimal pH foractivity.In these studies, proteinase production was correlated with primary growth, suggesting thatthe role of the secreted proteinases for sapstain fungi is to breakdown proteins to retrievenitrogen for growth and reproduction. The same may not be necessarily true for decay fungi(Micales, 1992; Venables and Watkinson, 1989). Whether decay and staining fungi produceproteinases as a means of nitrogen retrieval from the substrate or from older portions of themycelium, inhibition of proteinases in wood may slow fungal colonisation. Many of theextracellular proteinases produced by staining fungi are inhibited by the serine proteinaseinhibitor PMSF and are also affected by the chelating agent EDTA (Breuil and Huang, 1995).EDTA, amastatin, bestatin and 1,1 0-phenanthroline have been found to inhibitaminopeptidase activity (Banerjee et a!., 1995a).The in vifro properties and localisation of a proteinase (Hoffert et a!., 1995) may provide someindication of its function. However, with these data alone, it is difficult to draw more than153speculative conclusions about its in vivo role. In addition, alterations to proteinase levels duringphysiological responses or developmental changes allow us to predict functions. A more directapproach is dependent on the ability to manipulate proteinase activity in vivo. Many proteinaseinhibitors are now available but they are not always specific for individual proteolytic enzymes,and there is often the danger that processes other than those involving proteinase activity mightbe affected (North, 1982). Despite these limitations, these compounds were evaluated for theireffect on growth and proteinase production using the model organism 0. piceae.2 Thisevaluation is presented in the present chapter, as is the screening of proteinase production bydifferent staining fungi.2Some of the data concerning the testing of inhibitors were presented as a conference proceedings at theInternational Research Group of Wood Preservation conference in Helsingør, Denmark: Abraham, L.D.,Bradshaw, D.E., Byrne, T., Morris, P.1., and Breuil, C. 1995. Targeting fhngal proteinases to prevent sapstain onwood. IRG Doe No. IRG/WP/100971546.2 Materials and methods6.2.1 Fungal strains and culture conditionsThe fungal strains Ophiostomapiceae 387E, 387H, 3871, 387J and 387N, Ophiostoma ainoae701A, Ophiostoma piflferum 5511, Ophiostoma populinum 671A, Cephaloascus fragrans3071, Phialophora botulispora 707A, Alternaria tenuis 2G, Cladosporium cladosporioides273D, Aureobasidium pullulans var. melanogenus 132Q and Trichoderma harzianum E58were obtained from the Forintek Canada Corp. culture collection (Ste. Foy, Québec, Canada).Ophiostoma piceae 212735 was from the Agriculture Canada culture collection. All fungalstrains were maintained on malt extract agar slants at 4°C. The fungi were pre-grown in asynthetic medium containing ammonium nitrate as the nitrogen source and starch as carbonsource (section 2.2.2), before being inoculated on wood blocks (section 2.4.2) or in liquidsynthetic medium containing protein. Fungal biomass in wood was determined by ergosterol(section 2.3).6.2.2 Proteolytic activitiesProteinase activity was determined in infected wood powders or in culture supematant usingazocoll (section 2.5). Inhibitors were added during a 30 mm pre-incubation as describedpreviously (section 2.6). Aminopeptidase activity was determined using 50 mM L-Leu-pnitroanilide as substrate (Masuda et a!., 1975). The assays were carried out in Tris-Cl bufferpH 7.0 at room temperature for 24 hours on a platform shaker. After centrifugation the155supernatant was transferred to a 96-well plate and the absorbance was measured at 405 nm.One arbitrary unit of aminopeptidase activity was defined as the amount of enzyme whichproduced an increase absorbance reading of 0.01 O.D. units per mm.6.2.3 Testing inhibitors of the proteinase in artificial mediaThe synthetic medium (section 2.2) was supplemented with either NH4O3,2 g/l; or skimmilk, 10 g/l as the nitrogen source, and solidified with 20 g/l agar. Inhibitor solutions werefilter sterilised and added to the medium just prior to pouring the agar plates. Three replicateplates were used for each inhibitor at each concentration.The inoculum was prepared by growing 0. piceae in the synthetic medium containinginorganic nitrogen for 8 days. The biomass was harvested by centrifugation and resuspendedin phosphate buffered saline (PBS). Samples were homogenised (Omni Homogeniser model2000, Omni International) and centrifuged to obtain a washed pellet of cells. The cells weremade up to a final dry weight concentration of 1 mg/mi. Aliquots (10 jil) were used toinoculate the centre of each agar plate. The plates were incubated in the dark at roomtemperature for 5 days, and the diameter of each colony was measured. Percent inhibition ofgrowth was calculated relative to growth on media containing no inhibitor.1566.2.4 Testing inhibitors of the proteinase in woodThe ASTM D4445-84 test was used in these experiments. This is a standard laboratory testfor screening chemicals to control sapstain and mould on unseasoned lumber. The details ofthe testing procedure are specified, including a visual evaluation of stain of the wood samplesto provide a quick final assessment. Lodgepole pine sapwood blocks (5 cm x 2 cm x 0.5 cm)were labelled and sterilised as before (section 2.4.1). Six to eight replicate blocks weredipped in inhibitor solutions and placed on glass rods in Petri dishes to equilibrate for 24 h. Awashed 0. piceae inoculum prepared at 0.4 mg/ml was used to inoculate the wood blocks. Analiquot (250 p.1) was applied down the centre of the wood block. Plates were incubated asbefore (section 2.4.2). After 14 days incubation, the degree of pigmentation and surfacegrowthlsporulation was visually evaluated relative to untreated wood, and enumerated aspercent inhibition.1576.3 Results6.3.1 Proteolytic activity on solid mediaA number of staining fungi were screened for proteinase production on artificial mediacontaining skim milk as the nitrogen source. Zones of clearing, indicative of extracellularproteinase activity, were observed for all staining and mould fungi listed in section 6.2.1.6.3.2 Inhibition of proteolytic activity in culture filtratesSeveral of the strains tested on solid media were selected for liquid culture studies. As describedby Breuil and Huang (1994), all the selected strains secreted proteinases into culture filtrateswhen grown in liquid medium supplemented with different protein sources such as soy milk,skim milk, or BSA. After incubation with a range of proteinase inhibitors, activity in the culturefiltrates was reduced in all cases by PMSF (Table 6.1), although the inactivation was less forsamples from 0. populinum and C. cladosporioides. Enzyme activities in most samples werealso affected by EDTA, with the exception of the samples of A. tenuis and T harzianum. Theproteolytic activity of C. cladosporioides was strongly inactivated by 1,10 phenanthroline andEDTA. E-64, a cysteine proteinase inhibitor, did not affect any of the samples, while pepstatin,an aspartic proteinase inhibitor, only affected the sample from 0. populinum. The strong actionof PMSF suggested that most of the samples contained serine proteinases. However, sinceseveral inhibitors inactivated the samples, at least to some extent, it was necessary to determinewhether more than one proteinase was present in each sample.1586.3.3 Protein profilesElectrophoretic analysis of culture filtrates from eight of the sapstaining strains was conductedafter 3 to 5 days growth. X-ray film overlays following electrophoresis (section 2.7) showedthat all samples contained more than one proteolytic band after IEF between pH 3 to 9, with theexceptions of 0. populinum and A. tenuis which apparently produced only one proteinase withan alkaline p1 (p1>9). All four strains of 0. piceae produced a proteinase with p1 5.6, and minorproteinases with p1 5.2 and 8. Among the other proteolytic bands detected after IEF, thesamples from 0. ainoae showed two bands with acidic p1 values, and that from T harzianumshowed at least five active bands. After separation by native PAGE, multiple proteolytic bandswere again visible for most samples, but the number of bands was generally less, probably dueto the fact that proteins with higher p1 values would migrate to the cathode instead of the anodeduring separation.6.3.4 Fungal growth and proteolytic activity in woodAll staining fungi used in this work were mesophilic and grew actively on lodgepole pinesapwood at 23°C. After two weeks of fungal growth, all the wood blocks showed some visualdiscoloration. Fungal biomass was determined by subtracting the ergosterol content in thenon-inoculated wood from that in infected wood blocks. For most fungal strains, theergosterol content increased during the first week and reached a constant value after seven ornine days, indicating that the fungal growth had reached stationary phase (Figure 6.1 andTable 6.2).Table6.1Effects ofproteinase-inhibitorsonthehydrolysisofazocollbywoodpowdersandculturefiltrates’thathavebeeninoculatedwithdifferentstainingfungiFungalsamples%inhibition21,10-Phenanthroline3PMSF4EDTA5Pepstatin6E647woodfiltratewoodfiltratewoodfiltratewoodfiltratewoodfiltrate0.piceae387E+++++++++++-+-387J++++++++---387N-+++++++++---212735+++++++++---0.ainoae701A+++++++++++-0.populinum671AND8+ND+ND+ND+ND-A.tenuis2G-++++++----C.cladosporioides273DND+++ND+ND+++NDND-7’.harzianumE58+-++++++++-++-tbasedonBreuilandHuang,19942lnhibitionefficiencywascalculatedasfollows:+++inhibitionefficiencybetween75-100%;++inhibitionefficiencybetween75-50%; +inhibitionefficiencybetween50-25%;-inhibitionefficiencylessthan25%.i,10-phenanthroline(10mMwith1%methanol),4PMSF(2mMwith1%DMSO),5EDTA(10mM),6pepstatin(1jiMwith1%DMSO),7E-64(1OjiM)8not determined1.5001.0D>0.251600200 00.0 2 4 6 8 10 12 14 16Days of incubationFigure 6.1 Proteinase and aminopeptidase activities and ergosterolcontent in lodgepole pine sapwood inoculated with 0. piceae 212375.Bars indicate standard deviation (n=4).All strains produced proteolytic enzymes during growth on wood. Figure 6.1 shows theresults for 0. piceae 212375. Proteolytic activity, expressed per g dry wood, increased withtime and reached its maximum after three to seven days, shortly before or at the beginning ofthe stationary phase as shown by the ergosterol content. During the stationary phase a declinein the activity was generally observed. A similar trend was observed for all the fI.ingi grownin wood, although proteolytic activity peaked later at seven to nine days (Table 6.2). Allmeasurements were repeated in a second set of experiment. Due to the heterogeneity of woodthe values were slightly different, but the trend was the same in both sets of data. Theaminopeptidase activity increased with time and reached a maximum at days nine or twelve161(Figure 6.1 and Table 6.2). Contrary to the proteinase, the aminopeptidase activity oftenremained relatively constant or increased during the stationary growth phase.6.3.5 Inhibition of proteinases produced in woodThe pattern of inhibition for proteinase activities in wood was similar to that found in culturefiltrates. Cysteine and the aspartic proteinase inhibitors had no effect on the activity (Table6.1), whereas PMSF strongly inhibited the activity of all the different samples, suggesting thatthe majority of the proteinases secreted by the fungi were of the serine type. Since most werealso affected by the chelating agent, EDTA, but not by the metalloproteinase inhibitor, 1,10-phenanthroline, they were probably stabilised by calcium. The similarity of the inhibitionpatterns suggested that proteinases secreted during fungal growth on protein-supplementedliquid media were similar to those secreted during growth on wood in which protein was themajor nitrogen source. The detection of serine proteinases produced by all sapstaining fungiin both cultivation systems suggested that this group of proteinases should be targeted for theapplication of selective inhibitors on wood. All potentially inhibitory compounds werescreened against 0. piceae since this model system was better understood. Furthermore, theconcentrations of many of the inhibitors could be predicted by examining the data forinhibition of the purified subtilisin-like serine proteinase in vitro (section 4.3.4.4).Table6.2Proteolyticactivityandergosterol contentsinwoodinfectedwithstainingfungi.ProteinaseandaminopeptidaseactivitieswereassayedatpH8.0andpH7.0,respectivelyproteinase’aminopeptidase1 Ergosterol20.065±0.0280.130±0.0172.21.355±0.0870.250±0.02831.01.662±0.1780.370±0.03248.51.388±0.1070.260±0.02549.01.210±0.1000.320±0.02751.5proteinaseaminopeptidaseErgosterolproteinaseaminopeptidaseErgosterol0.057±0.0150.103±0.005ND30.285±0.0300.135±0.0208.50.962±0.0020.098±0.0 1830.00.210±0.1020.280±0.07017.20.883±0.1100.228±0.02534.90.278±0.0850.123±0.02715.23.175±00.212±0.01842.70.632±0.1280.427±0.015ND0.533±0.0430.172±0.028ND0.783±0.0700.233±0.032ND2.387±0.0020.342±0.03246.90.528±0.0850.707±0.02243.40.540±0.1150.143±0.02837.80.773±0.1330.270±0.018ND1.847±0.0030.255±0.02530.40.685±0.2 100.953±0.07551.90.690±0.0700.220±0.015ND0.483±0.0970.222±0.02794.01.742±0.0020.300±0.027NDFungalActivityandDaysafter inoculationstrainsBiomass3791215A.tenuis2G0.960±0.1270.087±0.01832.90.piceae387E0.piceae2123750.piceae387N0.ainoae701A7’.harzianumE581.100±0.1700.198±0.01883.5proteinaseaminopeptidaseErgosterolproteinaseaminopeptidaseErgosterolproteinaseaminopeptidaseErgosterol1.040±0.0880.330±0.032870.875±0.1270.243±0.025900.720±0.0900.288±0.027ND5-’ 10.022±0.0150.082±0.003trace1U/gdrywood(mean ±S.D.),2ergosterolrepresentsbiomassastgergosterolper gdrywood,3ND: Notdetermined1636.3.6 Effect of inhibitors on growth of 0. piceae in artificial mediaAn artificial medium was used for growth of 0. piceae in which nitrogen was available inorganic form as protein or inorganic form as NH4O3. Clearing zones were produced aroundfungal colonies in media supplemented with skim milk, indicating the production ofextracellular proteinases. Slightly slower growth rates were observed on media supplementedwith inorganic nitrogen (Figure 6.2). Therefore, when inhibitors were added to mediacontaining either inorganic or organic nitrogen, it was possible to measure the effect ofinhibitors on colony diameters and clearing zones due to protein hydrolysis.Figure 6.2 0. piceae strain 387N colonies (C) on mediasupplemented with skim milk (A) and NH4O3 (B). Clearing zones(arrow) were produced on organic medium due to the activity ofsecreted proteinase(s).164The four general groups of inhibitors tested were heavy metals, chelators, detergents andserine proteinase inhibitors. PQ-8, a currently used antisapstain formulation, was alsoincluded as a commercial control. Results for PQ-8 are presented separately because itcontains more than one possible active ingredient, and the results are therefore given for thediluted formulation. Concentrations for all inhibitors were either based on recommendationsin the literature or according to the results obtained using the purified enzyme (section 4.3.4.4and 4.3.4.5).The heavy metal salts ZnSO4•7H20,Pb(N03)2and CuCl2HO, inhibited growth on mediacontaining protein or inorganic nitrogen, suggesting that they were toxic to fungal growthrather than inhibitory to the proteinase (Table 6.3). EDTA, tartaric acid and SDS similarlyprevented growth on both media, while other chelators, TPEN and EGTA, were not aseffective. At the highest concentration EGTA caused a maximum inhibition of 15% onorganic medium. TPEN gave inconsistent results but with an inhibition of less than 1% at thehighest concentration on organic medium. Specific serine proteinase inhibitors, AEBSF andantipain, did not disrupt growth on either medium. In contrast, another serine proteinaseinhibitor, chymostatin, prevented fungal growth on the organic nitrogen medium butpermitted growth on inorganic nitrogen medium, suggesting specific inhibition of theproteolytic system. PQ-8 resulted in 82% and 11% inhibition on the organic and inorganicmedia, respectively, when the concentrate (5.4% Cu-8, 55-65% DDBSA) was used at 1000times dilution. This was the same dilution at which the purified proteinase was inhibited(section 4.3.4.5). Since growth was dramatically affected on organic medium, but only165inhibited to a lesser extent on inorganic medium, it suggested that at these low concentrations,PQ-8 was acting by specific inhibition of the proteolytic system.Table 6.3 Effect of heavy metals, chelators, serine proteinase inhibitors, anddetergents on growth of 0. piceae strain 387N on media supplemented withorganic and inorganic nitrogen.Type Compound Concentration % inhibition(mM) organic inorganicHeavy metals CuCl2HO 10 100.0 94.427.2 76.40.1 0.0 11.0Pb(N03)2 100 100.0 100.010 61.5 56.41 0.0 4.7ZnSO47H2O 100 100.0 92.010 87.0 56.416.9 13.70.1 0.0 0.0Chelators EDTA 10 87.8 89.11 49.6 45.20.1 5.9 11.0EGTA 10 15.5 11.01 0.0 1.90.1 0.0 8.3TPEN 0.13 0.6 11.00.013 87.8 0.00.0013 8.2 0.0tartaric acid 100 92.6 94.410 51.2 45.21 5.9 94.4Proteinase AEBSF 2.1 13.3 11.0inhibitors 0.21 0.0 1.9Chymostatin 0.01 60.0 13.70.00 1 0.0 8.3Antipain 0.007 0.0 4.7Detergent SDS 10 81.8 100.01 3.7 0.01666.3.7 Effect of inhibitors on growth of0. piceae on lodgepole pineCompounds applied to lodgepole pine samples inoculated with 0. piceae were visuallyevaluated for their effect on fungal growth, sporulation, and pigmentation. In general,chelators were not effective inhibitors of 0. piceae growth in wood, although EDTA did havesome effect on pigmentation at higher concentrations (Table 6.4). The detergent, SDS, wasmore promising because it affected growth and pigmentation at the higher concentrations.Similar to findings in artificial media, PQ-8 was an effective inhibitor of growth. However,specific serine proteinase inhibitors, AEBSF and antipain, did not prevent growth of 0. piceaeon wood. Chymostatin was not tested on wood due to its prohibitive cost and its instability.Table 6.4 Effect of chelators, serine proteinase inhibitors and detergents ongrowth and pigmentation of 0. piceae strain 387N grown on lodgepole pine.Type Compound Concentration % inhibition(mM) growth pigmentationChelators EDTA 134 3.48 75.013.4 1.7 16.61 0.0 2.7EGTA 134 0.0 0.013.4 1.1 0.01 0.5 0.0TPEN 1 0.0 0.00.1 40.9 10.70.001 28.2 29.7Protemase inhibitors AEBSF 0.1 23.1 18.90.01 2.6 0.0Antipain 0.01 5.0 0.0Detergent SDS 100 100.0 94.610 25.5 21.51 5.0 10.71676.4 DiscussionThe species obtained from the Forintek culture collection have been frequently isolated onlumber in both eastern and western Canada (Seifert and Grylls, 1991). All the selected strainssecreted some extracellular proteinases in solid agar media, liquid media, and wood. In liquidmedia supplemented with protein as a nitrogen source, proteinase production by staining fungihas been associated with fungal growth (Breuil and Huang, 1994). In this study, the level ofproteolytic activity varied among the different organisms, but the maximum proteinaseactivity was recorded when the fungi were actively growing, again suggesting that theenzymes played a major role in nutrition. The decline in proteinase activity during stationaryphase (Figure 6.1) suggested that conditions in wood were altered during the growth of thefungus such that the proteinase was inactivated, or unstable and degraded. The release of fattyacids during growth on lodgepole pine (Gao et a!., 1994) would result in a decrease in pHwhich may then cause instability or inactivation of the proteinase (section 4.3.4.3).Degradation of the proteinase may be part of the the recycling strategy of an organismgrowing in a nitrogen limited environment. Reuse of the nitrogen from extracellular enzymeswould also be mediated by proteinases. Therefore, it is possible that more than oneproteolytic enzyme is present at different stages of growth and that some of them may havedifferent roles.The aminopeptidase activity was measured throughout the different phases of fungal growth,with a maximum activity during stationary phase. It is likely that these enzymes are alsoinvolved in the terminal stages of peptide digestion. The first step in protein hydrolysis is168carried out by proteinases, converting proteins into peptide residues that may be furtherdegraded by peptidases into amino acids directly available for uptake and metabolism.Ergosterol could be used as a convenient measure of fungal biomass in wood. However, inliquid medium, the level of ergosterol per fungal biomass determined by dry weight, is notconstant during iIingal growth. For 0. piceae the ergosterol content in the mycelium at thestationary phase was twice the amount in the mid-exponential growth phase (Gao et al.,1993). Consequently, it seems more accurate to express the proteinase activity per unit drywood, and to correlate the activity to the growth phase of the fungus as indicated byergosterol. Our results thus showed that the proteinases were synthesised while the fungiwere actively growing. The highest proteinase activity in wood was recorded for the greenmould, T. harzianum, as had been previously found for liquid media when the activity wasexpressed as U/mg biomass (Breuil and Huang, 1994). Trichoderma spp. have been studiedextensively as producers of cellulases rather than proteinases. The existence of multipleenzymes of the same type in the cellulase complex of Trichoderma has been the subject ofnumerous debates (Nakayama et al., 1976; Labudova and Farkas, 1983; Luderer et at., 1991).Limited proteolysis of a common enzyme precursor has been suggested to explain themultiplicity of enzymes, and to explain the number of cellobiohydrolases with identical N-terminal sequences (Chen et a!., 1993).The fungal proteinases produced were classified by determining their susceptibility to alimited number of inhibitors. The inhibition pattern for proteinases secreted during growth onwood was almost identical to the pattern obtained for proteinases secreted in liquid media169(Breuil and Huang, 1994). All samples were strongly inhibited by the irreversible serineproteinase inhibitor PMSF. Electrophoretic analysis showed that many of the strainsproduced several proteinases. At least one other extracellular proteinase with an alkaline p1 wasobserved during growth of 0. piceae in wood and in media (section 3.3.5). Intracellularproteinases are almost certain to exist to fulfil other functions (section 1.7). Aminopeptidaseswere also present, associated with the cell pellet rather than as secreted enzymes (Banerjee et al.,1995a). Therefore, the selective inhibition of a single proteinase might not inhibit growthcompletely. However, if inhibition of proteinases causes a significant reduction in theavailability of nitrogen, the fungus may enter an exploratory-type of growth phase where itcommits energy to further exploration rather than consolidation and exploitation of the existingmycelial domain (Rayner and Boddy, 1988). The effect of an exploratory-type growth on woodmay be a reduction in mycelial biomass per unit surface area of wood which may result in littleor no discoloration. Therefore, since all strains produced at least one serine proteinases, it wasconsidered that targeted inhibition of this group may be a possible route for developingantisapstain compounds.The detailed inhibition characteristics of the serine proteinase secreted by 0. piceae showedthat it was sensitive to a range of serine proteinase inhibitors in vitro (section 4.3.4.4).However, many of the specific serine proteinase inhibitors were too expensive, toxic orunstable to be used in tests in artificial media or on wood. Inhibitors from Streptomyces spp.such as antipain and chymostatin have been cited as being of particular value in these types ofexperiments, since side effects have not been previously reported (North, 1982). Chymostatin, aspecific reversible inhibitor of serine proteinases, was effective in preventing growth on170protein-supplemented medium. Unfortunately, this compound was both too unstable andexpensive to test on wood samples.Chelating agents were also effective inhibitors when used in crude filtrates, wood powders, orapplied to the purified proteinase. The effect of EDTA could most likely be attributed to itschelation of calcium ions, which are important for maintaining the active folded form of theproteinase and preventing subsequent autolysis. However, the chelators did not consistentlyperform well in artificial media or when they were applied to wood. This apparentdiscrepancy in results may be attributed to the presence of many cations in media and wood(Fengel and Wegener, 1989) which could saturate the chelating capacity of the chelator at theapplied concentrations. It may also reflect the enhanced stability of proteinases when theywere secreted in a complex microenvironment, as compared to being tested in purified form.In contrast to the relative ease with which enzymes can be tested in purified form, practicaldifficulties arise when testing enzyme inhibitors in wood or culture. On wood it can bedifficult to assess the effectiveness of an inhibitor since many factors are unknown at theoutset of the experiment. For example, the distribution may not be uniform if the compoundis lipophilic or binds preferentially to certain wood components. The stability of the inhibitorsolution in wood and its bioavailability have generally not been determined.The ideal antisapstain product (section 1.1) would be affordable, effective and target fungicausing discoloration with no adverse effects on man or the environment. In Canada thewood treating industry has made an effort to use formulations containing environmentally171friendly chemicals. However, all existing formulations suffer from several of the followingproblems: high fish toxicity, skin reactions among workers, difficulties in handling in coldweather, corrosion of equipment, unwanted discoloration of the wood, and high costs. Thusnone of the currently used antisapstain chemicals fulfil all the demands of the ideal product asdefined earlier. PQ-8, a formulation containing 5.4% Cu-8 and 55-65% DDBSA, is one of thefew antisapstain chemicals that is approved for use on wood in contact with food. Therefore itwas included in the experiments as a currently used commercial control.Interestingly, PQ-8 appeared to function in these tests as a specific proteinase inhibitor. Themechanism of action of PQ-8 has not been definitively established (Mike Freeman, ISKBiosciences, personal communication), but the formulation does contain both a detergent anda chelating agent. Other detergents and chelating agents were identified as inhibitory to theproteinase (section 4.3.4.4) and to fungal growth (section 6.3.6). While these results are stillpreliminary, they point to the opportunities that exist for enhancing the performance ofantisapstain chemicals through knowledge of their interaction with wood, and with thestaining fungi which grow in wood.In summary, all staining fungi tested produced serine proteinases and many strains secretedmultiple proteinases. Although the secreted fungal proteolytic enzymes could be effectivelyinhibited in vitro, definitive testing on wood and commercial application were constrained bythe current lack of specific, cheap, stable, non-toxic proteinase inhibitors. Nevertheless, usingthe approaches described in this chapter, and related studies, it is possible to achieve a better172understanding of the physiology of sapstaining uiingi in order that control strategies can beformulated and developed at a later stage.173Chapter 7.Overview and future workThis project was directed at a significant economic problem for the lumber industry: sapstain.The discoloration of sapwood is caused by pigment-producing fungi which do not destroy thestructural integrity of lumber, but instead cause aesthetic damage to wood. The lack ofinformation available on the physiology of sapstaining fungi was the motivation for a researchprogramme. The overall approach was as follows: by examining the physiology andbiochemistry of staining fungi, specific enzyme systems important in the nutrition orpigmentation pathway could be identified. Once these systems were understood, they might bemanipulated or inactivated to disrupt growth or pigmentation. Targeting biocides that haveactivity only against identified systems could lead to highly specific protectants which have littleor no general toxicity. Mechanism-based inhibitors targeted at cellulases produced by decayfungi have been designed in accordance with this strategy (Namchuk et al., 1992).The project described in this thesis, a component of the research programme described above,was designed to address the issue of nitrogen utilisation by staining fungi. More specifically, itwas focused on examining the proteolytic enzymes required by staining fungi to retrievenitrogen from proteins in wood.Proteinases were produced by a range of staining fungi during growth in protein-supplementedliquid cultures. Detailed analyses of growth and proteolytic enzyme production by 0. piceaeindicated that proteinases were not produced during active growth when inorganic nitrogen was174supplied as the sole nitrogen source. These results suggested that the proteolytic enzymesplayed a role in primary retrieval of nitrogen rather than in recycling nitrogen during autolysis offungal mycelium. When the fungi were grown in wood, proteinases were detected during activegrowth, suggesting that protein was the major nitrogen source available in wood. This work alsoconfinned the low amounts of total nitrogen available in wood and the presence of proteins inpine sapwood.The proteolytic enzyme systems of staining fungi were further characterised in artificial media,where fungal and enzyme manipulation was considerably easier. Inhibition of proteinases byPMSF indicated that serine proteinases were produced by all staining fungi, although manyproduced several proteinases. In the model system examined, 0. piceae produced one majorproteinase in wood and in protein-supplemented cultures. This enzyme was classified as asubtilisin-like class II serine proteinase. It was similar in secondary and tertiary structure toother subtilisin class II enzymes such as proteinase K. However, in terms of its physiologicalrole, it appeared to be well suited to function in wood. In particular, the proteinase had broadspecificity, in that it was able to cleave a wide range of peptide bonds. This would beadvantageous if different protein substrates were available in wood at various times of the year.The inhibition pattern of the purified enzyme was studied in detail to determine the sensitivity toheavy metals, detergents, chelators, oxidising agents, reducing agents and specific proteinaseinhibitors. Chelation of calcium ions appeared to promote autoproteolysis or autolysis.Autolysis was the major factor affecting the stability of the enzyme. Factors such as pH and175temperature also appeared to affect the degree of folding and hence the sensitivity toautoproteolytic degradation.This study of this new proteinase has analysed some factors implicated in its stability andautolysis. Importantly, the site of autoproteolytic cleavage for subtilisin class II enzymes wasidentified. By changing the amino acid composition at this site, it may be possible to slow downor prevent autolysis in this group ofproteinases.Since serine proteinase production was a general feature of staining fungi growing in wood,inhibitory compounds were tested in preliminary studies in media and wood. Unfortunately,many of the commercially available proteinase inhibitors affected other physiological processes,rather than specifically inhibiting the proteinase. Therefore, when these compounds were testedon wood, conclusions were limited. Future definitive testing would require specific, stable andnon-toxic proteinase inhibitors. Possible areas where such compounds may be identified includeexisting pesticides, in nature, in the medical field, or in novel biotechnology approaches.An alternative approach may be to manipulate conditions to cause instability and autolysis of theproteinase. Compounds which affect the pH of wood may contribute to factors causingautolysis. Chelating agents were also tested for their effect on the enzyme activity in wood, butthe initial results were not encouraging. It may be necessary to use much higher concentrationssince wood contains significant quantities of ions. However, the results did suggest thatchelation affected physiological processes other than proteolytic activity. Therefore, this wouldnot be a selective or targeted approach.176The other possible protective action is the modification of proteins in wood such that theybecome unavailable for digestion by the proteinase. Research on chemical modification ofwood to improve its durability has intensified in the past decade due to increased environmentalconcern over the use of tropical hardwoods and broad spectrum pesticides (Beckers eta!., 1994).Acetylation has received more attention than any other modification process because of itsrelatively low cost, and the relatively low toxicity of the chemical materials and the finalmodified wood product (Wakeling eta!., 1992). The improved resistance of acetylated wood todecay fungi is well documented, compared to the poor performance against stain and mouldfungi (Wakeling et a!., 1992). Treatment of wood with the aim of specifically targeting proteinshas not yet been addressed. In the future this approach may be extended to the controlled releaseof a toxic chemical from its bonded site. Instead of permanent bonding, chemical could bereacted through labile bonds that would hydrolyse under conditions that occur during biologicalattack such as heat, moisture or changes in pH (Rowell, 1991).Existing organophosphorus pesticides developed against acetyicholine esterase (Fest andSchmidt, 1982) could be screened against isolated proteinases in vitro, and against fungalgrowth in media and in wood tests. The proteinase purified from 0. piceae was shown to haveesterase activity and was effectively inhibited by one of the earliest organophosphoruscompounds DFP synthesised as a nerve poison during the Second World War (Eto, 1974).About 140 phosphorus compounds are or were used as practical pesticides in the world, and lesstoxic organophosphates have been developed (Fest and Schmidt, 1982).Naturally occurring proteinase inhibitors may offer compounds with the desired specificityand little general toxicity. Proteinase inhibitors have been found in animals, plants and177microbes. Most of the well characterised inhibitors act as pseudo-substrates by combiningirreversibly with the enzymes without cleaving the peptide bond at the reactive site (Neurath,1984). In many cases it is not clear why proteinase inhibitors having substrate-like structuresare not degraded by the proteinase. In Streptomyces subtilisin inhibitor, structural featuressuch as a salt bridge are necessary for inhibitory action (Kojima et a!., 1994). The design ofinhibitors has benefited from the study of natural products, yet our knowledge of subtilisininhibitors is relatively poor compared with that of trypsin inhibitors (Terada eta!., 1994).Many proteases are involved in various human diseases, and these enzymes are targets for thedevelopment of inhibitors as therapeutic agents. Inhibition of HIV aspartic proteinase is anattractive target for therapy of AIDS which has met with some difficulties (Condra et a!., 1995)but is still considered a valid approach (Richman, 1995). Within the serine proteases, potentialtarget enzymes include neutrophil elastase, thrombin, dipeptidyl peptidase IV and grazymes. Inthe future, specific proteinase inhibitors will be tested clinically for the treatment of humandisease (Powers et al., 1993), and the requirements for these inhibitors in terms of stability, lowtoxicity and specificity may render them suitable candidates for application on wood.Another interesting and novel approach to controlling insects and flingal pathogens of woodyplants has been recently reported (Klopfenstein et a!., 1994). It involves the cloning of aproteinase inhibitor into poplar seedlings. Enzyme-linked immunosorbent assaysdemonstrated leaf expression of the proteinase inhibitor. Although using this approach toprotect lumber may seem impractical at the present time, molecular approaches of this naturewould require understanding the fungal enzymes involved in wood colonisation.178Although this thesis has addressed some aspects of the physiology of 0. piceae, many questionsremain unanswered:• The secretion of other proteinases by 0. piceae during other growth phases or morphologicalchanges has not been addressed and merits further investigation. Similarly, the occurrence,localisation and identification of intracellular proteinases would be essential to a completeunderstanding of the physiology of this fungus.• Interrelationships between the carbon and nitrogen cycle were not investigated. However,proteins were apparently used as a carbon and nitrogen source when starch and protein weresupplied in culture media. This may be an interesting issue in the regulation ofproteinases.• The assumption was made that proteins from wood constituted the major nitrogen sourceavailable to the sapstaining fungi. However, in wood colonisation, succession patterns havebeen shown, whereby the fungi colonise lumber after bacteria and actinomycetes. Therefore, thebiomass of these prior wood inhabitants may also serve as substrates.• A combined genetic and biochemical approach may be used to obtain more preciseinformation about the specific inactivation of a single proteinase. Several regions of theisolated subtilisin-like proteinase have been sequenced and shown to possess a high degree ofhomology (Siezen et a?., 1991). Therefore DNA probes could be created to screen cDNA orgenomic libraries of sapstaining fungi for the presence of subtilisin-like serine proteinase genes.Deletion mutants of 0. piceae defective in the production or activity of the subtilisin-likeserine proteinase could be generated. These mutants could be tested for growth in mediacontaining protein as the sole nitrogen source, and for their ability to colonise and discolourwood. This would illustrate unequivocally the key role of this enzyme. A similar approach has179been reported for examining the virulence ofAspergillusfumigatus mutants deficient in a serineproteinase (Kolattukudy eta!., 1993).• Regulation experiments could also be conducted using a genetic approach. For example,overexpression studies could be conducted on the proteinase in 0. piceae by the introduction ofa more efficient promoter (Kim et a!., 1995). Wild type and recombinant strains could then becompared for their growth characteristics on wood.In conclusion, this work has contributed to an understanding of the nutritional factors involvedin colonisation of wood by sapstaining fungi, particularly in terms of the nitrogen cycle. All thestaining fungi tested secreted proteinases and were able to utilise protein as a source of nitrogen.The study of the extracellular subtilisin-like serine proteinase from 0. piceae has providedinformation that may result in practical applications in the effort to improve existing methods tocontrol staining fungi. In addition, understanding differences in nitrogen utilisation may help toexplain succession patterns on wood. This may have important implications for the control ofother fungi which cause extensive damage to wood and wood products. Finally, this work mayprovide some additional data for the research effort to control other diseases caused byophiostomatoid fungi. Future consideration of physiological, genetic and ecological aspects ofsapstain fungi will further increase the knowledge required for more effective control.180ReferencesAltschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J. 1990. Basic localalignment search tool. J Mo!. 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