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Biodegradation of lipids by wood sapstaining OPHIOSTOMA SPP Gao, Yong 1996

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BIODEGRADATION OF LIPIDS B Y WOOD SAPSTAINING OPHIOSTOMA SPP. by Y O N G G A O B.Sc. 1985, M.Sc. 1988, Nanjing Agricultural University A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Wood Science) We accept this thesis as conforming the required standard THE UNIVERSITY OF BRITISH C O L U M B I A July, 1996 © Y o n g Gao, 1996 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholariy purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) 11 Abstract Canada is the largest exporter of softwood lumber in the world. Sapstain caused by fungi is a costly problem for the Canadian lumber industry. To maintain and increase future sales and exports of lumber requires not only effective, but also environmentally acceptable anti-sapstain methods. A thorough understanding of the physiological and biochemical features of sapstaining fungi should facilitate the development of new methods of protection. This work was carried out to determine the available lipid nutrients in wood and to understand the biodegradation mechanisms of lipids by sapstaining Ophiostoma species. Lodgepole pine (Pinus contorta Dougl. var. latifolia Engelm.) constitutes the greatest volume of wood harvested in British Columbia. As with all wood species, the freshly sawn sapwood is susceptible to sapstain. The total extractives content in the sapwood at the breast height of a 35-year-old lodgepole pine tree was 1.9-2.2% of oven dry weight, which included 1.1-1.3% triglycerides (TG), 0.02-0.03% free fatty acids (FA), 0.3-0.4% free resin acids (RA), and 0.15-0.21% steryl esters and waxes (SEAV). A considerably higher content of extractives was present in heartwood, i.e., 9.7-12.4%, including 0.6-0.7% TGs, 0.8-1.1% free FAs, 4.0-5.6% free RAs, and 0.25-0.33% SE/Ws. The wood TGs were composed mainly of oleic (18:1), linoleic (18:2), linolenic (18:3) and palmitic (16:0) acids. The dominant R A was palustric acid, followed by abietic, neoabietic, dehydroabietic, isopimaric, pimaric, and sandaracopimaric acids. The ability of three sapstaining fungal species, Ophiostoma piceae 387N, O. ainoae 701 A, and O. piliferum 55H to degrade and utilize the major lipids in the sapwood of lodgepole pine and trembling aspen (Populus tremuloides Michx.) was investigated. The fungal growth rate in I l l wood was monitored by quantifying ergosterol extracted from colonized wood. After two weeks colonization, the TGs in wood were degraded by 50% to 80%, which resulted in an accumulation of free FAs in the wood. Lipases (glycerol ester hydrolases, EC 3.1.1.3) are the enzymes responsible for hydrolyzing TGs into glycerol and FAs which are assimilable by fungal cells. Extracellular lipase activity of O. piceae 387N was detected both in colonized wood and in liquid culture. The effect of various factors (carbon sources, nitrogen sources, and medium pH) on the growth and lipase activity of O. piceae 387N were examined in liquid culture. The extracellular lipase secretion was enhanced in the presence of triglycerides. The composition of a medium was optimized for a high extracellular lipase production, which contained 2% olive oil as a carbon source and 0.5% ammonium sulfate and 3% peptone as nitrogen sources with an initial medium pH of 5.0. A major extracellular lipase was purified from the liquid culture filtrates of O. piceae 387N by hydrophobic interaction chromatography and anion exchange chromatography. This lipase was characterized as a monomer with a molecular weight of 35 kDa, and was glycosylated, containing 10.1% carbohydrates. It was resolved as a single band on SDS-PAGE (sodium dodecyl sulfate- polyacrylamide gel electrophoresis) gels, whereas 3 bands at pi's 4.3, 4.1 and 3.8 were observed on LEF (isoelectric focusing) gels. Lipolytic stain demonstrated that the three bands on IEF gels were lipolytically active. The 3 isoforms were found to have a same N -terminal sequence as D 1 - V 2 - S 3 - V 4 - T 5 - T 6 - T 7 - D 8 - I 9 - D 1 U 1 1 - L 1 2 - A 1 3 - F 1 4 - F 1 5 - T 1 6 - Q 1 7 - W 1 ^ A 1 9 -The purified O. piceae 387N lipase was stable at pH's 4 to 8 and at temperatures below 40°C. The pH and temperature optima for activity were approximately pH 5.2 and 30°C, respectively. iv Enzyme activity was not influenced by ./V-ethylmaleimide, P-mercaptoethanol, and dithiothreitol, was slightly enhanced by Ca and M n , and was severely inhibited by Hg and 3+ Fe , diethyl pyrocarbonate, diethyl /?-nitrophenyl phosphate, butyric acid, caproic acid, and SDS. The lipase showed high specificity toward substrates with intermediate and long chain F A residues, and belonged to a group of 1(3) positional specific lipases. The rate of hydrolysis of the lipase toward a triglyceride (l,3-dipalmitoyl-2-oleoyl-glycerol) was 25-50 fold higher than that toward the waxes (oleyl esters) and cholesteryl esters. Finally, it was conclusively shown that the purified lipase could effectively release fatty acid residues from the triglycerides isolated from wood. The data and information obtained in this work have contributed to the understanding of the physiological and biochemical features of sapstaining Ophiostoma species. The large amounts of various lipids in wood, in particular TGs and FAs, are important carbon and energy sources for the sapstaining fungi, which are capable of secreting extracellular lipases to degrade TGs. The information implies that the growth of sapstaining fungi may be hindered by disrupting the metabolic processes of lipid utilization, or by applying biological competitors which are more efficient in assimilating lipids and other nutrients in wood. Table of Contents Abstract ii Table of Contents v List of Tables ix List of Figures xi List of Abbreviations xiv Acknowledgements xviii Chapter 1 General Introduction and Research Objectives 1 1.1 Sapstain in the Canadian forest products industry: cost and control strategies 1 1.2 Fungi causing wood sapstain 7 1.3 Wood as a substratum for sapstaining fungi 9 1.3.1 Colonization of wood by sapstaining fungi 9 1.3.2 Environmental conditions of wood affecting the growth of sapstaining fungi 11 1.3.3 Non-structural wood components 12 1.3.3.1 Hy drophilic sub stances 13 1.3.3.2 Lipophilic substances 14 1.3.3.2.1 Triglycerides 14 1.3.3.2.2 Fatty acids 15 1.3.3.2.3 Resin acids 16 1.3.3.2.4 Sterols, steryl esters, fatty alcohols, and waxes 19 1.4 Degradation of lipophilic substances by fungi and other organisms 21 1.5 Fungal lipases 23 1.5.1 Secretion of lipases by fungi 24 1.5.2 Characteristics of lipase reaction and factors influencing lipolysis 26 1.5.3 Structural features and inhibition of fungal lipases 27 1.5.4 Substrate specificities of fungal lipases 29 1.6 Research objectives 32 Chapter 2 Identification and Quantification of Lipids in Pinus contorta var. latifolia wood 36 2.1 Introduction 36 2.2 Materials and Methods 38 2.2.1 Wood sample 38 2.2.2 Extraction of wood lipophilic extractives 38 2.2.3 Solid phase extraction (SPE) 40 2.2.4 Thin layer chromatography (TLC) , 42 2.2.5 Gas chromatography (GC) and combined gas chromatography -mass spectrometry (GC-MS) 42 2.2.6 Methylation 43 2.2.7 Saponification 44 2.2.8 Reagents 44 2.3 Results 45 2.3.1 Total extractive content 45 2.3.2 Different lipid classes in the extractives 47 2.3.3 Fatty acid composition in wood triglycerides 50 2.3.4 Fatty acid composition in wood steryl esters and waxes 52 2.3.5 Free fatty and resin acids 52 2.4 Discussion 58 Chapter 3 Changes of Major Lipids in the Sapwood of Pinus contorta var. latifolia and Populus tremuloides during colonization by Ophiostoma spp 64 3.1 Introduction 64 3.2 Materials and Methods 66 3.2.1 Fungal species and liquid culture 66 3.2.2 Wood materials and inoculation 67 3.2.3 Analysis of wood lipids 68 3.2.4 Extraction of ergosterol and quantification by HPLC 68 3.2.5 Assay of lipolytic activity in colonized wood 69 3.3 Results 70 3.3.1 Comparison of lipid composition in the sterilized lodgepole pine and trembling aspen sapwood 70 3.3.2 Measurement of fungal growth by the quantification of ergosterol 72 3.3.3 Changes of the total wood extractives during fungal colonization 75 3.3.4 Changes of triglycerides and free fatty acids in wood during fungal colonization 78 3.3.5 Changes of resin acids in lodgepole pine wood during colonization 83 3.3.6 Changes of waxes and steryl esters in trembling aspen wood during colonization 83 3.4 Discussion 88 Chapter 4 Production, Purification and Characterization of an Extracellular Lipase Secreted by Ophiostoma piceae 93 4.1 Introduction 93 4.2 Materials and Methods 95 4.2.1 Liquid media and culture conditions 95 4.2.2 Assay of lipase activity 96 4.2.3 Lipase Purification 98 4.2.4 Electrophoresis and lipolytic stain on gels 100 4.2.5 Determination of protein and glycosylation. 101 4.2.6 Molecular weight determination 101 4.2.7 Analysis of amino acid composition and N-terminal sequencing 101 4.2.8 Examination of the effects of pH and temperature on the activity and stability of the lipase 102 4.3 Results 103 4.3.1 Preliminary investigation of the profile and properties of the extracellular lipase(s) produced by O. piceae 103 4.3.2 Effect of carbon sources on the growth and lipase production 105 4.3.3 Effect of nitrogen sources and initial medium pH on the growth and lipase production 110 4.3.4 Purification of the major extracellular lipase 113 4.3.5 Molecular weight, isoelectric point, and glycosylation 119 4.3.6 Amino acid composition and N-terminal sequence 125 4.3.7 Effects of pH and temperature on the activity and stability of the lipase 125 4.4 Discussion 129 Chapter 5 Substrate Specificities of the Lipase Purified from O. piceae and Effects of Various Chemical Reagents on the Enzyme Activity 135 5.1 Introduction. 135 5.2 Materials and Methods 138 5.2.1 Lipase preparation and activity assay 138 5.2.2 Investigation of different chemical reagents on lipase activity 138 5.2.3 Examination of fatty acid specificity and positional specificity 139 5.2.4 Hydrolysis of synthetic triglycerides, waxes, and cholesteryl esters 140 5.2.5 Isolation, saponification, and hydrolysis of wood triglycerides 141 5.2.6 Isolation, identification and quantification of fatty acids released 141 5.3 Results 142 5.3.1 Effects of chemical reagents on the lipase activity 142 5.3.1.1 Organic solvents 142 5.3.1.2 Sulfhydryl agent and reducing agents 144 5.3.1.3 Metal ions and fatty acids 144 5.3.1.4 Ionic detergent and chelating agent 149 5.3.1.5 Enzyme inhibitors 149 5.3.2 Fatty acid specificity and positional specificity 152 5.3.3 Hydrolysis of synthetic triglycerides, waxes, and cholesteryl esters 152 5.3.4 Hydrolysis of the triglycerides isolated from trembling aspen wood 156 5.4 Discussion 159 Chapter 6 Concluding Remarks 165 Bibliography 170 ix List of Tables Table 1.1 Structures of some fatty acids present in wood 17 Table 2.1 Content of different lipid classes in lodgepole pine wood and their relative proportions to the total lipophilic extractives 49 Table 2.2 The composition (%) of fatty acid residues in the triglycerides of lodgepole pine wood 51 Table 2.3 The composition (%) of fatty acid residues in the steryl esters and waxes of lodgepole pine wood 54 Table 2.4 Contents of resin acids and fatty acids in the lodgepole pine wood and their relative proportion to the total amount of free lipophilic acids obtained by solid phase extraction 56 Table 2.5 Content of various free fatty acids in lodgepole pine wood and their relative proportion to the total free fatty acids identified 57 Table 2.6 Content of various free resin acids in lodgepole pine wood and their relative proportion to the total free resin acids identified 59 Table 3.1 Lipids in the sapwood of lodgepole pine and trembling aspen after sterilization by gamma irradiation 71 Table 3.2 Content of each resin acid in lodgepole pine sapwood before and after gamma irradiation 73 Table 3.3 The ergosterol content (%, w/w) in the cells of O. piceae during different growth stages in liquid culture 74 Table 3.4 Contents of total wood extractives in lodgepole pine and trembling aspen sapwood before and after fungal inoculation 79 Table 4.1 Effects of pH and temperature on the lipase activity in the culture filtrate of O. piceae 106 Table 4.2 Effect of the initial medium pH on the growth and extracellular lipase activity of 0. piceae 114 Table 4.3 Effect of peptone concentration on fungal growth and extracellular lipase activity of O. piceae 115 Table 4.4 Purification of the extracellular lipase produced by O. piceae 121 Table 4.5 Comparison of O. piceae lipase activity on olive oil and PNPP 121 Table 4.6 Amino acid composition of the extracellular lipase purified from 0. piceae compared with that reported for other fungal lipases (mole % ) 126 Table 5.1 Effects of N E M , DTT and B M E on the O. piceae lipase activity 146 Table 5.2 Hydrolysis of synthetic triglycerides, waxes, and cholesteryl esters by O. piceae lipase 155 Table 5.3 Hydrolysis of the triglycerides isolated from trembling aspen sapwood by saponification and by the lipase purified from O. piceae 157 List of Figures Figure 1.1 British Columbia log harvest in 1994 2 Figure 1.2 Quantity of anti-sapstain active ingredients used in BC in 1994 6 Figure 1.3 Structures of common resin acids in wood 18 Figure 1.4 Structures of some sterols in wood 20 Figure 1.5 Hydrolysis of triglycerides by lipases, (a) Reaction catalyzed by non-specific lipases, (b) Reaction catalyzed by 1,3-specific lipases 31 Figure 2.1 Diagram of wood sampling 39 Figure 2.2 Isolation of lipid classes from wood extractives by solid phase extraction 41 Figure 2.3 Content of total wood extractives in lodgepole pine wood 46 Figure 2.4 Thin layer chromatogram showing the total extractives and fractions after each step of solid phase extraction 48 Figure 2.5 Gas chromatogram showing fatty acids in the saponified products of the steryl esters and waxes of lodgepole pine inner heartwood 53 Figure 2.6 Gas chromatogram showing the free fatty and resin acids in the lodgepole pine sapwood 55 Figure 3.1 Ergosterol and fungal biomass of 0. piceae in liquid culture 74 Figure 3.2 H P L C chromatograms of ergosterol standard (A), extracts from non-colonized (B) and colonized lodgepole pine sapwood 8 days after inoculation (C) 76 Figure 3.3 Ergosterol levels of sapstaining fungi in lodgepole pine sapwood 77 Figure 3.4 Changes in triglycerides in the sapwood of lodgepole pine (A) and trembling aspen (B) after colonization by O. piceae, O. ainoae, or O. piliferum 80 Figure 3.5 The lipolytic enzyme activity of O. piceae in lodgepole pine sapwood 81 Figure 3.6 Changes in total free fatty acids in the sapwood of lodgepole pine and trembling aspen after colonization by O. piceae, O. ainoae, or O. pi lifer urn 82 Figure 3.7 Changes in the amount of various fatty acids in lodgepole pine sapwood after colonization by 0. piceae 84 Figure 3.8 Changes in the total free resin acid contents in the sapwood of lodgepole pine after colonization by O. piceae, O. ainoae, or O. piliferum 85 Figure 3.9 Changes in the amount of various resin acids in lodgepole pine sapwood after colonization by O. piceae 86 Figure 3.10 Changes in the waxes and steryl esters in trembling aspen sapwood after 14 days colonization by O. piceae, O. ainoae, or O. piliferum 87 Figure 4.1 Biomass, extracellular lipase activity, and pH in a culture of O. piceae 104 Figure 4.2 Electrophoretic profile of the extracellular proteins produced by O. piceae in olive oil supplemented medium 107 Figure 4.3 Growth of O. piceae in the medium with supplemented with starch, triolein, oleic acid or glycerol as carbon sources 108 Figure 4.4 Effect of carbohydrates on the growth (A) and extracellular lipase activity (B) of 0. piceae 109 Figure 4.5 Effect of plant oils on the growth (A) and extracellular lipase activity (B) of O.piceae I l l Figure 4.6 Effect of nitrogen sources on the growth (A) and extracellular lipase activity (B) of O. piceae 112 Figure 4.7 Separation of extracellular proteins of O. piceae by SDS-PAGE (PhastGel 12.5%) at various stages of purification 117 Figure 4.8 Fractionation of proteins in the culture filtrate of O. piceae by hydrophobic interaction chromatography 118 Figure 4.9 Fractionation of proteins partially purified by FLIC using anion exchange chromatography 120 Figure 4.10 Estimation of the molecular weight of the lipase purified from O. piceae by size exclusion chromatography and SDS-PAGE 122 Figure 4.11 Analysis of the purified O. piceae lipase by SDS-PAGE (PhastGel 8-25%) 123 Figure 4.12 Analysis of the purified O. piceae lipase by EEF gel electrophoresis (PhastGel 3-9) 124 Figure 4.13 Effects of pH on the purified O. piceae lipase activity (A) and stability (B) 127 Figure 4.14 Effects of temperature on the purified O. piceae lipase activity (A) and stability (B) 128 Figure 5.1 Effects of organic solvents (10%, v/v) on the 0. piceae lipase activity 143 Figure 5.2 Effects of DMSO concentrations on the O. piceae lipase activity 145 Figure 5.3 Effects of metal ions (20 mM) on the O. piceae lipase activity 147 Figure 5.4 Effects of fatty acids on the O. piceae lipase activity 148 Figure 5.5 Effects of SDS and E D T A on the 0. piceae lipase activity 150 Figure 5.6 Effects of different inhibitors on the 0. piceae lipase activity 151 Figure 5.7 Fatty acid specificity of O. piceae lipase 153 Figure 5.8 T L C chromatograms of the hydrolysis products obtained through the action of the O. piceae lipase on triolein 154 Figure 5.9 Gas chromatogram showing fatty acids in the hydrolysis products of the triglycerides isolated from trembling aspen sapwood 158 Figure 5.10 A proposed reaction sequence of O. piceae lipase on triglyceride lipolysis 163 List of Abbreviations 12:0 lauric acid 14:0 myristic acid 16:0 . palmitic acid 17:0 margaric acid 18:0 stearic acid 18:1 oleic acid (cis-9) 18:2 linoleic acid (cis-9,12) 18:3 linolenic acid (cis-9,12,15) 20:0 arachidic acid 20:1 eicosenoic acid (cis-11) 20:2 eicosadienoic acid (cis-11,14) 20:3 eicosatrienoic acid (cis-5,9,12) 22:0 behenic acid 22:2 docosadienoic acid (cis-13,16) 24:0 lignoceric acid A alanine A280 absorbance at 280 nm A404 absorbance at 404 nm Ala alanine A N O V A analysis of variance Arg arginine Asn asparagine Asp aspartic acid ATP adenosine triphosphate B M E P-mercaptoethanol B S A bovine serum albumin CAPS 3 -(cyclohexylamino)-1 -propanesulfonic acid CoA coenzyme A COFI Council of Forest Industries of British Columbia D aspartic acid D E A E diethylaminoethyl DEPC diethyl pyrocarbonate D G diglyceride Diazald A^-methyl-A'-riitroso-p-toluenesulfonamide DMSO dimethyl sulfoxide DTT dithiothreitol E glutamic acid E600 diethyl /?-nitrophenyl phosphate EC European Community E D T A ethylenediamine tetraacetate F phenylalanine F A fatty acid FPLC fast protein liquid chromatography G glycine g gram GC gas chromatography Gin glutamine Glu glutamic acid Gly glycine H histidine h hour FJJC hydrophobic interaction chromatography His histidine H P L C high performance liquid chromatography I isoleucine EEF isoelectric focusing He isoleucine IPBC 3-iodo-2-propynyl butyl carbamate K lysine kDa kilodalton L leucine xvi Leu leucine Lys lysine M methionine M e C N acetonitrile MES 2-(A^-morpholino)ethane sulphonic acid Met methionine M G monoglyceride min minute Mrad megarad MS mass spectrometry N asparagine N A D nicotiamide adenine dinucleotide N E M Af-ethylmaleimide NSERC Natural Sciences and Engineering Research Council of Canada od oven-dry P proline Phe phenylalanine pi isoelectric point PMSF phenylmethylsulfonyl fluoride PNPL /j-nitrophenyl laurate PNPP /?-nitrophenyl palmitate POP l,3-dipalmitoyl-2-oleoyl-glycerol PPO 1,2-dipalmitoyl-3 -oleoyl-rac-glycerol Pro proline psi pounds per square inches PVDF polyvinyldifluoride Q glutamine R arginine R A resin acid RP-HPLC reverse phase high performance liquid chromatography rpm revolutions per minute S serine SDS sodium dodecyl sulfate SDS-PAGE sodium dodecyl sulfate- polyacrylamide gel electrophoresis Ser serine SPE solid phase extraction spp. species T threonine T C A tricarboxylic acid T G triglyceride THF tetrahydrofuran Thr threonine T L C thin layer chromatography Tris tris-(hydroxymethyl)-1 -aminoethane Trp tryptophan Tyr tyrosine U V ultraviolet V valine v/v volume by volume Val valine var. varietas (Latin) vol volume W tryptophan w/v weight by volume w/w weight by weight Y tyrosine X V l l l Acknowledgements I wish to thank my research supervisor Dr. C. Breuil for providing the initial idea for this project, and for her support and effort in bringing this work to its fruition. I also appreciate her generous support which made me be able to attend two international conferences. I thank all the other members in my Supervisory Committee, Dr. J. N . Saddler, Dr. S. Withers, Dr. A . Potter, and Dr. S. Ellis for their advice and encouragement during the performance of this project. I am indebted to Dr. L . Paszner who reviewed a report generated from this project before submission for publication. I would also like to thank Dr. J. N . R. Ruddick and Dr. S. Withers for allowing me to access the facilities of GC-MS, and RP-HPLC in their laboratories, and Forintek Canada Corporation (Vancouver Laboratory) for the assistance in preparing wood samples. I thank my colleagues, in the NSERC/Industrial Chair of Forest Products Biotechnology at the University of British Columbia, Dr. T. Chen, Dr. K. K. Y . Wong, Dr. L . D. Abraham, Mr. A . Roth, Ms X . Feng, and Mr. K. L i , from whom I learned many laboratory skills, and with them I enjoyed valuable discussions. I gratefully acknowledge Dr. L . D. Abraham and Dr. A . Serreqi for reviewing the draft of this thesis and for their critical and constructive comments. I wish to express my sincere gratitude to various organizations which provided me with the financial support through several awards and scholarships, which allowed me to attend graduate school and conduct this research. They are: Forestry Canada (through the Network for Biorational Control of Forest Disease), the Science Council of British Columbia [Graduate Research, Engineering and Technology award (G.R.E.A.T)], the University of British Columbia (University Graduate Fellowship), VanDusen Graduate Fellowship Foundation (VanDusen Graduate Fellowship in Forestry), MacMillan Bloedel Ltd. (industrial sponsor for G.R.E.A.T), and Du Pont Canada Ltd. (Du Pont Canada Fellowship in Pulp and Paper). I also gratefully acknowledge the International Research Group on Wood Preservation (IRG) for granting me Ron Cockcroft award, which made me be able to present this work at IRG 26th Annual Conference in Helsingor, Denmark in June 1995. I am thankful to my wife Juan and my daughter Qiao, and all the other members in my family for their love, patience, and understanding. I thank my brothers You-Xiang and You-Jun and my friend Dr. Wen-Quan Sun, who financially helped me in 1991 in paying the high leaving-country head tax the 'education fee1, which, according to the then rules of the Chinese State Education Commission, was charged to those who quit their jobs in China going overseas to pursue higher education. Finally, I dedicate this thesis to my parents, my mother Chen Zong-Lan and my father Gao Chun-Rong, who raised six children during one of the hardest times in Chinese history, a time the country was in deep political and economical chaos, a time tens of millions of people died of starvation, poverty, and political suppression. 1 Chapter 1 General Introduction and Research Objectives 1.1 Sapstain in the Canadian forest products industry: cost and control strategies Forest products earn Canada's largest net export income, contributing $29.3 billion to the country's trade balance in 1994. This contribution from forest products was significantly greater than those made by products of crude or processed raw materials ($9.9 billion), energy ($14.6 billion), vehicles and parts ($10.6 billion) or agriculture and fish ($5.0 billion) (COFI 1995a). Among the total exports of forest products, 36.0% of the earnings came from lumber, 35.0% from newsprint and paper, and 21.0% from wood pulp (COFI 1995a). Canada is the world's largest lumber exporter, contributing 50.2% of the world's total export volume of 85.3 million cubic meters of softwood lumber in 1993. About 55.8% of the total Canadian softwood lumber export was from British Columbia (BC) (COFI 1995a). In 1994, the B C log harvest was 75.6 million cubic meters, of which more than two-thirds was from the interior regions, where the main softwood species are lodgepole pine (Pinus contorta var. latifolia), spruce (Picea spp.), and true firs (Abies spp.). The remaining one-third of the log harvest came from the coastal forest, where hemlock (Tsuga spp.) comprises the largest volume, followed by western red cedar (Thuja plicata), true firs, Douglas-fir (Pseudotsuga menziesii) and spruce. Figure 1.1 shows the log production of the major wood species in BC. Lodgepole pine Spruce Hemlock True firs Douglas-fir Western red cedar Others Hardwood - i — i — i — i — [ — i — i — i — i — | — i — i — i — i — r —1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 T -0 5 10 15 20 Million cubic metres Figure 1.1 British Columbia log harvest in 1994 (COFI 1995a). 3 The major international markets for BC's wood products (lumber, plywood, shingles/shakes, etc.) are the United States, Japan, and the European Community (EC). In 1994, the three destinations received 50.8%, 21.5% and 4.3% of BC's total shipments (by value), respectively, while the rest of Canada received 21.0% (COFI 1995a). Both overseas and domestic markets pay higher prices for clear, unseasoned, and defect-free lumber. In BC coastal sawmills, it is the clear and shop grades which effectively generate the operational profits, making cosmetic appearance critical. Sapstain, also called blue stain, is a long-standing problem for the Canadian lumber industry. It refers to the grey, black, or bluish discoloration of sapwood mainly caused by the presence of pigmented fungal hyphae, fruiting bodies, or asexual spores (Seifert 1993). The pigment which colours fungal hyphae has been characterized as melanin, or melanin associated with carbohydrates and proteinaceous components (Wheeler 1983; Zink and Fengel 1988). The damage to wood from sapstain is cosmetic, in contrast to structural damage caused by decay and soft rot fungi (Liese 1970; Eriksson 1981). The only potential mechanical concern is for applications requiring impact toughness, which can be reduced by up to 30% in heavily stained pine wood (Chapman and Scheffer 1940; Scheffer 1973; Subramanian 1983). Despite this fact, sapstain is of significant economic importance since the cosmetic discoloration is objectionable to wood buyers (Wilcox 1973; Zabel and Morrell 1992; Seifert 1993). Lumber with heavy sapstain may be refused by customers or may lead to monetary claims against the suppliers (Environment Canada 1994). Therefore, it is important for B C lumber exporters to keep wood 4 free of discoloration during processing of logs, storage of the lumber and transportation to market destinations. Sapstain control should be an integrated process involving expedient harvesting and storage of timber, judicious chemical treatment and efficient delivery to the customers. Several strategies have been used to prevent or reduce sapstain: (1) adopting preventive logging practices, such as avoiding storage of logs during high risk periods (e.g., in the warmer summer months), rapid handling of logs in warmer climates, storage in water and saturating log piles by sprinkling (Findlay 1959; Dickinson 1988); (2) kiln-drying; and (3) chemical treatment (Byrne and Smith 1987; Smith 1991; Environment Canada 1994). Kiln-drying can prevent sapstain by reducing the moisture content of wood below 20%, where fungi are unable to grow. However, kiln-drying is only feasible under certain circumstances because it can cause drying defects, and it is not suitable for lumber of large dimension which command premium prices in the export market. Furthermore, kiln-drying is expensive, and the cost cannot always be recovered by increasing lumber prices (Byrne and Smith 1987; Forestry Canada 1990). The success of kiln-drying is also dependent on ensuring that all the timber is adequately dried and remains so for the entire period of transportation and storage. However, during shipment, rainfall at the docks or humid conditions in the hold of a ship may rewet kiln-dried lumber, making it susceptible to sapstaining fungi attack (Environment Canada 1994). 5 Chemical treatment has been a major strategy for preventing sapstain. In 1994, approximately 3 billion board feet of softwood lumber, with an export value exceeding $2.9 billion, was treated with antisapstain chemicals in BC (COFI 1995b). The market for antisapstain chemicals in Canada is currently worth about $16 to $20 million annually (Abraham 1995). The demands on antisapstain chemicals have grown over the years. Concerns about safety with respect to the environment and the lumber treating process have become increasingly important. The ideal antisapstain chemicals should: (1) give a 12 month effective shelf life to treated lumber, (2) be amenable to safe use by mill workers and have no toxic effect on the environment, (3) be non-corrosive, non-leachable, water-soluble and easily handled, and (4) be cost effective to provide an affordable end product (Zabel and Morrell 1992; Abraham 1995). In Canada, increased emphasis has been placed on formulations containing relatively environmentally benign chemicals compared to the chlorinated phenols used for decades. Currently there are seven registered active ingredients: azaconazole, disodium tetraborate decahydrate, disodium octaborate tetrahydrate, D D A C (didecyl dimethyl ammonium chloride), IPBC (3-iodo-2-propynyl butyl carbamate), sodium carbonate, and T C M T B [2-(thiocyanomethylthio) benzothiazole] (Figure 1.2). These chemicals are prepared and marketed in various formulations under different names, such as Borax 10, Busan 1030; Ecobrite, F2, NP-1, Rodewod 200 EC, Timbercote II, Timbercote 2000, etc. (Smith 1991; Konasewich and St. Quintin 1994; Environment Canada 1995). However, none of the currently registered antisapstain chemicals have all the properties of an ideal protectant as defined above. 6 Kg 600,000 500,000 h 400,000 300,000 200,000 100,000 0 501,980 120,020 69,649 1,552 109,750 41,027 227 4? Figure 1.2 Quantity of anti-sapstain active ingredients used in BC in 1994 (Environment Canada 1995). DTD: disodium tetraborate decahydrate, DOT: disodium octaborate tetrahydrate, DDAC: didecyl dimethyl ammonium chloride, LPBC: 3-iodo-2-propynyl butyl carbamate, TCMTB: 2-(thiocyanomethylthio) benzothiazole. 7 1.2 Fungi causing wood sapstain Sapstaining fungi, in broad scope, include truly pathogenic staining organisms that occur in living trees, pathogenic fungi that grow on weakened trees and may also have a saprophytic phase, and truly saprophytic fungi (Gibbs 1993). Kaarik (1980) listed more than 250 fungal species associated with sapstained wood, 58 of them belonging to the genera Ceratocystis or Ophiostoma. However, many of these species are pathogenic fungi associated with stain in living trees. In lumber, discolorations are caused mostly by saprophytic fungi which grow in the sapwood after the wood is cut. Sapstain caused by pathogenic or endophytic fungi may be less important economically to the lumber industry because infected wood can be discarded before or during processing. Growth of saprophytic fungi or opportunistic pathogens is more insidious because colonization of the wood can occur at any time after the tree is felled (Seifert 1993). In a country-wide survey of sapstaining fungi in Canadian lumber conducted by Forintek Canada Corporation (Seifert and Grylls 1990), sapstaining fungi were divided into five groups: (1) Ophiostomatales (Ophiostomatoid fungi), (2) black yeasts, (3) dematiaceous moulds (dark moulds), (4) green moulds, and (5) fungi of unknown significance. The Ophiostomatales are generally recognized as Ascomycetes with long-necked perithecia, evanescent asci and hyaline ascospores lacking pores or slits (Malloch and Blackwell 1993). 8 Sapstaining Ophiostomatales includes species of Ophiostoma and Ceratocystis and their anamorphs (asexual states) classified as Graphium, Sporothrix, Leptographium and Hyalorhinocladiella. Ophiostoma piceae was found to be the most common species in both eastern and western Canada. The other Ophiostomatales species of importance were: O. minus (Ceratocystis pint) and C. adiposa, and to a lesser extent, O. olivaceum, O. sagmatospora, O. piliferum and C. coerulescens (Seifert and Grylls 1990). A previous survey carried out in BC by Chung and Smith (1986) also concluded that O. piceae and Sporothrix sp. were the predominant species. In U S A and Europe, the important sapstaining Ophiostomatales fungi reported include O. piceae, O. ips, O. minus, O. piliferum, O. pluriannulatum, C. coerulescencs, C. moniliformis, and C. virescens (Davidson 1935; Findlay and Pettifor 1937; Henningsson and Lundstrom 1974; Eslyn and Davidson 1976; Kaarik 1980). Green moulds, such as Penicillium spinulosum, P. brevicompactum and Trichoderma spp. produce abundant, dry, green asexual spores on the wood surface (Wilcox 1973; Seifert and Grylls 1990). The spores can be brushed or planed off without leaving any residual discoloration, therefore, these moulds are usually not considered sapstaining fungi. However, some species of Penicillium produce pigments that diffuse into the wood tissue (Seifert and Grylls 1990). Similarly, the darkly pigmented dematiaceous moulds, such as Alternaria alternata, Cladosporium sphaerospermum and C. cladosporioides produce abundant dry asexual spores (Seifert and Grylls 1990). Black yeasts refer to those darkly pigmented, yeast-like fungi, such as Hormonema dematioides, Aureobasidium pullulans, Rhinocladiella atrovirens and Phialophora spp., which can also stain wood (Seifert and Grylls 1990). 9 1.3 Wood as a substratum for sapstaining fungi This section begins with a discussion on the colonization of wood by sapstaining fungi in relation to structural features, followed by a description of micro-environmental requirements for sapstaining fungal growth in wood, and finally by an overview of wood chemical composition and the potential nutrients in wood for sapstaining fungi. 1.3.1 Colonization of wood by sapstaining fungi Most studies on sapstain have been conducted with softwoods, although the discoloration also occurs on hardwoods. Certain wood species, such as pine, hemlock, and Douglas-fir, are particularly prone to sapstain, while spruce species are less susceptible. Sapstaining fungi are among the initial microbial colonizers of wood. The colonization sequence of untreated lumber by different wood-inhabiting microorganisms is usually bacteria and sapstaining fungi first, followed by soft-rot, and finally by decay fungi (Butcher 1968; Clubbe 1980; Rayner and Boddy 1988). The sapstaining fungi may modify the wood substrate and make it susceptible to subsequent attack by decay fungi (Rayner and Boddy 1988). It has long been known that sapstaining Ophiostomatoid fungi are spread into the wood of dead or dying trees, or into timber by bark beetles or phoretic mites (Davidson 1935; Kaarik 1971; Bridges and Moser 1983; Christiansen and Ericsson 1986; Levieux et al. 1989; 10 Yamaoka et al. 1990; Levieux et al. 1991; Malloch and Blackwell 1993). With their insect vectors these fungi often form more or less firm associations that have been characterized as mutualistic symbiosis (Kaarik 1971). Both the fungi and the bark beetles can exist and develop fully without their associates, but they are always found together in nature. Once in wood, sapstaining fungi spread mainly through the natural passages of the wood structures. These passages occurs as two intercommunicating systems of wood cells, one axial and one transverse. Axial passages are provided by vessels, tracheids, fibres, and various forms of wood parenchyma cells (Liese 1970; Ballard et al. 1982; Blanchette et al. 1992a, b). An important feature of vessels and tracheids is the occurrence of pits in the lateral walls which provide the main opportunity for tangential passage between elements. Coniferous wood is often characterized by the presence of bordered pits between tracheids, which are usually larger but fewer in number than the simple or cross-lacunate pits typical in the lateral walls of hardwood vessels (Liese 1965; Schmid 1965; Tsoumis 1965). Although pits may also provide a limited degree of radial access, this is insignificant by comparison with the opportunities provided by the radially elongated parenchyma cells, which act both as nutrient depots and as radial passages for sapstaining fungi (Rayner and Boddy 1988). Liese and Schmid (1961) reported that in the pine and spruce wood colonized by O. piliferum and O. piceae, rays, tracheids and resin canals were all colonized by fungal hyphae, and ray cells were preferentially invaded. The apparent explanation for the prolific growth of sapstaining fungi within ray parenchyma cells is the availability of nutrients and the lack of 11 lignin within the cell walls (Kaarik 1971). Extensive growth was also observed in tracheids, and progress of the fungi from tracheid to tracheid was via bordered pits and rarely through direct penetration of the wall (Liese and Schmid 1961). In the medullary rays, growth from cell to cell was almost entirely via the pits. Blanchette et al. (1992b) also observed that the hyphae of an albino strain of O. piliferum were able to penetrate through simple and bordered pits, causing perforation of the pit membranes. The fungus also grew prolifically in longitudinal and transversely oriented resin canals and disrupted the epithelial cells surrounding the resin canals. 1.3.2 Environmental conditions of wood affecting the growth of sapstaining fungi Moisture, oxygen, temperature, and pH influence the growth and development of sapstaining fungi in wood. Sapstain generally occurs during seasoning or transportation of green lumber before the wood is dried. Fully water saturated wood wil l not stain because of the very low oxygen concentrations (Seifert 1993). The minimum moisture content reported as necessary for fungal growth in wood is 20% of oven-dry (od) wood (Colley and Rumbold 1930), and the optimum moisture content for maximum stain is 60% to 80% (Seifert 1993). For most softwood and hardwood species, the moisture content in freshly sawn lumber varies from 72% to 170%) in sapwood, and from 41% to 95% in heartwood (Rayner and Boddy 1988). In general, sapwood-ihhabiting fungi are less able to survive long periods without oxygen than are heartwood-inhabiting fungi (Seifert 1993). Scheffer (1986) studied the in vitro oxygen requirements of several Ophiostoma species, and found that O. piliferum and O. minus grew 12 fairly well at oxygen concentrations as low as 0.8%, but had much reduced growth at lower concentrations. Sapstaining fungi grow best on wood at temperatures between 22 to 30°C (Reynolds et al. 1972; Miller and Goodell 1981; Morrell and Sexton 1992). Although fluctuating temperatures reduce the rate of growth, there is evidence that serious stain can occur on wood stored at lower temperatures, e.g., in the range at 3 to 8°C (Miller and Goodell 1981). In addition, most fungi grow best within a pH range of 3 to 6 (Morrell and Sexton 1992; Zabel and Morrell 1992), compatible with most wood pH values (Rayner and Boddy 1988). 1.3.3 Non-structural wood components Wood is composed of major macromolecular cell wall structural components, and minor low molecular weight non-structural components. The structural components, typically accounting for more than 90% of wood dry weight, are cellulose, hemicelluloses, and lignin. A l l are present in wood cell walls, but a high concentration of lignin also accumulates in the middle lamella. A simplified picture of wood cell wall is that cellulose forms a skeleton which is surrounded by other substances functioning as matrix (hemicelluloses) and cementing (lignin) materials (Sj ostrom 1981). Although some sapstaining fungi have been reported to show slight cellulolytic activity in artificial media, no obvious cavity or erosion has been observed in colonized wood (Nilsson 13 1973; Federici 1982). In general, sapstaining fungi lack a complete enzyme system for degrading cellulose and lignin (Liese 1970; Sharpe and Dickinson 1992; Seifert 1993). Most of them have little effect on the strength properties of wood, and the loss of wood dry weight is restricted to a few percent (1% to 4%) after extended colonization (Eslyn and Davidson 1976; Tabirih and Seehann 1984). Therefore, to grow in wood, sapstaining fungi mainly assimilate non-structural wood components. The non-structural wood components are located in the cytoplasm of parenchyma cells, lumen of tracheid and vessels, and in some special structures like resin canals. These substances can be further categorized into two groups: (1) hydrophilic substances, such as proteins, amino acids, starch, soluble sugars, and minerals; and (2) lipophilic substances which are the so called wood extractives, resin, or pitch. Most wood contains 2% to 6% of wood extractives, which include a variety of lipids, such as glycerides, fatty acids, resin acids, sterols, steryl esters, and waxes (Sjostrom 1981; Fengel and Wegener 1984). 1.3.3.1 Hydrophilic substances Proteins and amino acids are the primary forms of nitrogen-containing compounds in wood (Fukuda 1963; Laidlaw and Smith 1965; Wetzel et al. 1989; Langheinrich and Tischner 1991; Sauter et al. 1989; Abraham and Breuil 1993). Fungi, like other organisms, require substantial amounts of nitrogen for the synthesis of proteins and other cell constituents. In mature wood only 0.03% to 0.1% of the dry weight is nitrogen, whereas the cambial tissues and newly 14 formed sapwood may contain ten times this amount. In the sapwood the nitrogen content can be 1.2 to 2.5 times higher than that in the heartwood (Cowling and Merrill 1966). Starch and soluble sugars in wood can be used by sapstaining fungi as carbon sources (Hudson 1986). The contents of these substances vary greatly among wood species (Kramer and Kozlowski 1960; Hillis 1987). Starch is present as grains in the ray and axial parenchyma cells (Nair et al. 1981), and the amount stored in sapwood is seasonal, falling to low levels early in the growing season with bud-swelling and unfolding of the leaves (Kramer and Kozlowski 1960). Glucose, fructose, sucrose, mannose, galactose, arabinose, and xylose are also present in sapwood in different amounts (Hillis 1962; 1987; Fischer and Holl 1992; Sauter and van Cleve 1994). For example, the highest contents of starch, sucrose, and other soluble sugars recorded in poplar branch wood (Populus x canadensis Moench crobusta') during the year were 1.5-1.8%, 1.0-1.5%, and 3.0%, respectively (Sauter and van Cleve 1994). 1.3.3.2 Lipophilic substances 1.3.3.2.1 Triglycerides Triglycerides are stored in small oleosomes of 0.2-1.0 um in diameter in the cytoplasm of the parenchyma cells (Sauter and van Cleve 1994). Triglycerides in wood may vary from 1% to 3%> of od weight. For example, triglyceride contents of 1.7% in Pinus sylvestris (Scots pine), 1.4% in Populus tremuloides (trembling aspen) and 2-3% in Tilia cordata (basswood) wood 15 have been reported (Holl and Priebe 1985; Saranpaa and Nyberg 1987a; Sithole et al. 1992). In Picea abies (Norway spruce) and trembling aspen, approximately half of the total extractives from sapwood were triglycerides (Ekman 1979; Sithole et al. 1992). In Scots pine the triglyceride content increased slightly from the cambial zone (1.0% of od wood) to the inner sapwood (1.5%), and then decreased to 0.03% in heartwood (Saranpaa and Nyberg 1987b). Triglycerides serve as an important form of food storage for growing trees. They may be hydrolyzed in the trunkwood in early spring, and their degradation products translocated to sites where they are needed (buds and leaves) or to metabolic sinks (such as roots). For example, in the trunk of a 29-year-old Tilia cordata tree, triglycerides were abundant at the beginning of December, while in March when the growing season began, they disappeared almost completely with a concomitant increase in free fatty acids (Holl and Priebe 1985). In contrast, in both a 30- and a 75-year-old Scots pine tree, sapwood triglycerides showed no major changes through the year, although in winter the content was slightly higher than in summer (Saranpaa and Nyberg 1987a; Fischer and Holl 1992). 1.3.3.2.2 Fatty acids In living trees or newly felled logs of many species, e.g., Norway spruce, Pinus radiata, Pinus muricata, Tilia cordata, and Scots pine, the sapwood contains very low amounts of free fatty acids, but the content increases toward the heartwood (Hemingway and Hillis 1971; Lloyd 1975; Holl and Pieczonka 1978; Ekman 1979; Holl and Priebe 1985; Saranpaa and Nyberg 16 1987a, b; Fischer and Holl 1992). For example, in Scots pine free fatty acids were below 0.01% in sapwood, and increased through the transition zone, to 0.5% in the heartwood. The high free fatty acid level in the heartwood may come from the degradation of triglycerides during heartwood formation (Saranpaa and Nyberg 1987b). More than 20 different fatty acids have been identified in wood (Fengel and Wegener 1984). They differ from each other in chain length, and in the number and position of double bonds (Table 1.1). In various wood species the most important fatty acids are oleic (18:1), linoleic (18:2), and linolenic (18:3) acids. These three fatty acids usually account for more than 70% of the total free fatty acids. Palmitic (16:0), stearic (18:0), 14-methyl hexadecanoic and 5,11,14-eicosatrienic acid (20:3) are detected in lower amounts (Ekman 1979; Saranpaa and Nyberg 1987a; Sithole et al. 1992). In addition, some uncommon fatty acids were identified from certain wood species. For example, pinolenic acid (18:3, cis-5,9,12) has been identified in several pine species, such as Scots pine, P. nigra and P. brutia (Holmbom and Ekman 1978; Yildirim and Holmbom 1977b, c), and a relatively high amount of triacontatrienoic acid (30:3 cis-5,11,14) was detected in Douglas-fir (Foster et al. 1980). 1.3.3.2.3 Resin acids Resin acids, a class of diterpenoid carboxylic acids, are found mainly in softwoods, while none or negligible amount occurs in hardwood species. The most common resin acids in softwoods are pimaric, sandaracopimaric, isopimaric, levopimaric, palustric, dehydroabietic, abietic and neoabietic acids (Figure 1.3). Free resin acids are mainly present in resin canals (Mutton 17 T a b l e 1.1 Structures of some fatty acids present in wood. Abbr.* Common name Systematic name Structure 14:0 Myristic Tetradecanoic CH3(CH2)i2COOH 14:19 Myristoleic cis-9-Tetradecenoic CH3(CH2)3CH=CH(CH2)7COOH 16:0 Palmitic Hexadecanoic CH3(CH2)i4COOH 16:19 Palmitoleic cis-9-Hexadecenoic CH3(CH2)5CH=CH(CH2)7COOH 17:0 Margaric Heptadecanoic CH3(CH2)i5COOH 18:0 Stearic Octadecanoic CH3(CH2)i6COOH 18:19 Oleic cis-9-Octadecenoic CH3(CH2)7CH=CH(CH2)7COOH 18:1U cis-Vaccenic cis-11 -Octadecenoic CH3(CH2)5CH=CH(CH2)9COOH 18:29-12 Linoleic cis-9,12-Octadecadienoic CH3(CH2)4CH=CHCH2CH=CH(CH2)7COOH 1 8. 39,12,15 Linolenic cis-9,12,15-Octadecatrienoic CH3CH2CH=CHCH2CH=CHCH2CH=CH(CH2)7COOH 18:35'9'12 Pinolenic cis-5,9,12-Octadecatrienoic CH3(CH2)4CH=CHCH2CH=CH(CH2)2CH=CH(CH2)3COOH 20:0 Arachidic Eicosanoic CH3(CH2)i8COOH 20:1U Gondoic cis-ll-Eicosenoic CH3(CH2)7CH=CH(CH2)9COOH 20:2H'1 4 cis-11,14-Eicosadienoic CH3(CH2)4CH=CHCH2CH=CH(CH2)9COOH 20:3s'11'14 cis-5,11,14-Eicosatrienoic CH3(CH2)4CH=CHCH2CH=CH(CH2)4CH=CH(CH2)3COOH 22:0 Behenic Docosanoic CH3(CH2)2 0COOH 24:0 Lignoceric Tetracosanoic CH3(CH2)2 2COOH * Abbr. abbreviation 18 Abietane Abietic Dehydroabietic Sandaracopimaric Isopimaric Pimaric Figure 1.3 Structures of common resin acids in wood. 19 1962). Although resin canals are a normal feature of pines, spruces, larches (Larix) and Douglas-fir, they reach their greatest development in the pines which are generally rich in resin acids. In other softwoods, like the true firs, hemlocks, and redwoods (Sequoia), resin canals are normally absent, but may be produced in response to injury. The content of resin acids varies between tree species. In some pine and spruce species the total resin acid content is 0.2% to 1.5% of od weight (Holmbom and Ekman 1978; Conner et al. 1980). Heartwood is generally higher in contents of resin acids than sapwood. For example, in the wood of Pinus edulis, P. monophylla and P. quadrifolia, resin acid contents were 0.7% to 1.2% in sapwood and 2.4% to 4.6% in heartwood (Zavarin and Snajberk 1980). High resin acid content was also reported in tree bark where these compounds can act against wood-boring insects and associated pathogenic microorganisms (Shrimpton 1973). 1.3.3.2.4 Sterols, steryl esters, fatty alcohols, and waxes Sterols are triterpenoids built from six isoprene units. Steryl esters are formed from the conjugation of sterols and fatty acids. The major sterols identified in wood are sitosterol, stigmasterol, campesterol, and cholesterol (Rowe 1965; Yildirim and Holmbom 1977a; Holl and Goller 1982; Saranpaa and Nyberg 1987b; Hafizoglu 1989), the structures of which are illustrated in Figure 1.4. In Norway spruce, Scots pine and P. nigra wood, sitosterol is the principal sterol, followed by stigmasterol and campesterol (Yildirim and Holmbom 1977a; Holmbom and Ekman 1978; Holl and Goller 1982; Saranpaa and Nyberg 1987b). 20 HO" HO" Sitosterol Stigmasterol HO" H( Campesterol Cholesterol Figure 1.4 Structures of some sterols in wood. 21 Saranpaa and Nyberg (1987b) reported that in Scots pine free sterols were 0.04% to 0.08% across the radial section, and highest in the cambial zone and in the heartwood. The amount of steryl esters were 0.06% to 0.09%. The heartwood contained more steryl esters than the sapwood. Similarly, the innermost heartwood in the trunk wood of Norway spruce contained the highest amounts of sterols and steryl esters (Holl and Goller 1982). In trembling aspen wood, higher contents of steryl esters (0.64%) were reported (Sithole et al. 1992). Fatty alcohols are long-chain alcohols with 16 to 24 carbon atoms. In Norway spruce, the content of fatty alcohols was 0.03% to 0.04%, with behenol (C22) and lignocerol (C24) representing the major components (Ekman 1979). Waxes are esters of long-chain fatty acids with fatty alcohols. The content of waxes in wood is usually quite low with a few exceptions like Ceroxylon andicola (wax palm) which can yield as much as 25 pounds of waxes from a single tree (Kramer and Kozlowski 1960). In Norway spruce and Scots pine wood, the wax content is 0.08% to 0.09% (Fengel and Wegener 1984). Some hardwoods like Populus species may contain higher contents of waxes and other unsaponifiables (Mutton 1958; Allen 1977; 1988; Chen et al. 1995). 1.4 Degradation of lipophilic substances by fungi and other organisms Among the above reviewed lipophilic substances found in wood, triglycerides, fatty acids, sterols and steryl esters are also present in fungal cells (Weete 1980), while resin acids 22 naturally are not detected in fungi. Sterols are one of the major components of fungal membranes, and may regulate the membrane permeability. The functions of steryl esters remain largely unknown, but they could be involved in the intracellular sterol transport (Weete 1980). From the nutritional standpoint, triglycerides and fatty acids are the most important lipids to fungi. Therefore, in this section the degradation process of triglycerides and fatty acids is discussed. Triglycerides represent the major lipid storage materials in fungal cells, which can be used for generating energy and carbon skeletons during fungal growth and development (Weete 1980; Griffin 1994). The catabolism of triglycerides begins with the action of lipases which hydrolyze the ester bonds releasing the three fatty acids and glycerol. The glycerol is converted with the input of ATP to ct-glycerolphosphate, which is then oxidized by N A D + to dihydroxyacetone phosphate that is coupled to glycolysis (Salisbury and Ross 1992). The fatty acids generated from triglyceride hydrolysis are subject to degradation through several pathways, of which the most important and widely distributed is P-oxidation (Weete 1980). The P-oxidation pathways comprise the same chemical reactions in all organisms. However, the proteins and enzymes involved in the catalysis vary greatly. In eukaryotic cells P-oxidation has been detected in mitochondria and/or in the microbodies of peroxisomes and glyoxysomes (Greville and Tubbs 1968; Lazarow and de Duve 1976). These two organellar systems of P-oxidation are clearly distinguished by at least two features. They differ by the type of enzyme which catalyzes the first reaction of the P-oxidation cycle, and by the structural organization of 23 the other enzymes. The mitochondrial (3-oxidation cycle starts with an acyl-CoA dehydrogenase-catalyzed reaction, while the peroxisomal counterpart depends on an acyl-CoA oxidase (Beinert 1962; Shimizu et al. 1979; Coudron et al. 1983; Kirsch et al. 1986). Moreover, in mitochondrial (3-oxidation systems the enzymes are discrete and separable protein entities, while peroxisomal (3-oxidation systems contain multifunctional proteins (Kunau et al. 1988; Hashimoto 1990; Schulz 1990). P-oxidation results in the successive removal of two carbon units from an acyl-CoA substrate derived from fatty acids. Acetyl-CoA is produced, which can be oxidized to C 0 2 and H 2 0 in mitochondria via the tricarboxylic acid (TCA) cycle. The complete oxidation of one acetyl-CoA molecule via the T C A cycle will generate 12 ATP molecules. 1.5 Fungal lipases Lipases (glycerol ester hydrolases, EC 3.1.1.3) are the first enzymes involved in triglyceride degradation. Lipases from many fungi, particularly Aspergillus, Candida, Geotrichum, Mucor, Penicillium, and Rhizopus have been studied extensively in the last three decades (see reviews by Brockerhoff and Jensen 1974;'Macrae 1983; Borgstrom and Brockman 1984; Antonian 1988; Lazar and Schroder 1992; Wolley and Petersen 1994). 24 1.5.1 Secretion of lipases by fungi Fungi typically grow by means of filamentous hyphae which extend only at their apices and form mycelia. By extending at their apices, hyphae can penetrate solid substrates such as wood. The success of fungi as heterotrophic organisms lies in their ability not only to scavenge readily available nutrients and invade new substrata but also to break down insoluble macromolecules or polymers into components which are small enough to be transported into the cells as nutrients (Das et al. 1979; Wood 1985; Eriksson et al. 1990; Lazar and Schroder 1992). Fungi can secrete numerous enzymes, e.g., lipases, to degrade the extraneous macromolecules. Secretion of extracellular lipases by fungi is influenced by nutritional and environmental factors. Most studies concerning lipase secretion have been performed with liquid cultures, in which the amount of lipase secreted depends on cultivation temperature, pH, nitrogen forms, carbon and lipid sources, concentration of inorganic salts and the availability of oxygen. As an example, for optimal production of extracellular lipases, Rhodotorula glutinis requires ammonium phosphate as a nitrogen source, palm oil as a carbon source, an initial medium pH of 8.0, and a growth temperature of 30°C (Papaparaskevas et al. 1992). For many fungi, lipase production is inducible, and the secretion is enhanced when oils or other lipids are present in the media (Iwai et al. 1973; Pal et al. 1978; Sztajer and Maliszewska 1989; Samad et al. 1990; Papaparaskevas et al. 1992; Ohnishie/a/. 1994a). 25 The synthesis and processing of extracellular enzymes begins in the lumen of the endoplasmic reticulum where post-translational processes are initiated. Vesicles carry molecules to the Golgi system, or its equivalent, where the processing continues. Vesicles and/or vacuoles then transfer the proteins to the tip of the growing hyphae where they fuse with the plasma membrane, releasing their contents into the periplasmic space. Enzymes released in the periplasmic space may be incorporated into the cell wall, or may be secreted across the cell wall into the external medium (Wessels 1993). Immuno-electron microscopy showed the presence of lipases in vesicles, periplasm, and cell wall of Rhizopus microsporus and Penicillium cyclopium (Diyorov et al. 1994; Abbadi et al. 1995). The cell bound lipase was shown to be identical to the extracellular lipase (Abbadi et al. 1995). The exact location for enzyme secretion across the cell wall has not yet been completely elucidated. Most secreted enzymes have a size larger than the pore size of hyphal walls (Trevithick and Metzenberg 1966; Money 1990). For example, the molecular weight of extracellular fungal lipases ranges from 21 to 360 kDa (Ishihara et al. 1975; Shaw et al. 1989). The paradox was highlighted by Chang and Trevithick (1974), who pointed out that ribonuclease, the smallest exoenzyme secreted by Neurospora crassa was 13.7 kDa, yet isolated walls do not permit penetration of polymers with molecular masses greater than 4.75 kDa. The authors suggested that the passage of extracellular enzymes is likely to occur more easily through the more plastic and porous nascent cell wall in the hyphal apex. It is implicit in this hypothesis that the porosity of the hyphal apex does not represent that of the wall of 26 mature mycelia. As the apical wall develops, its structure becomes less porous, causing some exoenzymes to be trapped in transit and thus become bound to the wall. 1.5.2 Characteristics oflipase reactions and factors influencing lipolysis The natural substrates of lipases are triglycerides, monoglycerides, or diglycerides of long-chain fatty acids. These compounds have no or very low solubility in water. Lipases are characterized by their ability to rapidly catalyze the hydrolysis of ester bonds at the interface between an insoluble substrate phase and an aqueous phase in which the enzyme is dissolved (Brockerhoff 1968; Entressangles and Desnuelle 1974). Although lipases can catalyze the hydrolysis of a wide range of water-insoluble fatty-acid esters, triglycerides are normally the best substrates. The hydrolysis of water-soluble carboxylic acid esters by many lipases is usually very slow. The ability of lipases to catalyze hydrolysis of insoluble fatty acid esters distinguishes them from esterases (EC 3.1.1.1), which are highly active toward short-chain fatty acid esters or water soluble molecules (Petersen and Drablos 1994). The occurrence of the lipase reaction at an interface between the substrate and aqueous phases causes difficulties in the assay and kinetic analysis of the reaction. In such cases, normal Michaelis-Menten kinetics does not apply, since factors affecting the amount or properties of the interface between the two phases will affect the reaction rate (Patkar and Bjorkling 1994). For example, partial glycerides and free fatty acids, which are formed during lipase reactions, are surface active and tend to accumulate at the interface. This may prevent access of the lipase 27 to substrate (Yamaguchi et al. 1985; Markweg-Hanke et al. 1995). Metal ions can have a pronounced effect on lipolysis by influencing the ionization of the fatty-acid product (Muraoka et al. 1982; Muderhwa et al. 1985). In particular, calcium ions often stimulate the reaction by removing, as calcium soaps, the inhibitory fatty-acid anions (Iwai et al. 1964; Yamaguchi et al. 1985). Lipases from most mesophilic fungi are reasonably stable in neutral solutions at room temperature, assuming that there is no proteolytic degradation. Most fungal lipases lose activity at temperatures above 40°C, but some, especially those secreted by thermophilic fungi, are more resistant to heat inactivation. For example, the thermophilic fungus Humicola lanuginosa secretes a lipase which is stable at 60°C (Liu et al. 1973a). In general, lipases from different fungi have a broad pH-activity profile, showing highest activity between pH 5 and 9. The optimum pH for lipase activity has been reported as 5.6 for Aspergillus niger (Fukumoto et al. 1963), 6.3 for Geotrichum candidum (Tsujisaka et al. 1973), and 8.0 for Humicola lanuginosa (Liu et al. 1973 a). This may reflect the natural adaptation of fungi to their growing environments. Some fungi have been reported to produce several lipases with different pH optima (Iwai and Tsujisaka 1974; Iwai et al. 1975). 1.5.3 Structural features and inhibition of fungal lipases Since the first complete amino acid sequence of a lipase was reported by de Caro et al. (1981), at least 50 lipase sequences had been published by 1993 (Derewenda and Sharp 1993). 28 Although the degree of sequence homology between different lipases is very low, practically all known lipase amino acid sequences share the so-called consensus pentapeptide Gly-X-Ser-X-Gly (where X represents any amino acid). This consensus sequence is also observed in esterases, and has frequently been named as the substrate-binding site (Svendsen 1994). Crystallographic studies of the structures of the lipases from Geotrichum candidum (Schrag et al. 1991; Schrag and Cygler 1993), Rhizomucor (formerly called Mucor) miehei (Brady et al. 1990; Derewenda and Derewenda 1992), Pseudomonas glumae (Noble et al. 1994) and human pancreas (Winkler et al. 1990) revealed that these enzymes share a very similar topology in their mixed P sheet cores, with the catalytic sites buried under one or more surface helical loops (or lids). The active sites of these lipases possess a triad of residues similar to the catalytic triad in serine proteases. In the lipases of Rhizomucor miehei, Pseudomonas glumae, and human pancreas, the triad contains Ser-His-Asp (Brady et al. 1990; Winkler et al. 1990; Noble et al. 1994), and in Geotrichum candidum lipase the triad contains Ser-His-Glu (Schrag etal. 1991). The most common active-site-directed inhibitors of proteases are reactive phosphorus compounds. They react with the active-site serine, forming a stable transition-state-like complex (Salvesen and Nagase 1989). Similarly, phosphorus-containing compounds such as diethyl /?-nitrophenyl phosphate (E600), «-hexylphosphonate ethyl ester, bis-p-nitrophenyl phosphonate, and 3-guanidopropyl /?-riitrophenyl methyl phosphonate have been used as inhibitors for lipases. A breakthrough in understanding the lipase-substrate relationship was the 29 resolution of the crystal structures of Rhizomucor miehei lipase complexed with the inhibitors ft-hexylphosphonate ethyl ester (Brzozowski et al. 1991) and diethyl />-nitrophenyl phosphate (Derewenda et al. 1992). It was found that the active site is exposed by movement of a helical lid, and that the inhibitor is linked covalently to the active site serine. This movement also increased the hydrophobicity of the surface surrounding the catalytic site, strengthening the interactions with the lipid substrates. This structure of the enzyme-inhibitor complex was suggested to be equivalent to the activated state generated by the oil-water interface. 1.5.4 Substrate specificities of fungal lipases Fungal lipases can show specificity with respect to either the fatty acyl (fatty acid specificity) or alcohol (positional specificity) parts of their substrates. Iwai et al. (1980) reported that Penicillium cyclopium lipase is more active on short-chain triglycerides than on long-chain triglycerides. Lipases from Aspergillus niger and Rhizopus delemar display a high activity toward triglycerides of medium chain length, whereas Geotrichum candidum lipase is fairly specific for triolein. Some fungi produce two or more extracellular lipases with different fatty acid specificities. Nagaoka and Yamada (1973) purified two extracellular lipases from Mucor lipolyticus which differed in both their substrate specificities and physical properties. The lower-molecular-weight enzyme catalyzed the hydrolysis of triglycerides of medium- and long-chain fatty acids but showed almost no activity with tributyrin, while the higher-molecular-weight lipase catalyzed the hydrolysis of tributyrin in addition to the medium- and long-chain fatty acid triglycerides. Sugihara et al. (1994) purified four lipases from 30 Geotrichum candidum, and found that lipase I showed high activity on tricaprylin and triolein while lipase III was less active on the same substrates. Fungal lipases can be divided into two groups on the basis of their positional specificity. The first group, such as lipases from Geotrichum candidum and Penicillium cyclopium (Okumura et al. 1976), is non-specific and releases fatty acids from all three ester linkages of the glycerol moiety. These lipases catalyze complete breakdown of triglycerides to free fatty acid and glycerol, although 1,2 (2,3)-diglycerides, 1,3-diglycerides and monoglycerides appear as intermediates (Figure 1.5 a). The second group, such as lipases from Aspergillus niger, Humicola lanuginosa, Pyihium ultimum, and Rhizopus delemar (Okumura et al. 1976; Omar et al. 1987; Mozaffar and Weete 1993), releases fatty acids only from the outer 1 and 3 positions of the glycerol moiety. Under the action of such lipases, triglycerides are hydrolyzed to free fatty acids, 1,2 (2,3)-diglycerides and 2-monoglycerides (Figure 1.5b). However, 1,2 (2,3)-diglycerides and especially 2-monoglycerides are chemically unstable and undergo acyl migration to form 1,3-diglycerides and 1-monoglycerides, respectively. Therefore, under prolonged incubation, complete breakdown of triglycerides can take place with the formation of glycerol and fatty acids (Macrae and Hammond 1985). As well as fatty acid specificity and positional specificity, some lipases may show specificity toward the types of glycerides. Several lipases which are strictly specific to mono- and diglycerides have recently been purified from Penicillium camembertii (Yamaguchi and Mase 31 (a) O 0 C H 2 O C R C H 2 O H +3 H 2 O 3 R C 0 0 H + HOCH - 3 H 2 0 RCOCH O C H 2 O C R C H 2 O H Triglyceride Fatty acid Glycerol o 0 C H 2 O C R RCOCH 0 CH 2 OCR (b) O C H 2 O H + H 2 O - H 2 0 ^ RCOCH O CH 2 OCR Q C H 2 O H + H 2 O 11 + RCOOH •» RCOCH + 2 RCOOH - H 2 0 C H 2 O H 1,2(2,3)-Diglyceride 2-Monoglyceride Figure 1.5 Hydrolysis of triglycerides by lipases (Macrae and Hammond 1985). (a) Reaction catalyzed by non-specific lipases, (b) Reaction catalyzed by 1,3-specific lipases. 32 1991; Isobe et al. 1992), Aspergillus oryzae (Toida et al. 1995), and Fusarium sp. (Mase et al. 1995a). 1.6 Research objectives Although much research and technology development have been conducted on chemical protection of lumber against sapstain, maintaining future sales and exports requires that effective and non-toxic (to human and animals) control methods be developed. Future control strategies against sapstain may target either fungal growth or fungal pigmentation pathways, including using biological control agents. A thorough understanding of the organisms responsible for sapstain and their physiological and biochemical features would facilitate the development of biorational and environmentally friendly approaches. The Ophiostomatoid fungi are of considerable economic importance. Besides the saprophytic species which cause wood sapstain problems, there are many pathogenic species causing diseases in trees and field crops, of which the Dutch elm disease (caused by Ophiostoma ulmi) is an example. Through over a century of research, although abundant information has been obtained on the taxonomy, ecology, and pathogenicity of Ophiostomatales (Wingfield et al. 1993), work conducted on the physiology of these fungi has been sparse, with most comprehensive investigations being performed on the pathogenic species of 0. ulmi (Holmes and Heybroek 1990; Sticklen and Sherald 1993). A detailed study on the physiology of the saprophytic sapstaining Ophiostomatales species was conducted by Kaarik in the 1950s and 33 1960s with isolates predominantly from Swedish forest products. The work examined the carbon, nitrogen and vitamin requirements for growth and sporulation of 18 different species of Ophiostoma in artificial media (Kaarik 1960). Little work has been conducted on the physiology of sapstaining fungi when they grow in solid substrates such as wood. Five years ago, with the creation of the NSERC/Industrial chair of Forest Products Biotechnology at the University of British Columbia, a research program was initiated on the physiology and biochemistry of the Ophiostomatales species and strains frequently isolated from Canadian forest products. Studies have been carried out on the characterization of available nitrogen nutrients in wood, and on the biochemistry and enzymology of nitrogen utilization by Ophiostomatales species, especially O. piceae. In sapwood, nitrogen is mainly in the form of proteins and peptides (Abraham and Breuil 1993). To retrieve nitrogen, sapstaining fungi secrete various proteinases and aminopeptidases (Abraham et al. 1993; Breuil and Huang 1994; Breuil et al. 1995). A subtilisin-like proteinase produced by O. piceae was characterized, and its functions elucidated (Abraham et al. 1995; Abraham and Breuil 1995a; 1996). Immunogold labeling of the proteinase revealed that the enzyme was secreted into the cell wall and released in a- sheath surrounding the hyphae when the fungus grew in wood or in liquid media (Hoffert et al. 1995; Gharibian et al. 1996). As an integrated part of the research program investigating the nutritional physiology of sapstaining fungi, the present project was aimed at generating information on the utilization of lipophilic carbon nutrients by these fungi. Sapstaining fungi have been called non-cellulolytic 34 'sugar' fungi, as it was believed that these organisms utilized mainly the more readily assimilable carbon compounds (soluble sugars and starch) present in wood (Hudson 1986). As reviewed in previous sections of this chapter, most wood contains 2% to 6% total lipophilic substances,- which are composed mainly of a variety of lipids, including triglycerides and fatty acids. The information on the ability of sapstaining fungi to degrade and utilize wood lipids as carbon sources for growth was limited. Pioneer investigations by Blanchette and his colleagues have shown that an albino strain of O. piliferum could decrease the total wood extractives and the esterified fatty acids in southern yellow pine wood chips (Blanchette et al. 1992b; Brush et al. 1994). However, no information was available on the secretion and properties of lipolytic enzymes produced by the sapstaining Ophiostomatales. The objectives of this thesis were to determine the major lipids in wood, to examine the biodegradation and utilization of the lipids by Ophiostoma, and to characterize one of the key enzymes, lipases, involved in lipid degradation. Several Ophiostoma species were selected as model sapstaining fungi. Particularly, O. piceae (Munch) H. & P. Sydow was used in several experiments since it was most frequently isolated from Canadian lumber mills (Seifert and Grylls 1990). Lodgepole pine, one of the most important wood species harvested in BC, was chosen as the major wood material because little information was available about the lipophilic extractives of this species. Trembling aspen was also used in some experiments since it is being increasingly utilized in wood composite products and paper manufacturing in Canada, and it can be subject to heavy sapstain. 35 The results of this thesis are presented in four separate chapters: (1) Identification and quantification of the major lipids in lodgepole pine wood, including the contents and composition of glycerides, fatty acids, and resin acids, and the distribution across the log cross section (chapter 2 ) . (2) The changes of major lipids in lodgepole pine and trembling aspen sapwood during the colonization by three Ophiostoma species (chapter 3) . (3) The effects of lipids, carbohydrates and nitrogen compounds on the growth and extracellular lipase production of O. piceae in liquid culture, purification of one major lipase, and characterization of the enzyme properties (chapter 4 ) . (4) Examination of the factors affecting the activity of the lipase purified from O. piceae, its substrate specificities, and the hydrolysis of the substrates from both synthetic and natural sources by the lipase (chapter 5) . 36 Chapter 2 Identification and Quantification of Lipids in Pinus contorta var. latifolia wood 2.1 Introduction Lipids in wood are commonly extracted with neutral organic solvents, such as petroleum ether, acetone, benzene, ethanol, dichloromethane, or their mixtures. The extracts, commonly called wood extractives, contain glycerol esters (mono-, di- and triglycerides), fatty acids, resin acids, sterols, steryl esters, waxes, fatty alcohols, and volatile compounds (Sjostrbm 1981). Lipophilic extractives have been studied extensively in some wood species, such as Pinus sylvestris (Yildirim and Holmbom 1977a; Hafizoglu 1983; Saranpaa and Nyberg 1987a, b; Fischer and Holl 1992; Lange and Janezic 1993), Pinus brutia (Yildirim and Holmbom 1977c; Hafizoglu 1983), Pinus nigra (Yildirim and Holmbom 1977a, b; Hafizoglu 1983), Pinus elliottii (Quinde and Paszner 1991; 1992), Pinus tabulaeformis (Weissmann and Lange 1988), Picea abies (Ekman 1979; Holl and Goller 1982; Lorbeer and Zelman 1988), Cedrus libani (Hafizoglu 1987, Hafizoglu and Holmbom 1987a, b), Platycladus orientalis (Chen et al. 1984) and Populus tremuloides (Yanchuk et al. 1988; Sithole et al. 1992). Lodgepole pine (Pinus contorta Dougl. var. latifolia Engelm.) is a major commercial softwood species in western North America. It is distributed from Alaska to Baja, California, and from the Pacific Coast to the eastern edge of the Rocky Mountains (Elias 1980). It is the most 37 abundant commercial tree species in British Columbia, with an average annual log harvest of more than 18 million cubic meters from 1985 to 1994 (COFI 1995a). Research on lodgepole pine extractives was carried out in the 1960s and 1970s at the Forest Products Laboratory in Madison, Wisconsin (Rowe and Scroggins 1964; Rowe 1965; Rowe et al. 1972) and at the University of California at Berkeley (Anderson et al. 1969). Some sterols and terpenes were identified from the bark (Rowe 1965; Rowe et al. 1972), and some fatty and resin acids were determined in the wood (Anderson et al. 1969). In the 1980s, Kim (1988) determined the contents of total wood extractives in lodgepole pine trees of different ages at various latitudes and elevations. However, the differences in the contents of total wood extractives and the chemistry of major lipid classes between the sapwood and heartwood of lodgepole pine remain poorly defined. The cross section of a lodgepole pine log contains sapwood, which is darker in color, high in moisture content, and contains some living cells, as well as heartwood, which is lighter in color, low in moisture content, and consists of all dead cells. The sapwood of freshly sawn lodgepole pine is susceptible to fungal stain. Determination of the quantity and distribution of the lipophilic substances in the wood may provide information on the potential lipid nutrients available for sapstaining fungi. The objective of this chapter was to obtain detailed information on the distribution, quantity and the nature of non-volatile lipophilic extractives in freshly cut lodgepole pine wood1. 1 The results were published: Gao, Y., T. Chen and C. Breuil. 1995. Identification and quantification of nonvolatile lipophilic substances in fresh sapwood and heartwood of lodgepole pine (Pinus contorta Dougl.). Holzforschung 49:20-28. 38 2.2 Materials and Methods 2.2.1 Wood sample Two lodgepole pine trees were felled in November 1991 at the Alex Fraser Forest Research Centre (located near Gavins Lake, BC) of the University of British Columbia. At the breast height of the stem, tree A was 35 years old (based on the number of growth rings) and 18 cm in diameter. Heartwood represented about 37% of the cross section area. Tree B was 45 years old, the trunk diameter was 25 cm at breast height and the proportion of heartwood was about 32%> of the cross section area. After felling, a 1.6 m log was debarked, cut into uniform small blocks (30x10x5 mm), and frozen at -20°C. The samples were stored separately, according to the longitudinal and horizontal positions within the log (Figure 2.1). 2.2.2 Extraction of wood lipophilic extractives Frozen wood blocks were ground to fine sawdust powder (40 mesh) in liquid nitrogen with a Micro-Mil l (Bel Arts, Pequannock, NJ, USA). The sawdust was stored at -20°C. The sample moisture content was determined gravimetrically after drying at 105°C overnight. The moisture contents of the sapwood and heartwood were 108% and 48% (based on od weight), respectively. About 1.5 g of fresh wood powder was extracted in a Soxhlet apparatus (Pyrex brand, V W R Scientific of Canada Ltd., Toronto, Canada) with 120 ml acetone for 8 h at the rate of 6-8 syphonings per hour. No increase in the extractive amount was found with 39 160cm Figure 2.1 Diagram of wood sampling. The 1.6 m long log was divided into four sections in the longitudinal direction (I, II, III, and IV), and six sections in the horizontal direction (OS: outer sapwood, MS: middle sapwood, IS: inner sapwood, OH: outer heartwood, M H : middle heartwood, IH: inner heartwood). 40 extracting beyond 8 hours. After extraction, the solvent was evaporated in a rotary evaporator (Model: R E 51, Brinkman Instruments Canada Ltd., Rexdale, Ontario, Canada) at 35°C. The extract in each flask was re-dissolved in 6x2 ml chloroform. The chloroform solution was run through an anhydrous sodium sulfate cartridge column to remove trace amounts of water and possible solid particles. The eluate was saved in a clean, pre-tared bottle, then evaporated to dryness under a stream of N 2 and weighed to determine the total extractives. A l l extractive determinations were repeated three to six times. 2.2.3 Solid-phase extraction (SPE) SPE was used to isolate the different nonvolatile lipid classes. This method, originally developed by Kaluzny et al. (1985) to separate the lipid classes from an extract of adipose tissue, was modified by Chen et al. (1994) to isolate the lipid classes from acetone extracts of wood and pulp. The latter protocol was used in this study with slight modifications. Total extractives (10-15 mg per batch) were loaded onto a Bond Elut aminopropyl silica cartridge column (bed vol. 3 ml, Analytichem International and Varian, Harbor City, CA, USA). Each lipid class was successively eluted with a sequence of solvent combinations (Figure 2.2). Each fraction was collected in a pre-tared clean bottle, and dried under a stream of N 2 gas, and weighed with an accurate electronic balance (accuracy 0.01 mg). Using this technique the following fractions were isolated and quantified: (1) fatty and resin acids; (2) steryl esters and waxes; (3) triglycerides; (4) sterols, fatty alcohols, and diglycerides; (5) monoglycerides; and (6) other high polar compounds. B Fatty and resin ac ids ( 2 ) Steryl esters and waxes ( 3 ) Tr iglycerides (T) Sterols, fatty alcohols, & diglycerides (IT) Monoglycer ides ( ? ) Others C H rVi N H , © Elution Solvents: A : Chloroform : hexane (1:5) B: E t h e r : hexane (8:1) C : E t h e r : acet ic acid (98:2) D: Hexane E: Ether F: E t h e r : hexane (1:4) G : E t h e r : methanol (2:1) H: Ace tone or methanol Figure 2 .2 Isolation of lipid classes from wood extractives by solid phase extraction. Columns with NH 2 labels are new columns. 1-6 are different classes of compounds; A ~ H are solvents used to elute each class. Fraction 6 can not be completely eluted from the column. 42 2.2.4 Thin layer chromatography (TLC) T L C was used to monitor the separation and the purity of the lipid classes during the SPE process. Samples were spotted on silica gel 60 F 2 5 4 pre-coated plates (thickness: 0.2 mm, Merck, Germany), which were then developed with a solvent mixture of hexane:diethyl ethenacetic acid (70:30:1, v/v) and air dried for a few minutes. The separated spots were visualized after spraying with molybdate oxidizing solution (prepared by dissolving 5 g ammonium molybdate in 10 ml concentrated sulfuric acid and 90 ml ethanol) and heating the plate at 105°C. Representative standards used were: cholesteryl linoleate; oleic acid oleyl ester, triolein, oleic acid, dehydroabietic acid, linolenyl alcohol, diolein, stigmasterol, and monoolein. 2.2.5 Gas chromatography (GC) and combined gas chromatography-mass spectrometry (GC-MS) A Hewlett Packard HP5890 Series II gas chromatograph (Hewlett-Packard Co., Palo Alto, CA, USA), equipped with a flame ionization detector (FLD) and an HP7673 automatic sampler, was used. Methylated fatty and resin acids were separated on a DB-5 fused silica capillary column (length: 30 m; diameter: 0.25 mm; J & W Scientific, CA, USA). The temperature program started at 150°C and was held for 3 min, then raised at 1.2°C/min to 170°C, then 0.6°C/min to 190°C, and finally increased at 3.3°C/min to 270°C. The total 43 ranning time was 77.24 min. The detector temperature was set at 290°C. Helium was used as the carrier gas at a flow rate of 1.4 ml/min, and the split ratio was 71.4:1. The injection volume was 1.0 ul. The equipment performance, data collection and processing were controlled and carried out by the Hewlett-Packard software HP3365 Series II. The mass spectra were obtained using the same gas chromatograph equipped with a VG-TR10 1000 mass selective detector. Authentic fatty acids and resin acids as methyl esters were used as references for identification and calibration. 2.2.6 Methylation Fatty and resin acids were methylated with fresh diazomethane in diethyl ether solution, which was prepared by reacting Diazald (Aldrich Chemical Co., Milwaukee, MI, USA) with NaOH in a mixture of water, ethanol and diethyl ether, in a sealed flask (Chen et al. 1994). The diazomethane released in the reaction was bubbled into a cold diethyl ether solution with a nitrogen carrier gas stream. Three ml of the diazomethane in diethyl ether solution and one drop of anhydrous methanol (as catalyst) were added to the dried fatty and resin acid fraction (1-1.5 mg). After 10 min, while the color of the solution was still yellow, the diethyl ether was gently evaporated under a stream of N 2 gas, and the fatty and resin acid methyl esters were re-dissolved in 2 ml of ethyl acetate. The sample solutions were kept at -20°C and analyzed by GC or GC-MS within two weeks. 44 2.2.7 Saponification Triglycerides, steryl esters and waxes were saponified according to the procedure of Saranpaa and Nyberg (1987b). The eluted SPE fractions (4-12 mg triglycerides or 1.5-4 mg steryl esters and waxes) were heated in 5 ml 0.5 M K O H dissolved in 90% ethanol for 3 h at 70°C in a water bath. Afterwards, the solutions were diluted with one volume distilled water and acidified to pH 2.0 with 1 M HC1. The saponification residues were extracted with 3x20 ml hexane:diethyl ether (1:1, v/v), then dried under a stream of N 2 gas and methylated with diazomethane in diethyl ether solution. 2.2.8 Reagents Pentadecanoic acid (15:0) (99%), palmitic acid (16:0) (99%), margaric acid (17:0) methyl ester (99%), stearic acid (18:0) (99%), oleic acid (18:1, cis-9) (99%), linoleic acid (18:2, cis-9, 12) (99%), linolenic acid (18:3, cis-9, 12, 15) (98%), arachidic acid (20:0) methyl ester (99%), eicosenoic acid (20:1, cis-11) (99%), eicosadienoic acid (20:2, cis-11, 14) (99%), behenic acid (22:0) methyl ester (99%), docosadienoic acid (22:2, cis-13, 16) methyl ester (99%), lignoceric acid (24:0) methyl ester (99%) were obtained from Sigma Chemical Co., St. Louis, M O , USA. Abietic acid (90-95%), dehydroabietic acid (99%), levopimaric acid (98%>), neoabietic acid (99%), sandaracopimaric acid (85-90%), palustric acid (90-95%), isopimaric acid (99%), and pimaric acid (85-90%) were obtained from Helix Biotech Corp. 45 Richmond, BC, Canada. Diazald (A^-methyl-A'-nitroso:p-toluenesulfonamide) (99%), ammonium molybdate (VI) (99.98%), and all solvents (in HPLC grade, >99.8%) were obtained from Aldrich Chemical Co. 2.3 Results 2.3.1 Total extractive content Preliminary investigations with trees A and B showed that, although the levels of extractives decreased with the increase in height in the four longitudinal sections along each log, the differences between the longitudinal sections of each log were not significant (tested by one-way A N O V A with the software 'Origin', Microcal Software, Inc., Northampton, M A , USA, a at 0.05 level). Similarly, no significant differences were found between the extractive levels in the sapwood of tree A (2.0%) and tree B (2.3%). In contrast, significant differences in extractive contents were found between the sapwood and heartwood of each tree. Following the preliminary examination, detailed analytical work was focused on section IV of tree A as shown in Figure 2.1. The wood extractive contents in Section IV of tree A are shown in Figure 2.3. Although the inner sapwood seemed to contain more extractives (2.2%) than the outer sapwood (1.9%), the difference was not significant (tested by one-way A N O V A with Origin, a at 0.05 level). However, five times more extractives were present in the heartwood, with I S O H Wood sections Figure 2.3 Content of total wood extractives in lodgepole pine wood. OS: outer sapwood; IS: inner sapwood; OH: outer heartwood; LH: inner heartwood. The growth rings that IH, OH, IS and OS contained were 1-4, 11-15, 18-24 and 30-35. A l l data points are means of six determinations with standard deviations shown by error bars. 47 concentrations varying from 12.4 ± 0.4% in the outer heartwood to 9.7 ± 0.8% in the inner heartwood. 2.3.2 Different lipid classes in the extractives A T L C chromatogram shows total extractives and their fractions separated by SPE (Figure 2.4). Table 2.1 lists the amounts of different lipid classes in each section of the wood, and the relative proportion of each class in the total extractives. Triglycerides (TG) in the sapwood represented 1.1% to 1.3% of od weight. The T G content decreased towards the center of the tree, to 0.6% to 0.7% in the heartwood. In contrast, the free fatty and resin acids, which were low in the sapwood (0.35% to 0.42%), increased more than 15 fold in the heartwood, reaching 5.4% to 6.7%. Minor amounts of steryl esters, waxes, sterols, fatty alcohols, diglycerides and monoglycerides were found in both sapwood and heartwood. Some unidentified yellowish substances, in the range of 0.2% in the sapwood and 2.8-4.2%) in the heartwood, were observed. Important differences were observed between the relative proportion of the different classes of lipids in the sapwood and heartwood. In the sapwood, the most abundant fraction was the triglycerides (58%> of the total extractives), followed by the free fatty and resin acids (19%) and the colored components noted above (11-13%). In the heartwood, however, the free fatty and resin acids were the dominant fraction (54-56%), followed by the colored components (28-34%). 48 SE, FE - m m TG -• * FA, RA - • Fal, DG • S- f M G - L L .n Solvent front Mix A B C D E F G Figure 2.4 Thin layer chromatogram showing the total extractives and fractions after each step of solid phase extraction. SE: steryl esters; FE: waxes; TG: triglycerides; FA: fatty acids; RA: resin acids; Fal: fatty alcohols; DG: diglycerides; S: sterols; M G : monoglycerides; Mix: total extractives; A ~ G : eluted fractions as shown in Figure 2.2. 49 Table 2.1 Content of different lipid classes in lodgepole pine wood and their relative proportions to the total amount of lipophilic extractives. OS1" IS OH EH Class'1' Content8 Proportion* Content Proportion Content Proportion Content Proportion (mg/g) (%) (mg/g) (%) (mg/g) (%) (mg/g) (%) FA+RA 3.50 ±0 .55 18.5 4.20 ± 0.06 19.3 66.83 ± 10.1 54.1 53.84 ± 3 . 0 9 55.5 SE+FE 1.52 ± 0 . 3 7 8.0 2.14 ± 1.04 9.8 2.54 ±0 .21 2.1 3.31 ± 1.96 3.4 T G 11.03 ±0 .41 58.3 12.76 ± 0 . 5 8 58.5 7.27 ±0.11 5.9 5.77 ± 0 . 4 3 6.0 S+DG+Fal 0.22 ±0 .01 1.2 0.27 ± 0 . 1 0 1.2 1.02 ± 0 . 7 7 0.8 0.76 ± 0.08 0.8 M G 0.22 ±0 .01 1.2 0.15 ± 0 . 0 4 0.7 3.93 ± 2 . 5 7 3.2 5.34 ± 2 . 7 7 5.5 Others 2.45 ±0 .91 12.9 2.29 ±0 .84 10.5 42.05 ±6 .31 34.0 27.93 ± 1 1 . 4 28.5 f Wood sections are: OS: outer sapwood; IS: inner sapwood; OH: outer heartwood; IH: inner heartwood. X FA: fatty acids; RA: resin acids; SE: steryl esters; FE: waxes; TG: triglycerides; S: sterols; DG: diglycerides; Fal: fatty alcohols; M G : monoglycerides, Others: mainly phenolic and colour substances. § Based on oven-dry weight. A l l data points are means of six determinations with standard deviations shown by uncertainties. * Proportion refers to the relative percentage (%) of each class to the total extractive amount. 50 2.3.3 Fatty acid composition in wood triglycerides After isolation by SPE, the T G fraction was saponified to release the fatty acid residues, which were extracted, methylated and analyzed by GC. While great differences in fatty acid (FA) composition were noted between the sapwood and the heartwood, no major differences were observed between the outer and inner sapwood, or between the outer and inner heartwood (Table 2.2). The most abundant F A residues in the sapwood T G fraction were oleic (18:1) and linoleic (18:2), which represented 31-35% and 36-37% of the total F A residues, respectively. Together, they accounted for about 67% to 72% of the total residues. The other F A residues were linolenic (18:3) and palmitic (16:0). Minor amounts of stearic (18:0), eicosenoic (20:1) and eicosadienoic (20:2) acids were also detected (less than 2% each). The major F A residues in the heartwood T G fraction were 18:2 (30%), 18:1 (18%) and 18:3 (12%). Between 33% to 36 %> of the acid residues in the saponified components of the heartwood T G appeared not be common FAs, and were not identified (Table 2.2). 51 T a b l e 2.2 The composition (%) of fatty acid residues in the triglycerides of lodgepole pine wood. Sapwood Heartwood Abbr. Fatty acid Retention time (min) Outer* Inner* Outer* Inner* 16:0 Palmitic 22.914 4.1 ±0 .7 4.3 ±0 .3 2.1 ±0 .3 2.3 ± 0 . 4 17:0 Margaric 27.538 1.2±0.1 1.8 ± 0.1 nd1" 0.6 ± 0 . 4 18:0 Stearic 37.654 1.5 ±0 .6 1.4 ±0 .5 nd nd 18:1 Oleic 35.265 35.0 ±2 .7 30.7 ± 1.5 18.4 ± 0 . 7 18.9 ± 2 . 8 18:2 Linoleic 34.708 37.4 ± 1.8 35.6 ± 1.3 29.4 ± 1.2 30.7 ±3 .3 18:3 Linolenic 33.109 9.9 ± 1.1 13.5 ±0 .1 11 .6±0 .8 12.6 ± 2 . 5 20:1 Eicosenoic 53.804 0.3 ± 0 . 2 0.3 ± 0 . 2 nd nd 20:2 Eicosadienoic 53.196 1.8 ±0 .1 1.9 ±0 .1 2.5 ± 0 . 1 2.0 ± 1.5 Others* Unknown 9.2 ± 2 . 0 10.6 ± 2 . 4 36.1 ± 3 . 5 33.1 ± 13 t nd: not detected. X Others refer to unidentified components. * A l l data points are means of three determinations with standard deviations shown by uncertainties. \ 52 2.3.4 Fatty acid composition in wood steryl esters and waxes The F A composition of the steryl esters and waxes was determined by GC (Figure 2.5). The F A compositions in the outer and inner sapwood were similar, and so were those in the outer and inner heartwood, but large differences in quantity were observed between the sapwood and heartwood (Table 2.3). The 18:2 was the most common residue found in both sapwood (50-53%) and heartwood (27-28%), followed by 18:3, 18:1 and 16:0. However, nearly 50% of the acids in the saponified components of the steryl esters and waxes from the heartwood were not common fatty acids, and were not identified (Figure 2.5, Table 2.3). 2.3.5 Free fatty and resin acids Twelve free FAs and seven free resin acids (RAs) were identified from lodgepole pine wood (Figure 2.6). The relative composition of free FAs and free RAs in the total free lipophilic acid fraction varied in the different sections (Table 2.4). The content of free FAs increased from 0.02-0.03% (i.e., 0.2-0.3 mg/g od wood) in the sapwood to 0.8-1.1% in heartwood (Table 2.4). Table 2.5 shows the identity and the concentration of each individual free FA. Comparing the relative proportion of the various FAs in the different sections of the wood (Table 2.5), it was found that 18:2 and 18:1 were most abundant, accounting for 32-41% and 24-28% of the total F A content, respectively. 18:3 and 16:0 were also present in high amounts, while others, including 17:0, 18:0, 20:0, 20:1, 20:2, 22:0, 22:2 and 24:0 were found in trace amounts (Table 2.5). 53 3.0e4 H 2.0e4 H 1.0e4 A 20 ' ' ' 40 ' " ~60 Retention time (min) Figure 2.5 Gas chromatogram showing fatty acids in the saponified products of the steryl esters and waxes of lodgepole pine inner heartwood. The steryl esters and waxes fraction was obtained from SPE elution. A l l acids were detected as methyl ester derivatives. Column: DB-5. Peaks: 1 palmitic, 5 linolenic, 6 linoleic, 7 oleic, 15 eicosadienoic, 2, 3, 4, 8, 9, 10, 11, 12, 13, 14, 16: unidentified. 54 Table 2.3 The composition (%) of fatty acid residues in the steryl esters and waxes of lodgepole pine wood. Sapwood Heartwood Abbr. Fatty acid Retention time (min) Outer* Inner* Outer* Inner* 16:0 Palmitic 22.920 5.9 ±0 .1 5.6 ± 0 . 4 9.6 ± 1.3 7.6 ± 0 . 7 17:0 Margaric 27.526 0.7 ±0 .5 nd t nd nd 18:0 Stearic 37.642 0.5 ± 0 . 4 0.5 ±0 .3 nd nd 18:1 Oleic 35.270 16.3 ± 7 . 8 16.9 ± 6 . 9 5.5 ± 1.0 7.1 ±0 .6 18:2 Linoleic 34.714 52.5 ± 9 . 8 48.9 ±9 .3 25.6 ± 3 . 5 28.2 ± 1.6 18:3 Linolenic 33.102 18.2 ±5 .1 14.3 ± 1.9 9.7 ± 0 . 9 8.2 ± 0 . 1 20:1 Eicosenoic 53.797 nd 0.3 ± 0 . 2 nd nd 20:2 Eicosadienoic 53.201 0.7 ±0 .6 0.8 ± 0 . 6 nd nd Others* Unknown 5.2 ± 1.9 12.9 ± 7 . 8 49.6 ± 2 . 8 48.9 ± 3 . 2 f nd: not detected. $ Others refer to unidentified components. * A l l data points are means of three determinations with standard deviations shown by uncertainties. 55 2.0e4 A 1.6e4 A 1.2e4 A 8000 A 4000 A I2 |3 I5 I6 22 VAj IO 14 y 19 18 23 24 lOJJ La 2 0 4 0 6 0 Retention time (iriin) Figure 2.6 Gas chromatogram showing the free fatty and resin acids in the lodgepole pine sapwood. The free acid fraction was obtained from SPE elution. A l l acids were detected as methyl ester derivatives. Column: DB-5. Peaks: 1 palmitic, 2 margaric, 3 linolenic, 4 linoleic,'5 oleic, 6 stearic, 7-8 unidentified, 9 pimaric, 10 sandaracopimaric, 11 isopimaric, 12 palustric, 13 dehydroabietic, 14 arachidic, 15 abietic, 16 neoabietic, 17-19 unidentified, 20 docosadienoic, 21 behenic, 22-23 unidentified, 24 lignoceric. 56 Table 2.4 Contents of resin acids and fatty acids in the lodgepole pine wood and their relative proportion to the total amount of free lipophilic acids obtained by solid phase extraction. Sapwood Heartwood Outer Inner Outer Inner Content^  Proportion''' Content Proportion Content , Proportion Content Proportion (mg/g) (%) (mg/g) (%) (mg/g) (%) (mg/g) i Resin acids 3.2 91.6 3.8 90.4 55.8 83.5 40.1 74.5 Fatty acids 0.2 6.0 0.3 7.2 8.4 12.6 10.8 20.1 Unidentified 0.1 2.4 0.1 2.4 2.6 3.9 2.9 5.4 f Based on oven-dry wood; all data points are means of three determinations with standard deviations less than 5% of the means. X Relative proportion (%) to the total lipophilic acids. 57 Table 2.5 Content of various free fatty acids in lodgepole pine wood and their relative proportion to the total free fatty acids identified. Sapwood Heartwood Outer Inner Outer Inner Abbr. Fatty acid RT^ ug/g od wood* %^  ug/g od wood % ug/g od wood % ug/g od wood % 16:0 17:0 18:0 18:1 18:2 18:3 20:0 20:1 20:2 22:0 22:2 24:0 Palmitic Margaric Stearic Oleic Linoleic Linolenic Arachidic Eicosenoic 53.817 Eicosadienoic 53.181 Behenic 65.853 Docosadienoic 64.505 22.908 26.3 ±3.9 16.1 27.542 4.7 ±0.5 2.9 37.660 12.2 ±4.6 7.5 35.249 40.5 ± 3.6 24.9 34.701 52.9 ±7.0 32.5 33.219 11.2 ±1.4 6.9 56.283 8.1 ±3.4 5.0 nd^  nd nd nd nd nd nd nd Lignoceric 71.807 7.0 ± 0.9 4.3 31.9 ± 3.8 11.6 8.8 ±0.5 3.2 13.8 ±1.6 5.0 66.1 ±3.8 24.0 87.4 ±3.0 31.7 23.3 ±1.1 8.5 10.9 ±1.8 4.0 nd nd nd nd 17.3 ±1.1 6.3 7.2 ±0.9 2.6 8.8 ±0.8 3.2 290 ± 16 3.5 376 ±27 3.5 190 ± 5 2.3 255 ± 5 2.4 93 ±13 1.1 44 ±25 0.4 2274 ±43 27.5 3009 ±48 28.2 3050 ±66 36.9 4346 ± 63 40.7 1116 ±21 13.5 1496 ±68 14.0 125 ± 18 1.5 161 ±28 1.5 186 ± 16 2.3 43 ±61 0.4 640 ±77 7.7 578 ± 143 5.4 118 ± 9 1.4 190 ± 17 1.8 162 ±11 2.0 171 ±25 1.6 26 ± 8 0.3 nd nd f RT: retention time (min). * A l l data points are means of three determinations with standard deviations shown by uncertainties. X %: relative proportion to total fatty acids. § nd: not detected. 58 The content of RAs which was 0.3-0.4% in sapwood, increased sharply in the outer heartwood to 5.6% and then decreased slightly in the inner heartwood (Table 2.4). Seven common RAs were identified. Table 2.6 lists each individual free RA, its content and the relative proportion of each R A to the total R A amount. Palustric acid was the most abundant R A in freshly cut lodgepole pine wood. It accounted for 52-50% of the total RAs in the sapwood, 43%) in the outer and 29% in the inner heartwood, whilst the relative composition of abietic, dehydroabietic and isopimaric acids increased from the sapwood towards the heartwood (Table 2.6). 2.4 Discussion The identification and quantification of nonvolatile wood extractives was normally achieved by first extracting them with neutral solvents; second, separating the free fatty and resin acids from the neutral substances by ion exchange chromatography; third, saponifying the neutral fraction to hydrolyze the triglycerides and steryl esters into free acids which are separated from the unsaponifiables by ion exchange chromatography, and finally methylating the two acid fractions for detailed analysis by GC (Zinkel 1975; 1983). Although this method could provide information on the composition of the wood extractives, it is time consuming. Also the triglycerides could not be separated from other fatty esters by this procedure. To shorten the procedure, preparative TLC, short capillary columns and reverse phase high performance 59 Table 2.6 Content of various free resin acids in lodgepole pine wood and their relative proportion to the total free resin acids identified. Sapwood Heartwood Resin acids R.T1 uj Outer Inner Outer Inner >/g od wood* %* ug/g od wood % ug/g od wood % ug/g od wood % Pimaric 45.748 150 ± 1 4.7 213 ± 4 5.6 2902 ± 23 5.2 2926 ±95 7.3 Sandaracopimaric 47.273 40 ± 1 1.3 49 ± 1 1.3 771 ± 2 1.4 646 ± 21 1.6 Isopimaric 51.388 264 ± 2 8.3 355 ± 8 9.4 5459 ±55 9.8 5063 ±391 12.6 Palustric 53.335 1663 ±60 52.4 1918 ± 79 50.5 23501 ±1153 42.1 11548 ±2083 28.8 Dehydroabietic 55.743 224 ± 25 7.1 298 ± 47 7.9 6773 ± 312 12.1 5588 ±442 13.9 Abietic 58.575 459 ± 12 14.5 504 ± 9 13.3 9898 ± 972 17.7 9683 ±1258 24.1 Neoabietic 61.388 373 ±31 11.8 461 ±41 12.1 6505 ± 477 11.7 4668±1916 4.6 f RT: retention time (min). * A l l data points are means of three determinations with standard deviations shown by uncertainties. X %: relative proportion to total resin acids. 60 liquid chromatography (RP-HPLC) have been used with different degrees of success (Ekman 1979; Anas et al. 1983; Saranpaa and Nyberg 1987a; Suckling et al. 1990; Sithole et al. 1992). The current study adopted the SPE technique, which has recently received interest from pulp and wood researchers (Sweeney 1988; Backa et al. 1989; Chen et al. 1994). SPE is fast, it does not require expensive equipment, and allows the direct separation of wood extractives into major lipid classes (Chen et al. 1994). Another great advantage of SPE over conventional ion exchange chromatography in the analysis of wood extractives is that the triglycerides and fatty/resin acids, the two major classes of nonvolatile lipids, can be isolated rapidly and in high purity. Furthermore, each purified fraction is in its native state, no modification of the compounds occurs during the separation. This makes the further identification and quantification of the individual components in the isolated fractions more convenient and accurate. However, this procedure did not permit the analysis of extremely polar compounds such as phenolics, lignans or mono- and disaccharides that could also be extracted with aqueous acetone. Many factors have been known to affect the content of wood lipophilic extractives. For different wood species, the values reported in the literature vary from 1.5% to more than 10% of the dry weight (Holl and Poschenrieder 1975; Yildirim and Holmbom 1977a; Holmbom and Ekman 1978; Torul and Olcay 1984; Hafizoglu 1987; Hafizoglu and Holmbom 1987a; Yanchuk et al. 1988; Hafizoglu 1989; Sithole et al. 1992). Within a log, great variations have 61 also been observed between bark, sapwood and heartwood (Holl and Poschenrieder 1975; Yanchuk et al. 1988; Sithole et al. 1992). The results of this study also displayed this variation across the radius. Lodgepole pine heartwood contained higher levels of extractives than the sapwood. Similar observations have been reported for shortleaf pine (Posey and Robinson 1969), trembling aspen (Yanchuk et al. 1988), Cedrus libani (Hafizoglu 1987); Tilia cordata, Betula verrucosa and Robiniapseudoacacia (Holl and Poschenrieder 1975). It was found that the amount of TGs in lodgepole pine decreased from sapwood to heartwood, while FAs, RAs, monoglycerides, steryl esters and waxes increased. The TGs accounted for more than half (58%) of the total sapwood extractives. Similar results were reported for other wood species. For example, in fresh Norway spruce sapwood and trembling aspen fresh wood, TGs represent 52% and 59% of the total extractives, respectively (Ekman 1979; Sithole et al. 1992). The results of this study also showed that FAs and RAs were significantly higher in heartwood than in sapwood. This trend has been described in species such as Norway spruce (Holl and Pieczonka 1978; Ekman 1979), Pinus radiata (Hemingway and Hillis 1971), Pinus muricata (Lloyd 1975), Tilia cordata (Holl and Priebe 1985), and Scots pine (Saranpaa and Nyberg 1987a, b; Fischer and Holl 1992). Saranpaa and Nyberg (1987a) suggested that TGs were hydrolyzed during heartwood formation. A similar phenomenon may occur in lodgepole pine, and that could explain the high level of free fatty acids and monoglycerides in the heartwood. Holl and Goller (1982) noted that the heartwood of Norway spruce contains higher amounts of free sterols and steryl esters. A similar trend was observed in lodgepole pine. 62 Little variation was observed in the composition of the F A residues in the TGs between the outer and inner sapwood, as well as between the outer and inner heartwood. However, the relative proportions of the major FAs (18:1 and 18:2) were different between sapwood TGs and heartwood TGs. Similar observations were reported for Pinus radiata (Hemingway and Hillis 1971), Pinus banksiana and Pinus strobus (Conner et al. 1980), and Scots pine (Saranpaa and Nyberg 1987a; Fischer and Holl 1992). The dominant F A residues in TGs were 18:1 and 18:2, and the proportions of both 18:1 and 18:2 decreased from the sapwood to the heartwood. Among the F A residues in the steryl esters and waxes, 18:2 was the most abundant, followed by 18:3 and 18:1; however, they all decreased towards the heartwood. The similar abundance of these three F A residues in TGs and steryl esters was also found in Norway spruce and Scots pine (Ekman 1979; Anas et al. 1983; Saranpaa and Nyberg 1987a). It should be pointed out that in lodgepole pine heartwood about 33-36% F A residues of the TGs and 50% acid residues of the waxes and steryl esters were not common fatty acids. The chemistry of these acid residues remains to be studied further. Free FAs 18:1 and 18:2 were reported as the dominant fatty acids in most wood species including Scots pine (Saranpaa and Nyberg 1987a; Holmbom and Ekman 1978; Fischer and Holl 1992), Pinus nigra (Yildirim and Holmbom 1977b), Norway spruce (Ekman 1979), Cedrus libani (Hafizoglu 1987), Pinus pinea (Hafizoglu 1989), Pinus elliottii (Quinde and Paszner 1991) and Populus tremuloides (Sithole et al. 1992). The lodgepole pine data from the current study were in agreement with those results. 63 The most common RAs found in softwoods (Abies, Larix, Picea, Pinus, Pseudotsuga and Cedrus) include pimaric, sandaracopimaric, isopimaric, levopimaric, palustric, dehydroabietic, abietic and neoabietc acids (Hafizoglu and Holmbom 1987b). Anderson et al (1969) previously reported that isopimaric, abietic, dehydroabietic, palustric and levopimaric acids were present in lodgepole pine. The current study showed that the heartwood of lodgepole pine contained much higher RAs than the sapwood. Although seven RAs were present, palustric acid was the dominant one. In summary, the content and composition of lipophilic wood extractives, including triglycerides, fatty acids, resin acids, steryl esters, waxes, etc., in lodgepole pine wood were investigated mainly by SPE and GC. Although the lipid compositions in the sapwood and heartwood of lodgepole pine were qualitatively similar, the total content was five times higher in heartwood than in sapwood. Triglycerides (1.1-1.3%) were the dominant lipids in sapwood, while resin acids (4.0-5.6%) were most abundant in heartwood. Resin acids are toxic to most organisms (Leach and Thakore 1977), and the large amount of free resin acids in the heartwood could help to explain why sapstaining fungi do not colonize the heartwood of lodgepole pine. Furthermore, the high content of triglycerides in the sapwood could be a potential nutrient supply for sapstaining fungi. 64 Chapter 3 Changes of Major Lipids in the Sapwood of Pinus contorta var. latifolia and Populus tremuloides during Colonization by Ophiostoma spp. 3.1 Introduction Sapstain caused by Ophiostomatales occurs on both softwoods and hardwoods. Unlike decay fungi, sapstaining fungi generally do not to utilize cellulose or lignin as nutrient sources (Liese 1970; Tabirih and Seehann 1984; Sharpe and Dickinson 1992; Seifert 1993). These initial wood colonizers seem to assimilate the easily available carbon and nitrogen nutrients present in wood parenchyma cells (Clubbe 1980; Ballard et al. 1982; Gibbs 1993; Harrington 1993). Therefore, the non-structural compounds in wood are crucial to the survival of sapstaining fungi. However, most studies on nutrient requirements of these wood-inhabiting fungi have been conducted in synthetic media, where it is relatively easy to quantify changes in nutrient levels and fungal biomass. For example, Lagerberg et al. (1927) and Kaarik (1960) reported that glucose, fructose, maltose, starch, glycerol, and sorbitol were good carbon sources for the growth and sporulation of Ophiostoma species. Some strains are vitamin-heterotrophic, and require thiamine, pyridoxine and biotin for growth (Robbins and Ma 1942; Kaarik 1960). Others studies showed that unsaturated fatty acids stimulated the production of perithecia in some Ophiostoma species (Neumann and Hubbes 1972; Dalpe and Neumann 1977). More recently, Abraham et al. (1993) reported that 0. piceae in liquid culture was able to use 65 ammonium, amino acids and proteins, but not nitrate. Nutrient requirements for other staining fungi, e.g., Pullularia pullulans and Aureobasidium pullulans have also been reported (Troya etal. 1990; Sharpe and Dickinson 1992). Soluble sugars, starch, proteins, and lipids, the most important non-structural components in wood, are stored mainly in the parenchyma cells as food reserves. The nitrogen content in mature wood is usually 0.03% to 0.1% (Cowling and Merrill 1966). The contents of lipids in wood can be comparable to or higher than starch, soluble sugars, and proteins. Most coniferous trees, in particular, are known to be rich in triglycerides and low in starch as food reserves (Sinnott 1918; Ishibe 1935; Kramer and Kozlowski 1960; Hillis 1987). The sapwood of lodgepole pine contained 1.1% to 1.3% triglycerides, which represented about 58% of the total lipophilic extractives in the wood (section 2.3.2). While numerous studies have been done on the utilization of carbohydrates and proteins by sapstaining fungi, little attention has been given to the availability and usability of wood lipids by these fungi. To determine how efficiently sapstaining fungi degrade and utilize nutrients in wood, it is necessary to measure fungal growth or biomass accurately. When fungi grow on solid substrates like wood, their hyphae are tightly intermingled with the substrate, rendering the direct determination of biomass impossible. Several indirect measurements based on quantification of fungal proteins, chitin, and enzyme activities have been used, but many of these measures have disadvantages or limitations (Sharma et al. 1977; Matcham et al. 1985; 66 Breuil et al. 1988; Boyle and Kropp 1992; Freitag and Morrell 1992). In the past decade, ergosterol has been the preferred biomass index for fungi (Seitz et al. 1977; Grant and West 1986; Salmanowicz and Nylund 1988; Gessner et al. 1991). Ergosterol is a dominant sterol in fungal cell membranes, and it is absent or present in negligible amounts in most plant tissues (Nes 1977; Yokokawa and Mitsuhashi 1981). However, ergosterol has been rarely used for measuring fungal growth in wood (Nilsson and Bjurman 1990). In this study, the ergosterol analysis procedure used by Nilsson and Bjurman (1990) was modified to improve the extraction yield from wood and the resolution by H P L C 2 . Then, using ergosterol to follow the fungal growth and the analytical techniques described earlier (chapter 2), we examined the ability of O. piceae3, O. ainoae, and O. piliferum to degrade the major lipids in the sapwood of lodgepole pine and trembling aspen. 3.2 Materials and Methods 3.2.1 Fungal species and liquid culture Three species of sapstaining fungi, 0. piceae 387N, O. piliferum 55H, and O. ainoae 701A were obtained from Forintek Canada Corp., Ste Foy, Quebec, Canada. When received, the 2 The results on the methodology were published: Gao, Y., T. Chen and C. Breuil. 1993. Ergosterol - a measure of fungal growth in wood for staining and pitch control fungi. Biotechnology Techniques 7:621-626. 3 The results were published: Gao, Y., C. Breuil and T. Chen. 1994. Utilization of triglycerides, fatty acids and resin acids in lodgepole pine wood by a sapstaining fungus Ophiostoma piceae. Material und Organismen 28:105-118. 67 fungi were grown on 2% malt extract agar plates, and cores 3 mm in diameter were taken from the edge of the colonies and stored in 10% sterile glycerol at -70°C. For liquid culture, the fungi were grown in 300 ml Erlenmeyer flasks with 60 ml of culture medium per flask, at 23°C on a rotary shaker at 250 rpm. The liquid medium contained 2% starch, 0.16% N H 4 N 0 3 , 0.04% CaCl 2 2H 2 0 , 0.1% K H 2 P 0 4 , 0.08% N a 2 H P 0 4 , 0.05% M g S 0 4 7 H 2 0 , 0.3%) potassium hydrogen phthalate, and Vogel's micronutrients (Vogel 1956). The initial pH of the medium was adjusted to 6.1 with NaOH. After autoclaving, the filter-sterilized vitamin stock solution (Montenecourt and Eveleigh 1977) was added. The fungal biomass in liquid culture was determined by filtering and washing a known volume of culture through pre-tared glass microfibre filters, which were then dried in a microwave to constant weight. 3.2.2 Wood materials and inoculation Sapwood blocks of lodgepole pine (from the Section IV as shown in Figure 2.1) and sapwood chips (approximately 20x20x3 mm) from a 32-year-old trembling aspen (felled in winter 1992 from Alberta, Canada) were used. The moisture contents of the lodgepole pine and trembling aspen sapwood, determined gravimetrically after drying at 105°C, were 108% and 84% (based on od wood), respectively. The wood samples were sterilized by gamma irradiation (2.5 Mrad) according to Morrell and Sexton (1993) and stored at -20°C. 68 For inoculation, the wood blocks or chips were aseptically transferred into sterile Petri dishes, in which the wood samples were placed on plastic supports above three layers of moist Whatman filter paper. The preinoculum was prepared by transferring a core (3 mm) of the colony to the liquid medium (section 3.2.1). After four days of growth, the culture was gently homogenized by an Omni Homogenizer (Model 2000, Omni International, Waterbury, CT, USA) and centrifuged. The fungal pellet was rinsed, and re-suspended in sterile water to obtain a slurry of 1 ug dry mycelia per ul. The upper surface of each wood block or chip was inoculated with 100 ul of the fungal slurry. Both the inoculated and uninoculated control wood samples were incubated at 23°C and harvested periodically. 3.2.3 Analysis of wood lipids Extraction and determination of total wood extractives, and the isolation of lipid classes (triglycerides, fatty acids, and resin acids, waxes and steryl esters) were carried out as described in sections 2.2.2 to 2.2.6. 3.2.4 Extraction of ergosterol and quantification by HPLC Two grams of ground wood powder, or 10-30 mg lyophilized dry mycelia from liquid culture, was weighed into round bottom flasks. The sample was extracted and saponified by refluxing for 1 h in a mixture of 50 ml methanol, 13 ml ethanol and 6.3 g K O H . After refluxing, the sample was cooled to room temperature and filtered to remove wood debris. The filtrate was 69 transferred into a.separatory funnel, to which 26 ml petroleum ether and 13 ml nanopure H 20 were added and vigorously mixed. After partitioning, the aqueous phase (bottom) was removed and the organic phase (upper) was saved. The aqueous phase was extracted once more with 26 ml petroleum ether. The two organic extracts were combined and evaporated to dryness at 35°C in a rotary evaporator (Brinkman). The residue was redissolved in 15 ml petroleum ether, transferred to a clean bottle, dried with a N 2 stream, and finally redissolved in an accurate volume of HPLC grade methanol (1.0 ml for each wood sample and 1.5 ml for dry mycelia). The solution was filtered through a 0.45 urn Millipore filter, and stored in the dark at 4°C before analysis by HPLC. The identification and quantification of ergosterol was carried out using a Waters HPLC system (Millipore Corp., Milford, M A , USA), which includes a 625 L C pump, 600 system controller, 700 satellite WISP autosampler and 486 UV/VIS detector. The operation was controlled through the Waters software Baseline 815 (version 3.3). A reverse phase Nova-pak C-18 column (150x3.9 mm, Millipore) was used, and the detection wavelength was 283 nm. The mobile phase was methanol plus 0.005 N H 2S0 4 (98:2, v/v), at a flow rate of 2.2 ml/min. Standard ergosterol (Sigma Chemical Co.) was run as reference, and the ergosterol peak was eluted at approximately 8.3 min. 3.2.5 Assay of lipolytic activity in colonized wood Lipolytic activity in colonized wood was measured photometrically using /?-nitrophenyl laurate (PNPL) as substrate. The wood samples, harvested at different days after inoculation, were 70 ground into sawdust under liquid nitrogen. Two hundred mg of the sawdust was added to 4 ml of 0.05 M acetate buffer (pH 5.5) containing 0.4% (v/v) Triton X-100 and 0.2% (w/v) gum arabic. The reactions were started by adding 200 til of 15 m M PNPL dissolved in dimethyl sulfoxide (DMSO). The mixtures were incubated at 30°C for 10 min before being stopped by adding 40 ul of 500 m M diethyl pyrocarbonate. After centrifugatidn the absorbance of the supernatant was recorded at 404 nm, and the uninoculated control wood was used as blank. 3.3 Results 3.3.1 Comparison of lipid composition in the sterilized lodgepole pine and trembling aspen sapwood Table 3.1 shows the total wood extractives and different classes of lipids in the sterilized sapwood of lodgepole pine and trembling aspen. The total extractive contents were 2.2% and 3.1% of od wood in the pine and aspen, respectively. TGs were the dominant lipids, representing 59% and 47% of the total extractives in lodgepole pine and trembling aspen, respectively. Very low amounts of free FAs, about 0.03%, were present in both wood species. RAs were not detectable in trembling aspen, but were present at 0.38%> in lodgepole pine sapwood. Waxes and steryl esters were higher in trembling aspen (0.62%) than in lodgepole pine (0.13%>). Minor amounts of diglycerides, monoglycerides, sterols and fatty alcohols were also detected in both species. Finally, the unidentified color substances were three times higher in trembling aspen (0.94%) than in lodgepole pine (0.3%). 71 Table 3.1 Lipids in the sapwood of lodgepole pine and trembling aspen after sterilization by gamma irradiation. Lodgepole pine Trembling aspen Content1" Proportion''" Content1" Proportion1" Triglycerides 1.31 ± 0 . 0 6 59 1.45 ± 0 . 1 5 47 Fatty acids 0.03 ± 0 . 0 0 1 0.03 ± 0 . 0 0 1 Resin acids 0.38 ± 0 . 0 1 17 0 0 Steryl esters and waxes 0.13 ±0 .03 6 0.62 ± 0 . 2 1 20 Sterols, diglycerides and fatty alcohols 0.03 ± 0 . 0 1 1 0.02 ± 0 . 0 0 1 Monoglycerides 0.04 ± 0 . 0 2 2 0.03 ± 0 . 0 1 1 Unidentified substances 0.30 ±0 .15 14 0.94 ± 0 . 2 8 30 Total extractives 2.22 ±0 .19 100 3.09 ±0 .12 100 f % of od wood, all data points are means of three determinations with standard deviations shown by uncertainties. X the relative percentage (%) of each class to the total extractive amount. 72 The F A composition in lodgepole pine wood TGs and the contents of various free FAs were not changed by gamma irradiation (data not shown). However, some variations were observed among several RAs after gamma irradiation. Palustric and neoabietic acids decreased by more than 50%, while dehydroabietic acid increased by about 3 fold. The contents of pimaric, sandaracopimaric, isopimaric and abietic acids showed no detectable changes (Table 3.2). 3.3.2 Measurement of fungal growth by the quantification of ergosterol The recovery of ergosterol during the extraction process was examined. The effect of a saponification step on the yield of ergosterol extracted from fungal mycelia or colonized wood was assessed. Saponifying the sample during the refluxing extraction increased the ergosterol yield by 25%>. Refluxing the sample for 1 h gave over 90%> recovery, but increasing the refluxing time to 16 h did not significantly increase the ergosterol yield. About 99% of the ergosterol added to an uninoculated wood sample was recovered with the extraction procedure incorporating saponification and 1 h refluxing. Using the modified extraction method (section 3.2.4), the ergosterol content and the mycelial dry weight of O. piceae grown in liquid culture were determined each day. Figure 3.1 shows that the total amount of ergosterol increased from day 1 to day 4 and then remained relatively constant. The shape of the ergosterol curve was similar to that obtained for fungal dry weight. Table 3.2 Content of each resin acid in lodgepole pine sapwood before and after gamma irradiation. Acids Content (yig/g od wood)* Freshly cut gamma irradiated^ Pimaric 213 ± 4 212 ± 2 0 Sandaracopimaric 49 ± 1 51 ± 4 Isopimaric 355 ± 8 364 ± 2 7 Palustric 1918 ± 7 9 891 ± 5 5 Dehydroabietic 298 ± 47 1036 ± 8 3 Abietic 504 ± 5 9 440 ± 19 Neoabietic 461 ± 4 1 156 ± 14 t The irradiation dosage was 2.5 Mrad. * A l l data points are means of three determinations with standard deviations shown by uncertainties. 74 0 1 2 3 4 5 6 D a y s after inoculat ion Figure 3.1 Ergosterol and fungal biomass of O. piceae in liquid culture. A l l data points are means of three determinations with standard deviations shown by error bars. Table 3.3 The ergosterol content (%, w/w) in the cells of Ophiostoma piceae during different growth stages in liquid culture. Days after inoculation^ 1 2 3 4 5 6 Ergosterol (%)* 0.05 +0.01 0.24 + 0.08 0.42 + 0.01 0.46 ±0.02 0.51 +0.02 0.49 + 0.02 f Day 1: lag phase; Days 2-4: exponential phase; Days 5-6: stationary phase. * A l l data points are means of three determinations with standard deviations shown by uncertainties. 75 There was a positive correlation between the two parameters, with a correlation coefficient of 0.97. Ergosterol content expressed per unit of fungal biomass varied during the different growth phases, from 0.05% at the lag phase, increasing sharply during the exponential phase, reaching 0.51% when the stationary phase began (Table 3.3). After inoculation, the fungi grew and spread over the sapwood blocks (lodgepole pine) or chips (trembling aspen), first as a white hyphal mat, then after 10 to 14 days the hyphae became dark grey to black, causing wood discoloration. No ergosterol was detected in the uninoculated control wood (Figure 3.2B), while an ergosterol peak was observed in fungal colonized wood (Figure 3.2C). The fungal growth rate, indicated by ergosterol level (Figure 3.3), increased steadily during the first two weeks of colonization, which indicated an exponential growth phase. After two weeks, the amount of ergosterol extracted became relatively stable, indicating that the fungal growth had reached a stationary phase. Although the pattern of growth was similar for the three fungal species, higher amounts of ergosterol were obtained with O. ainoae than with the other two species. 3.3.3 Changes of the total wood extractives during fungal colonization Major changes in wood extractives in lodgepole pine and trembling aspen sapwood occurred during the first two weeks after inoculation of fungi. The three fungal species, O. piceae, O. ainoae, and O. piliferum, showed similar effects in each of the two wood species. In lodgepole 76 £ c CO 0 0 £ L CD O c CO _Q i o w . Q < 0.030 L 0.025 L A B 4 6 8 10 Retent ion t ime (min) Figure 3.2 HPLC chromatograms of ergosterol standard (A), extracts from non-colonized (B) and colonized lodgepole pine sapwood 8 days after inoculation (C). Notes: e: ergosterol peak; fungal strain: Ophiostoma piceae 387N; column: Nova-pak C-18 (150x3.9 mm); detector: UV/VIS; elution solvent: methanol and 0.005 N sulfuric acid (98:2, v/v). 77 Days after inoculation Figure 3.3 Ergosterol levels of three sapstaining fungi in lodgepole pine sapwood. A l l data points are means of three determinations with standard deviations shown by error bars. 78 pine the total extractives were reduced by 16-17%, and in trembling aspen by 30-33% in 14 days (Table 3.4). The uninoculated wood, which was incubated under the same conditions as the fungal-treated wood, showed a negligible decrease in total extractives (Table 3.4). The absence of fungi in the uninoculated wood was confirmed by the lack of ergosterol (Figure 3.2B). 3.3.4 Changes of triglycerides and free fatty acids in wood during fungal colonization The rapid increase in fungal growth in wood corresponded to a sharp decrease of the TGs. In lodgepole pine, O. piceae was most efficient, followed by O. piliferum, while O. ainoae was less effective at decreasing TGs. More specifically, in 14 days 0. piceae, 0. piliferum, and 0. ainoae hydrolyzed the TGs in wood by 86%, 79%, and 48%, respectively (Figure 3.4A). In trembling aspen between 70%> to 80% of the TGs were removed after two weeks colonization, however, a major decrease of approximately 50% to 60% occurred during the first week (Figure 3.4B). In another experiment, the lipolytic enzyme activity in lodgepole sapwood inoculated with 0. piceae was determined. Lipolytic activity increased steadily up to 3 weeks after a lag phase (Figure 3.5). As the TGs in wood decreased, the free FAs increased. Figure 3.6 shows that more free FAs accumulated in lodgepole pine than in trembling aspen. O. piceae colonization of lodgepole pine resulted in higher free FAs increase than O. piliferum and O. ainoae. The pattern 79 Table 3.4 Contents of total wood extractives in lodgepole pine and trembling aspen sapwood before and after fungal colonization. Extractives content (% of od wood)* Time after inoculation (days) Lodgepole pine Trembling aspen Untreated^ 0 2.31 ±0.03 3.09 ±0.07 Control 1 14 2.26 ±0.02 2.88 ±0.05 0. piceae 14 1.92 ±0.01 2.08 ±0.05 0. ainoae 14 1.92 ± 0.11 2.07 ±0.04 0. piliferum 14 1.94 ±0.03 2.15 ±0.01 t Untreated: fresh wood frozen at -20°C. X Control: wood incubated aseptically at 23°C without fungal inoculation. * A l l data points are means of three determinations with standard deviations shown by uncertainties. 80 Figure 3.4 Changes in triglycerides in the sapwood of lodgepole pine (A) and trembling aspen (B) after colonization by O. piceae, 0. ainoae, or 0. piliferum. A l l data points are means of three determinations with standard deviations shown by error bars. Days after inoculation Figure 3.5 The lipolytic enzyme activity of 0. piceae in lodgepole pine sapwood. A l l data points are means of three determinations with standard deviations shown by error bars. 82 Days after inoculation —#— O. piceae (in the pine) — A — O. ainoae (in the pine) —•— O. piliferum (in the pine) —O— O. piceae (in the aspen) —A— O. ainoae (in the aspen) —•— O. piliferum (in the aspen) Figure 3.6 Changes in total free fatty acids in the sapwood of lodgepole pine and trembling aspen after colonization by O. piceae, O. ainoae, or O. piliferum. A l l data points are means of three determinations with standard deviations shown by error bars. 83 of F A change was closely related to the decrease of TGs. For example, the total TGs in lodgepole pine colonized by O. piceae dropped from 13.1 to 3.7 mg/g od wood in 10 days, while the total free FAs increased from 0.9 to 9.2 mg/g od wood during the same period. Analyses by GC showed that these FAs were predominantly 18:1 and 18:2, followed by 18:3 and 16:0, while 18:0, 17:0, and 20:2 were found in small amounts (Figure 3.7). 3.3.5 Changes of resin acids in lodgepole pine wood during colonization O. piceae, O. ainoae, and O. piliferum were able to decrease RAs in lodgepole pine sapwood (Figure 3.8). About 55% to 60% of the total RAs were removed by each of the three fungi, although O. ainoae appeared to be more effective. The various RAs in the O. piceae inoculated wood were quantified, and results indicated that all the seven RAs decreased to some extent (Figure 3.9). Dehydroabietic acid and palustric acid, which accounted for 60% of the initial total RAs, dropped by 50% and 85%, respectively. 3.3.6 Changes of waxes and steryl esters in trembling aspen wood during colonization Waxes and steryl esters accounted for 20% of the total wood extractives in trembling aspen sapwood. O. piceae, O. ainoae, and O. piliferum decreased 40%, 55%, and 31% of the waxes and steryl esters in the aspen wood in 2 weeks (Figure 3.10). Figure 3.7 Changes in the amount of various fatty acids in lodgepole pine sapwood after colonization by 0. piceae. A l l data points are means of three determinations with standard deviations shown by error bars. 85 Figure 3.8 Changes in the total free resin acid contents in the sapwood of lodgepole pine after colonization by O. piceae, 0. ainoae, or 0. piliferum. A l l data points are means of three determinations with standard deviations shown by error bars. 0.08 CD E 0.04 "5 10 1"5 20 25 D a y s after inoculat ion Figure 3.9 Changes in the amount of various resin acids in lodgepole pine sapwood after colonization by O. piceae. A l l data points are means of three determinations with standard deviations shown by error bars. 87 Control O. piceae O. ainoae O. piliferum Figure 3.10 Changes in the waxes and steryl esters in trembling aspen sapwood after 14 days colonization-by O. piceae, O. ainoae, or O. piliferum. A l l data points are means of three determinations with standard deviations shown by error bars. 88 3.4 Discussion In order to examine the utilization of potential nutrients in wood by sapstaining fungi, it was necessary to sterilize the wood samples to prevent endogenous microflora from changing the wood components and thus giving erroneous data. The selection of an appropriate sterilization method is important since some sterilization methods alter not only microflora but also the nutritional quality of wood (Morrell and Sexton 1993). Gamma irradiation was used in this study because it usually causes less effects on the composition of nutrients than autoclaving (Joshi et al. 1990; McAllister et al. 1991). When the lipid composition of wood was compared before and after gamma irradiation, no obvious changes in the TGs and FAs were observed. Although the total RAs concentration was not markedly affected, the contents of four RAs changed. Palustric, neoabietic and abietic acids decreased, while dehydroabietic acid significantly increased. A similar phenomenon among resin acids has been reported during tall oil distillation (Holmbom et al. 1974), wood seasoning (Quinde and Paszner 1991), and the storage of wood extracts (Arrabal and Cortijo 1994). The exact mechanisms by which these RAs are converted is not fully understood. Takeda et al. (1968) reported that isomerization could occur among the RAs with two conjugated double bonds, such as palustric, abietic, neoabietic, and levopimaric acids. Quinde and Paszner (1991) suggested that these RAs could be converted to dehydroabietic acid by the processes of isomerization and dehydrogenation. These processes might have taken place during the gamma irradiation of the lodgepole pine wood. 89 To compare how efficiently sapstaining fungi utilize non-structural components in wood, it is necessary to correlate fungal growth with the consumption of nutrients. However, most fungal physiological activities measured on wood have not been reported per unit of fungal biomass, because it is difficult to accurately determine fungal biomass in wood. In this work, a positive correlation was found between fungal dry weight and ergosterol in liquid culture, but the ergosterol content per unit of fungal biomass varied during the different growth phases. In the literature it has also been reported that ergosterol contents in fungal cells vary with species, strains and culture conditions (Seitz et al. 1979; Matcham et al. 1985; Nout et al. 1987). These factors complicate the calculation of fungal biomass in solid substrates based on ergosterol content obtained from artificial media. Consequently, it is inaccurate to convert ergosterol values directly to fungal biomass, since the content of ergosterol per unit dry weight is not constant at different fungal growth stages. Therefore, it is more appropriate to use ergosterol as an index for fungal growth phases and to associate various biochemical events of fungi in wood with the different growth stages. In the current study, the three Ophiostoma fungi showed an exponential growth phase in the first two weeks after inoculation in wood, and this phase corresponded to the rapid decrease in wood extractives, particularly triglycerides, suggesting that these fungi were efficient in degrading wood extractives during the early active colonization stage, in which large amount of new hyphae were forming and penetrating wood. Although the content of total wood extractives decreased moderately in both wood species after fungal colonization, significant changes occurred in TGs, FAs, and RAs. Between 50% to 80%) of the TGs in the sapwood disappeared within the first two weeks, while free FAs 90 increased during the same period, especially in the lodgepole pine wood. Similarly, an albino strain of O. piliferum was reported to decrease the total fatty acid esters in southern yellow pine wood (Blanchette et al. 1993; Brush et al. 1994). The results of this study further showed that the free FAs accumulating in the pine sapwood were identical to the major F A residues in the wood TGs (section 2.3.3), suggesting that the fungi secreted extracellular lipase(s) to hydrolyze the TGs liberating the FAs and glycerol, which can be assimilated by fungal cells. FAs can be completely metabolized through P-oxidation inside fungal cells, releasing twice as much as energy as the equivalent amount of carbohydrates (Weete 1980). The accumulation of FAs during the early colonization stage implies that these fungi might be able to assimilate the released glycerol before the free FAs. During the stationary growth phase, when the majority of TGs in wood were hydrolyzed, free FAs were seen to decline, which can most likely be attributed to fungal assimilation. In fact, the F A utilization by the fungi might have been slightly higher than that measured, since the total FAs determined might include some fungal FAs. The lipid content in fungal cells is highly variable, depending on growth conditions and species. Except for a few species which are lipid producers, lipid contents between 3% to 30% have been reported in various fungal species and strains (Weete 1980). Based on these data and the ergosterol content at the stationary growth phase (section 3.3.2), it was estimated that the amount of lipids contributed by O. piceae could be 0.15 to 1.5 mg per gram of dry wood. Linoleic, palmitic and oleic acids are the major FAs reported from Ophiostoma species (Koch et al. 1993), and they might account for a small fraction of the total free FAs detected. 91 It is interesting to note the difference in the concentrations of free FAs in the two wood species after fungal colonization. Much higher free FAs accumulated in lodgepole pine (about 6 to 9 mg/g od wood after the first week) than in aspen (about 1 to 3 mg/g od wood), although the initial T G contents and the rate of T G degradation by the fungi were similar in the two wood species. This suggested that the fungi assimilated the free FAs more efficiently in trembling aspen than in lodgepole. pine. It is not clear what factors caused the difference in F A assimilation by the fungi in the two wood species. The differences between the two wood species in concentrations of other components (such as soluble sugars, nitrogen sources, and resin acids), moisture contents, or pH may affect the time and selection of F A assimilation and utilization by fungi. This study showed that the three Ophiostoma species could substantially decrease the level of RAs in lodgepole pine sapwood. Other researchers have reported similar results with an albino strain of O. piliferum in southern yellow pine wood (Farrell et al. 1993; Brush et al. 1994). It should be pointed out that although the present work has shown the association between the increase of fungal growth in wood and the decrease of RAs, it is not clear whether the RAs could be used as a carbon source by these fungi. The lack of oxygen atoms and the high degree of saturation in their composition make RAs very stable and difficult to degrade. Moreover, high concentrations of resin acids are toxic to many organisms (Leach and Thakore 1977). None of the Ophiostoma species were able to grow in the heartwood of lodgepole pine where the resin acids were as high as 4.0-5.6% (section 2.3.5). At this point, it was not determined 92 whether the RAs were completely degraded or only modified to form less toxic metabolites, as reported for the fungus Mortierella isabellina (Kutney et al. 1981). Because of difficulties in the isolation, purification, and radioactively labeling of individual RAs, very little information is currently available on the degradation pathways of RAs. In summary, this study provided direct information on the degradation of the major lipophilic substances in sapwood by three sapstaining Ophiostoma species. It was suggested that the wood lipids, in particular triglycerides and fatty acids, could be used by these fungi as nutrient sources during colonization in wood. Since triglycerides were the dominant lipids in the sapwood of both lodgepole pine and trembling aspen, and they were rapidly hydrolyzed by all the three fungal species investigated, further study is necessary to characterize the enzyme activities responsible for this effect. 93 Chapter 4 Production, Purification and Characterization of an Extracellular Lipase Secreted by Ophiostoma piceae 4.1 Introduction Lipases (glycerol ester hydrolase, EC 3.1.1.3) are an important group of lipolytic enzymes with the biological function of breaking down triglycerides into glycerol and fatty acids (Macrae and Hammond 1985). In the previous chapter, O. piceae and two other species were shown to degrade the triglycerides in lodgepole pine and trembling aspen sapwood, indicating that these fungi were capable of secreting extracellular lipases in wood. Further characterization of the properties and mode of action of the lipase(s) is an integral part of understanding the biochemical features of O. piceae. However, it is very difficult to purify sufficient enzyme for characterization studies from the colonized wood because various wood components interfere with the purification procedures. Therefore, it is more practical to purify the enzymes from a liquid medium even though our goal is to understand the role of the lipase(s) produced by the fungus when it colonizes wood. Thus, it is essential to use a medium with a composition similar to the lipid substrates in wood, so that the same lipases are likely to be produced by O. piceae in both liquid media and wood. 94 To obtain a high yield of extracellular lipases in liquid culture for purification requires the optimization of medium composition and culture conditions. Carbon and nitrogen sources, medium pH, and incubation temperature all affect lipase yield. Different fungal species often require quite different media and cultivation conditions for high lipase production (Sztajer and Maliszewska 1989; Papaparaskevas et al. 1992). Some of the nutrient requirements and culture conditions in liquid culture have been reported for 0. piceae. For optimal growth in a medium with starch as carbon source and ammonium nitrate as nitrogen source, the optimum temperature and initial medium pH were 23°C and pH 6.1, respectively (Abraham et al. 1993). The initial work of the current study used such a medium composition and incubation temperature, but focused on the effects of various carbon and nitrogen sources, and the initial medium pH's, on the production of extracellular lipase(s). In particular, this study examined the effects of various plant oils which have a similar fatty acid composition to wood triglycerides, and starch and soluble sugars which are generally present in wood (Fischer and Holl 1992; Sauter and van Cleve 1994) on lipase production. The first stage in the purification of extracellular enzymes or proteins from a liquid culture is generally clarification, including the removal of fungal cells from the culture by filtration or centrifugation, followed by isolation of total proteins from the culture filtrate by precipitation (with a salt or solvent) or ultrafiltration. The separation of the enzyme or protein of interest from the total proteins is then achieved by successively using various chromatography techniques such as ion exchange, hydrophobic interaction, affinity, size exclusion, and chromatofocusing (Harris and Angal 1989). During the purification process, enzyme activity 95 assays and protein determinations wil l indicate whether the yield is acceptable, whether the enzyme is stable, and whether one technique is more effective than another. Analysis of proteins by gel electrophoresis wil l indicate the purity of the protein and how many contaminants are present. The objectives of this chapter were first to optimize the liquid media composition to increase the yield of extracellular lipase production by O. piceae4, and then to purify one major lipase and study the enzyme characteristics including the molecular weight, isoelectric point (pi), amino acid composition, degree of glycosylation, N-terminal sequence, pH and temperature optima for activity5. 4.2 Materials and Methods 4.2.1 Liquid media and culture conditions The fungal strain used in this study was 0. piceae 387N. The basic culture medium was slightly modified from that described previously (section 3.2.1) in that the concentration of nitrogen was increased to 0.5% (w/v). The carbon sources tested (2%) included glucose, fructose, arabinose, galactose, xylose, sucrose, raffinose, dextrin, olive oil, soybean oil, corn 4 The results on lipase production were published: Gao, Y. and C. Breuil. 1995. Extracellular lipase production by a sapwood-staining fungus Ophiostoma piceae. World Journal of Microbiology and Biotechnology 11:638-642. 5 The results on lipase purification and characterization have been submitted for publication: Gao, Y. and C. Breuil. 1996. Purification and characterization of an extracellular lipase produced by the sapstaining fungus Ophiostoma piceae. 96 oil, sunflower seed oil, sesame oil, cotton seed oil, and peanut oil. Nitrogen sources examined included NH4CI, NH4NO3, (NFL^SC^, peptone, tryptone, casamino acids, and urea. Agitated liquid cultures were prepared as described previously (section 3.2.1). Sampling was carried out daily by aseptically withdrawing 5 ml of each culture to measure the fungal biomass and extracellular lipase activity. Fungal biomass was determined by dry weight (section 3.2.1) and the filtrate was used for the assay of extracellular lipase activity. For lipase purification, O. piceae was grown in a 2 litre flask containing 500 ml of the optimized medium containing 2% (v/v) olive oil as carbon source, 0.5% (w/v) ammonium sulfate and 3% (w/v) peptone as nitrogen sources. A l l other constituents were as described earlier (section 3.2.1) with the initial pH of 5.0. Each flask was inoculated with 4 mg of fungal cells from a preculture grown in the basic medium (section 3.2.1). The cultures were incubated at 23°C on a rotary shaker (250 rpm) for 72 h before harvesting. 4.2.2 Assay of lipase activity Two substrates (olive oil and^-nitrophenyl palmitate) were used to assay the lipase activity: When olive oil was used as substrate, the lipase activity was determined by measuring the amount of fatty acids released with a colorimetric procedure according to Duncombe (1963) and Lippi et al. (1972). A substrate emulsion was prepared by mixing 10 ml highly purified olive oil (Sigma Chemical Co.) with 90 ml 0.1 M acetate buffer (pH 5.5) containing 0.5% 97 (w/v) gum arabic and 0.2% (w/v) sodium benzoate with an Omni homogenizer for 10 min. A mixture of 2 ml substrate emulsion and 2 ml culture filtrate was incubated at 37°C in a reciprocal water bath at 325 rpm. The reaction was carried out for 30 to 60 min, and then, 2 ml copper reagent (0.27 M copper nitrate in 0.45 M triethanolamine buffer, pH 7.8) and 5 ml chloroform were added. The mixture was vortexed vigorously and the partition between organic and aqueous phases was facilitated by centrifugation at 5000 x g for 5 min. Half a milliliter of the lower organic phase was transferred to a clean tube, mixed with 2.5 ml chloroform and 0.5 ml 9 m M sodium diethyldithiocarbamate dissolved in butanol. Absorbance of the mixture was measured at 435 nm. The blank was prepared by using boiled culture filtrate and following the same procedure. A standard curve was made with palmitic acid (16:0). One unit of lipase was defined as the amount of enzyme that liberates 1 umol equivalent of fatty acids per minute under the assay conditions. When /7-nitrophenyl palmitate (PNPP) was used as substrate, the lipase activity was measured by the amount of /?-nitrophenol liberated (Winkler and Stuckmann 1979; Kordel et al. 1991). The reaction mixture comprised 100 ul of 15 m M PNPP dissolved in DMSO, 1.8 ml of 0.1 M acetate buffer (pH 5.5) containing 0.4% (v/v) Triton X-100 and 0.2% (w/v) gum arabic, and 100 ul of an appropriate dilution of the enzyme preparation. The mixture was incubated at 37°C in a water bath for 10 to 15 min before the absorbance was recorded at 404 nm. One unit of lipase activity was defined as the amount of enzyme that liberated 0.1 umol of p-nitrophenol from PNPP per minute under the assay conditions. 98 4.2.3 Lipase purification The major purification steps included ammonium sulfate and acetone precipitation, hydrophobic interaction chromatography (Phenyl-Sepharose CL-4B), and anion exchange chromatography (DEAE-Sepharose CL-6B). Procedures were carried out at 4°C, unless otherwise specified. Triton X-100 and phenylmethylsulfonyl fluoride (PMSF) were immediately added to the harvested whole culture at final concentrations of 0.1% (v/v) and 2 mM, respectively. The culture was agitated for 40 min, and then centrifuged at 2100 x g for 15 min. The lipid layer formed on the top of the supernatant and the fungal cell pellet at the bottom were removed. The supernatant was then filtered, and solid ammonium sulfate was added to the filtrate at 80% saturation. After equilibration, a solid sticky mass aggregate which formed on the surface of the solution was collected, while the remaining solution was centrifuged. The precipitate was combined with the sticky aggregate and resuspended in 20 m M Tris-HCl (pH 8.0). The combined fractions gave a cloudy enzyme solution which was centrifuged at 5000 x g for 10 min. The supernatant was cooled to 0°C before adding cold acetone (-20°C) to 60% (v/v). The precipitated proteins were re-suspended in 20 m M Tris-HCl buffer (pH 8.0), and solid ammonium sulfate was added to this preparation at 30% saturation. The above enzyme solution (556 mg protein per batch) was applied to a Phenyl-Sepharose C L -4B column (1.5x30 cm, bed volume 53 ml, Pharmacia, Uppsala, Sweden) which was pre-99 equilibrated with 20 m M Tris-HCl buffer (pH 8.0) containing ammonium sulfate at 30% saturation. The column was washed with two bed volumes of this buffer, and then subjected to 3 stages of elution: (1) 250 ml of a linear gradient of 30% to 0%> ammonium sulfate in 20 m M Tris-HCl (pH 8.0); (2) 100 ml of 20 m M Tris-HCl (pH 8.0); and (3) 150 ml of 1% Triton X -100 in water. The flow rate was 60 ml/h, and fractions in 5 ml were collected. The fractions in which lipolytic activity was detected, were pooled and concentrated. The concentrated fractions were transferred into an ultrafiltration cell (Model 8400, cell volume: 180 ml, Amicon Division, W. R. Grace & Co., Beverly, M A , USA) and washed with 6 cell volumes of 20 m M Tris-HCl buffer (pH 8.0) using a Y M 10 membrane (MW cut-off at 10 kDa, Amicon) under the pressure of 55 psi. Afterwards, the enzyme preparation in smaller volumes was further concentrated with Filtron Microsep (size: 3 ml, M W cut-off at 10 kDa, Eastern Container Corp., Springfield, M A , USA). Following Filtron Microsep concentration, the enzyme preparation in 20 m M Tris-HCl buffer (pH 8.0) was loaded onto a DEAE-Sepharose CL-6B column (1.5x30 cm, Pharmacia) which was pre-equilibrated with the same buffer. After washing with the buffer until the U V absorbance (280 nm) became stable, the enzyme was eluted with 250 ml of a linear gradient of NaCl from 0 to 0.6 M in the buffer with a flow rate of 27 ml/h. The lipolytically active fractions were pooled, desalted, and concentrated with an ultrafiltration cell and Filtron Microsep as described above. 100 4.2.4 Electrophoresis and lipolytic stain on gels Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was performed with the PhastSystem (Pharmacia), using pre-cast gels and SDS buffer strips (Pharmacia). The sample buffer consisted of 10 m M Tris-HCl (pH 8.0), 2.5% SDS, 1 m M ethylenediamine tetraacetate (EDTA), 5% (3-mercaptoethanol (BME), and 0.02% bromophenol blue. Low-molecular-weight standard proteins from Pharmacia were used as references. The mixture contained phosphorylase b (94 IcDa), albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa) and a-lactalbumin (14.4 kDa). The separation was performed at a constant current of 10 mA for a total of 70 Vh. The gels were silver-stained in the PhastSystem development unit (Pharmacia) according to the manufacturer's recommendations. Isoelectric focusing (EEF) gel electrophoresis was performed with pre-cast gels of pi 3 to 9 in the PhastSystem. The separation was carried out at a constant current of 5 mA for a total of 510 Vh. The gels were run in duplicate: one gel was silver-stained, and the other was used for lipolytic visualization according to Baillargeon and Sonnet (1988). The gels were agitated in freshly prepared 0.1 M acetate buffer (pH 5.5) containing 0.03% a-naphthyl acetate and 0.05% Fast Blue RR salt (Aldrich Chemical Co.) at 30°C until reddish lipolytic bands appeared. 101 4.2.5 Determination of protein content and glycosylation Protein contents in enzyme preparations were measured with the DC protein assay kit (Bio-Rad Laboratories Ltd., Mississauga, Ontario, Canada) using bovine serum albumin (BSA) as the standard. The degree of glycosylation of the purified lipase was determined by the phenol-sulfuric acid method according to Dubois et al. (1956) with D-glucose as the standard. 4.2.6 Molecular weight determination The molecular weight was determined by both SDS-PAGE and size exclusion chromatography. For size exclusion chromatography, 45 ug purified lipase was loaded onto a Superose 12 high performance size exclusion column (Pharmacia), which was then eluted with 0.1 M acetate buffer (pH 5.5) at a flow rate of 0.25 ml/min. This process was controlled through the fast protein liquid chromatography system (FPLC, Pharmacia). Gel filtration standards from Bio-Rad, bovine thyroglobulin (670 kDa), bovine gamma globulin (158 kDa), chicken ovalbumin (44 kDa), horse myoglobin (17 kDa), and vitamin B-12 (1.35 kDa) were employed for calibration. 4.2.7 Analysis of amino acid composition and N-terminal sequencing The amino acid composition of the purified lipase in nanopure water was analyzed with an automated amino acid analyzer (Model 420 A / H , Applied Biosystems Inc., Forster City, CA, 102 USA). The analysis was performed by Dr. Krystyna Piotrowska at the Protein Service Laboratory of the University of British Columbia. For sequencing, the lipase sample was incubated in 70 m M Tris-HCl buffer (pH 6.8) containing 11% glycerol, 2.2% SDS, 5.7% B M E , and 0.02% bromophenol blue at 37°C for 15 min, then loaded onto 12.5% SDS-polyacrylamide gel which was cast and aged for 24 h prior to the sample application. The gel was run under a constant voltage of 110 V using a Mini-P R O T E A N II cell (Bio-Rad). Separated proteins were electro-blotted onto an Immobilon-PS Q PVDF protein sequencing membrane (Millipore Corp.) using the Trans-Blot electrophoretic transfer cell (Bio-Rad) for 3.5 h at 45 V . The transfer buffer used was 10 m M CAPS buffer (pH 11) containing 10% methanol. Proteins on the membrane were stained with 0.02% Ponceau S, and the lipase band was cut out and sent for analysis. The N-terminal sequence was determined by Sandy Kielland at the Protein Microchemistry Facility of the University of Victoria, Victoria, BC, Canada, with a pulsed liquid sequenator (Model 473, Applied Biosystems Inc.). 4.2.8 Examination of the effects of pH and temperature on the activity and stability of the lipase The effect of pH on the lipase activity was determined using 0.1 M acetic acid/MES/Tris buffer mixture (Ellis and Morrison 1982). The reaction was carried out at 35°C for 30 min with olive oil emulsion as substrate. The effect of pH on the lipase stability was determined 103 by incubating the enzyme in 0.1 M acetic acid/MES/Tris buffer at different pH values at 4°C for 24 h. After treatment, the remaining lipolytic activity was determined with olive oil emulsion at pH 5.4 and 35°C. The optimal temperature for the lipase activity was determined by carrying out the reaction for 30 min at 15 to 60°C, with 5°C increments. The thermostability was determined by keeping the enzyme in 0.1 M Bis-Tris buffer, pH 6 at different temperatures for 15 min. After incubation, the enzyme was immediately transferred to ice and kept for 30 min, and the remaining activity was then assayed at pH 5.4 and 35°C. 4.3 Results 4.3.1 Preliminary investigation of the profile and properties of the extracellular lipase(s) produced by O. piceae The growth and extracellular lipase activity of O. piceae grown in a liquid medium containing 2% olive oil as carbon source and 0.5% ammonium sulfate as nitrogen source were monitored (Figure 4.1). The extracellular lipase activity in the filtrate increased when the fungus was actively growing, and reached a maximum of 2.4 U/ml at the end of the exponential growth phase at day 3. Lipase activity decreased substantially at day 4 and 5, while the biomass remained relatively constant as the stationary growth phase was reached. The pH of the medium decreased from an initial level of 6.1 to 4.8 on day 3, and to 2.7 on day 5 (Figure 4.1). 104 D a y s after inoculat ion Figure 4.1 Biomass, extracellular lipase activity, and pH in a culture of O. piceae. The medium with an initial pH of 6.1, contained 2% (v/v) olive oil as carbon source, 0.5% (w/v) (NH 4 ) 2 S0 4 as nitrogen source, and other constituents as described in section 3.2.1. Lipase activity was assayed with olive oil emulsion at 37°C. A l l data points are means of three determinations with standard deviations shown by error bars. 105 The optimal pH and temperature for lipolytic activity was determined with crude culture filtrates of liquid culture (Table 4.1). The optimal pH for the activity was 5.5, and the relative activity decreased by about 50% at pH's 4.0 and 6.5. The optimal temperature was about 35°C, and the lipase(s) retained more than 90% of its relative activity between 30 and 40°C. Lipase activity reached its maximum at day 3, but protein concentrations in the culture filtrate were very low (about 0.005 mg/ml). After concentration by ultrafiltration and ammonium sulfate precipitation, the profile of the total extracellular proteins was analyzed by SDS-PAGE and IEF. SDS-PAGE showed that the proteins had molecular weights between 14 to 100 kDa (Figure 4.2A). IEF revealed that all the major proteins had a pi's less than 7. Major bands were detected at pi's of 6.8, 6.6, 6.2, 5.1, 4.7, 4.3, 4.1, and several minor bands at pi's below 4 (Figure 4.2B). The lipolytic activity stain showed that the bands below pi 4.5 were lipolytically active (Figure 4.2B). 4.3.2 Effect of carbon sources on the growth and lipase production In preliminary experiments, O. piceae grew well in media supplemented with triolein, oleic acid, glycerol, or starch as carbon sources. The biomass was higher in media containing triolein or oleic acid than in media containing starch or glycerol (Figure 4.3). The effects of different carbohydrates on fungal growth and extracellular lipase production was examined. Figure 4.4A shows that in media supplemented with glucose, fructose, sucrose, starch Table 4.1 Effects of pH and temperature on the lipase activity in the culture filtrate of O. piceae. pH Relative activity (%) 2.5 26.6 3.5 35.2 4.5 71.5 5.5 100.0 6.5 53.6 7.5 29.5 Temperature (°C) Relative activity (%) 20 62.2 25 81.0 30 92.6 35 100.0 40 94.3 45 60.2 50 12.7 O. piceae was grown in the medium containing 2% (v/v) olive oil as carbon source, 0.5% (w/v) (NFL^SC^ as nitrogen source, and other constituents as described in section 3.2.1. The culture filtrate was obtained by removing the fungal cells 3 days after inoculation. The lipase activity assay was carried out with olive oil emulsion at 37°C when studying the pH effect, and at pH 5.5 when studying the temperature effect. Relative activity was expressed by taking the highest activity as 100. 107 Figure 4.2 Electrophoretic profiles of the extracellular proteins produced by O. piceae in olive oil supplemented medium. O. piceae was grown in a medium supplemented with olive oil (2%) and (NH 4 ) 2 S0 4 (0.5%) and other constituents as described in section 3.2.1. A : SDS-P A G E (PhastGel 12.5%), lane 1: molecular weight markers; lane 2: extracellular proteins of O. piceae. B: IEF (PhastGel 3-9), lane 3: pi markers; lane 4: the extracellular proteins. Lanes 1, 2, 3, and 4 were silver-stained to indicate proteins; and lane 5 is the same sample as lane 4, but detected by a lipolytic stain to indicate enzymes with lipolytic activity. 108 E CO CO 0 3 E o in Tr io le in O l e i c ac id G lyce ro l Carbon Sources Starch Figure 4.3 Growth of O. piceae in the medium supplemented with starch, triolein, oleic acid or glycerol as carbon sources. The medium, initially at pH 6.1, contained 1% (w/v) carbon source, 0.5% (w/v) NH4CI as nitrogen source and other constituents as described in section 3.2.1. The dry weight determinations were taken after 4 days incubation at 23°C. A l l data points are means of three determinations with standard deviations shown by error bars. 109 T 1 1 1 1 1 1 r Glu Fru Ara Gal Sue Raf Dex Sta Carbohyd ra tes F i g u r e 4.4 Effect of carbohydrates on the growth (A) and extracellular lipase activity (B) of O. piceae. The medium, initially at pH 6.1, contained 2% (w/v) carbon source, 0.5% (w/v) NH 4 C1 as nitrogen source and other constituents as described in section 3.2! 1. Lipase activity was assayed with olive oil emulsion at 37°C. Glu: glucose, Fru: fructose, Ara: arabinose, Gal: galactose, Sue: sucrose, Raf: raffinose, Dex: dextrin, Sta: starch. A l l data points are means of three determinations with standard deviations shown by error bars. 110 or dextrin, similar biomass (5-6 mg/ml) was produced, while less biomass was obtained in media containing arabinose, galactose or raffinose. Extracellular lipase activity was very low with all the carbohydrates tested, less than 0.2 U/ml (Figure 4.4B). When plant oils were used as carbon sources, both higher biomass and lipase activity were obtained (Figure 4.5). The lipase activity was about 10 to 15 fold higher in the media supplemented with plant oils than in the media supplemented with carbohydrates (Figures 4.4B; 4.5B), while the biomass was only 2 times higher in oil media than in carbohydrate media (Figures 4.4A; 4.5 A). The highest activity, 2.5-2.7 U/ml, was obtained in media supplemented with olive oil or peanut oil (Figure 4.5B). When a plant oil and a carbohydrate were mixed in the same medium as carbon sources, the yield of lipase was lower than when an oil was used as a sole carbon source (data not shown). 4.3.3 Effect of nitrogen sources and initial medium pH on the growth and lipase production The effect of seven inorganic and organic nitrogen sources on the growth and extracellular lipase activity of O. piceae was examined using olive oil as a carbon source (Figure 4.6). The results showed that the highest biomass, 11-12 mg/ml, was obtained in media supplemented with tryptone and casamino acids, whereas the highest lipase activity, 2.8 U/ml, was detected in media containing ( N H ^ S C ^ . However, the pH of the media supplemented with inorganic nitrogen sources decreased rapidly during incubation (Figure 4.1), and the low pH appeared to be detrimental to lipase stability. Ill E E CO CO ca E o CD A Olv Soy Cor Sun Ses Cot Pea E > o CO CU CO 03 Q. B Olv Soy Cor Sun Ses Cot Pea Plant oils Figure 4.5 Effect of plant oils on the growth (A) and extracellular lipase activity (B) of 0. piceae. The medium, initially at pH 6.1, contained 2% (v/v) oil as carbon source, 0.5% (w/v) NH 4C1 as nitrogen source and other constituents as described in section 3.2.1. Lipase activity was assayed with olive oil emulsion at 37°C. Olv: olive oil, Soy: soybean oil, Cor: corn oil, Sun: sunflower seed oil, Ses: sesame oil, Cot: cotton seed oil, Pea: peanut oil. A l l data points are means of three determinations with standard deviations shown by error bars. 112 co E CO CO 03 E g E ;> o CO CD CO CO C L C L N 0 3 S 0 4 P e p Trp C a a Ure Ni t rogen s o u r c e s A B Figure 4.6 Effect of nitrogen sources on the growth (A) and extracellular lipase activity (B) of 0. piceae. The medium, initially at pH 6.1, contained 2% (v/v) olive oil as carbon source, 0.5% (w/v) nitrogen source and other constituents as described in section 3.2.1. Lipase activity was assayed with olive oil emulsion at 37°C. CL: ammonium chloride, N 0 3 : ammonium nitrate, S 0 4 : ammonium sulfate, Pep: peptone, Trp: tryptone, Caa: casamino acids, Ure: urea. A l l data points are means of three determinations with standard deviations shown by error bars. 113 To investigate the effect of initial medium pH, 5% peptone was added to the medium supplemented with 0.5% ( N F L ^ S C M since peptone has high buffering capacity. Table 4.2 shows that in this medium, when the initial pH was 4, 5 or 6, one fold more fungal biomass was obtained than when the initial pH was 7. The highest extracellular lipase activity (8 U/ml) was obtained when the initial medium pH was 5. In the media containing peptone, the pH of the media increased rather than decreased with fungal growth (Table 4.2). Finally, the effect of peptone concentration on both fungal growth and lipase production was studied. While the highest biomass was obtained with the highest concentration of peptone used, the enzyme activity did not markedly increase when peptone concentrations were increased above 1.5% (Table 4.3). 4.3.4 Purification of the major extracellular lipase Lipase purification started from the culture grown for 3 days in a medium supplemented with 2%o olive oil as carbon source, 0.5% ( N F L ^ S C M and 3% peptone as nitrogen sources. Washing the fungal cells with 0.1% Triton X-100 after harvesting showed that up to 60% of the extracellular lipase activity was cell-bound. Previous work showed that O. piceae produces serine proteinases which can be effectively inhibited by PMSF (Breuil and Huang 1994; Abraham and Breuil 1996). Therefore, Triton X-100 and PMSF were added to the culture immediately after harvesting to dissociate the lipase from fungal cells and to inhibit proteolytic activity, respectively. 114 Table 4.2 Effect of the initial medium pH on fungal growth and extracellular lipase activity of O. piceae\ Medium pH Biomass (mg/ml) * Lipase activity (U/ml) Initial Final* 4 5.25 21.1 ±0 .4 3.3+0.1 5 ' 5.85 22.8 ± 1.2 8.0 + 0.3 6 6.70 20.6 + 2.2 2.1 +0.3 7 7.34 10.2 + 1.8 1.8 + 0.4 f The medium contained 2% olive oil as carbon source, 0.5% (HELi^SC^ and 5% peptone as nitrogen sources, and other constituents as described in section 3.2.1. % A l l measurements were taken after 4 days' incubation, and all data points are means of three determinations with standard deviations shown by uncertainties. Lipase activity was assayed with olive oil emulsion at 37°C. 115 Table 4.3 Effect of peptone concentration on fungal growth and extracellular lipase activity of O. piceae\ Peptone (%) Biomass (mg/ml) * Lipase activity (U/ml) * 0.5 20.2 ±0 .4 2.1 ±0.6 1.0 27.2 ± 0.2 5.8 ±0 .6 1.5 27.6 + 0.2 9.3 ±0 .6 2.0 27.8 ±0.7 10.4 ±0 .6 5.0 29.4 ±0.2 10.7 + 0.5 t The medium contained 2% olive oil as carbon source, 0.5% ( N L L ^ S C M and peptone as nitrogen sources and other constituents as described in section 3.2.1. The initial medium pH was 5.0. % A l l measurements were taken after 3 days' incubation, and all data points are means of three determinations with standard deviations shown by uncertainties. Lipase activity was assayed with olive oil emulsion at 37°C. 116 When ammonium sulfate was added to the culture filtrate at 80% saturation, a sticky aggregate was formed on the surface of the solution, and up to 70% of the original lipase activity was detected in the aggregate. Part of the remaining activity was recovered from the pellet obtained by centrifugation of the solution. When the sticky aggregate and the precipitate were re-suspended in 20 m M Tris-HCl buffer (pH 8.0), a cloudy solution was obtained, which was subjected to 60% (v/v) acetone precipitation. It was found that the proteins with lipase activity precipitated at the bottom. Analysis by SDS-PAGE revealed that this precipitate mainly contained low molecular weight proteins. Except for faint discrete banding, a smear of dark background appeared after silver-staining (Figure 4.7 lane 2). The acetone precipitate was subjected to purification with hydrophobic interaction chromatography (HIC) on a Phenyl-Sepharose CL-4B column (Figure 4.8). The first elution stage consisted of a linear gradient of 30% to 0% ammonium sulfate. No lipase activity was detected in the eluted fractions (1 to 50). A small proportion of the activity was eluted by applying two bed volumes of 20 m M Tris-HCl (pH 8.0), however, most dark colored components were washed out of the column at this stage. Finally, most lipase activity was eluted from the column by applying 1% Triton X-100 in water. Analysis by SDS-PAGE showed that this step removed abundant amounts Of unwanted proteins in the acetone precipitate, and there were only a few proteins remaining in the preparation (Figure 4.7 lane 3). After clarification and concentration, the enzyme preparation was further purified by anion Figure 4.7 Separation of extracellular proteins of 0. piceae by SDS-PAGE (PhastGel 12.5%) at various stages of purification. The gel was silver-stained. Lane 1: molecular weight standards (Pharmacia), kD: kilodalton; Lane 2: acetone precipitate of the culture filtrate; lane 3: pooled fractions (80 to 85) after Phenyl-Sepharose CL-4B chromatography; lane 4: pooled fractions (24 to 33) after DEAE-Sepharose CL-6B chromatography. 118 i—i—•—i—i—i—•—i—i—i—i—i—•—i—1—i—1—i—1—i—1—i—1—i—r Fract ion number Protein; —o— Activity; Ammonium sulfate Figure 4.8 Fractionation of proteins in the culture filtrate of O. piceae by hydrophobic interaction chromatography. The enzyme solution was applied to a Phenyl-Sepharose CL-4B column (1.5x30 cm) pre-equilibrated with 20 mM Tris-HCl buffer (pH 8.0) containing 30% ammonium sulfate. The elution process consisted of 3 stages: (1) 250 ml linear gradient of 30% to 0% ammonium sulfate in 20 mM Tris-HCl buffer (fractions 1 to 50); (2) 100 ml 20 mM Tris-HCl (fractions 51 to 67); and (3) 150 ml 1% Triton X-100 in water (fractions 68 to 87). The fractions in which lipase activity was detected (80 to 85) were pooled and concentrated. Since Triton X-100 has strong absorbance at 280 nm, the protein concentration was not monitored during stage 3. The flow rate was 60 ml/h, and fraction size was approximately 5 ml. Lipase activity was assayed with PNPP at 37°C. 119 exchange chromatography (Figure 4.9). The fractions containing lipase activity were pooled, concentrated, desalted, and the proteins examined by SDS-PAGE. The final preparation showed a single band on SDS-PAGE gels at approximately 35 kDa (Figure 4.7 lane 4). The overall purification procedure is summarized in Table 4.4. The lipase was purified about 5200 fold with a final yield of 26% based on lipase activity. The F£IC step increased the purification by 75 fold, while the ammonium sulfate/acetone precipitation, and anion exchange chromatography increased the purification factor by 9 and 8 fold, respectively. The specific activity of the enzyme preparations toward the two substrates (olive oil and PNPP) before and after purification was shown in Table 4.5, which implies that each of the two assays showed consistent results. 4.3.5 Molecular weight, isoelectric point, and glycosylation The molecular weight of the purified lipase was 37 kDa as determined by size exclusion chromatography (Figure 4.1 OA), and 35 kDa as measured by SDS-PAGE (Figure 4.1 OB). In contrast to SDS-PAGE where only one protein was resolved on the gel (Figure 4.7 lane 4; Figure 4.11), 3 protein bands at pi's 4.3, 4.1 (major), and 3.8 (minor) were seen after isoelectric focusing (Figure 4.12A). A l l the bands appeared to be lipolytically active (Figure 4.12B). The purified lipase was glycosylated containing 10.1% carbohydrates, as determined by the phenol-sulfuric acid method with D-glucose as reference. 120 0 10 20 30 4 0 Fract ion number Protein; —o— Activity; NaCl Figure 4.9 Fractionation of proteins partially purified by FfJC using anion exchange chromatography. The enzyme preparation following FfJC was applied to a DEAE-Sepharose CL-6B column (1.5x30 cm) pre-equilibrated with 20 mM Tris-HCl buffer (pH 8.0). The elution was carried out with 250 ml linear gradient of 0 to 0.6 M NaCl in the buffer at a flow rate of 27 ml/h. The fraction size was 5 ml. The fractions in which lipase activity was detected (24 to 33) were pooled, concentrated, and desalted. Lipase activity was assayed with PNPP at 37°C. 121 T a b l e 4.4 Purification of the extracellular lipase produced by O. piceae. Steps Total Total Specific activity Purification Yield1^ protein (mg) * activity (U) (U/mg) fold (%) Culture filtrate 26800 6680 0.24 0 100 (NH 4 ) 2 S0 4 /acetone 2220 4680 2.11 9 70 Phenyl-Sepharose CL-4B 12.9 2040 158 658 31 DEAE-Sepharose CL-6B 1.4 1750 1250 5208 26 * Lipase activity was assayed with PNPP at 37°C. One unit (U) of lipase activity was defined as the amount of enzyme that liberated 0.1 umol of p-nitrophenol from PNPP per minute under the assay conditions. f Yield was calculated based on total activity. T a b l e 4.5 Comparison of O. piceae lipase activity on olive oil and PNPP. Specific activity PNPP Olive oil (umol p-nitrophenol/mg.min) (umol fatty acids/mg.min) Crude enzyme in culture filtrate 0.024 0.426 Purified lipase 125 2105 *A11 data points are means of three determinations with the standard deviations less than 10% of the means. 122 Figure 4.10 Estimation of the molecular weight of the purified 0. piceae lipase by size exclusion chromatography (A) and SDS-PAGE (B). A: Size exclusion chromatography by FPLC system using Superose 12 column (1x30 cm), eluted by 0.1 M acetate buffer (pH 5.5) at the flow rate of 0.25 ml/min. The elution volume was plotted against logalithum of molecular weight. Calibration standards: a: vitamin B-12 (1.35 kDa), b: horse myoglobin (17 kDa), c: chicken ovalbumin (44 kDa), d: bovine gamma globulin (158 kDa), e: bovine thyroglobulin (670 kDa). B: SDS-PAGE (PhastGel 12.5%) by PhastSystem. The relative mobility (Rf, expressed relative to bromophenol blue) was plotted against logalithum of molecular weight. Calibration standards: a: albumin (67 kDa), b: ovalbumin (43 kDa), c: carbonic anhydrase (30 kDa), d: trypsin inhibitor (20.1 kDa), e: ot-lactalbumin (14.4 kDa). The equations of linear regression were obtained using the software 'Origin' (Microcal Software Inc.). Figure 4.11 Analysis of the purified O. piceae lipase by SDS-PAGE (PhastGel 8-25%). The gel was silver-stained. Lane I: molecular weight standards (Pharmacia), kD: kilodalton; Lanes 2 to 4: purified lipase at the loading amount of 0.025, 0.2, and 1 ug, respectively. pi 93 . 8.7 -85" 8.2" 7.4-6.6- -5.9-5.2-4.6-1 2 A 1 2 B Figure 4.12 Analysis of the purified O. piceae lipase by IEF gel electrophoresis (PhastGel 3-9). A and B are identical gels, A was silver-stained, and B was detected by lipolytic stain. Lane 1: pi standard markers (Pharmacia), lane 2: 0.2 ug purified lipase preparation. 125 4.3.6 Amino acid composition and N-terminal sequence The major amino acids of the purified lipase were glycine, alanine, aspartic acid/asparagine, serine, leucine, valine, threonine, glutamic acid/glutamine, and proline (Table 4.6). The composition was compared with lipases from Geotrichum candidum, Candida cylindracea and Penicillium camembertti. The O. piceae lipase had comparable percentage of total polar/charged, nonpolar, or aliphatic amino acids to the lipases purified from G. candidum, C. cylindracea and P. camembertti, but slightly less aromatic amino acids (Table 4.6). Automated protein sequence analysis of the lipase purified from O. piceae yielded the following sequence for the first 20 amino acids: D 1 - V 2 - S 3 - V 4 - T 5 - T 6 - T 7 - D 8 - I 9 - D 1 0 - A 1 1 - L 1 2 - A 1 3 -F 1 4 _ F 1 5 _ T 1 6 - Q 1 7 - W 1 8 - A 1 9 - G 2 0 , which was compared with that of the proteins in the "non-redundant" protein database [National Center of Biotechnology Information, National Library of Medicine, National Institute of Health (NTH), Bethesda, Maryland, USA] through the B L A S T electronic mail server. No homology was found to the proteins listed in the database. 4.3.7 Effects of pH and temperature on the activity and stability of the lipase When olive oil was used as substrate, the optimum pH and temperature for the activity of the purified lipase was about pH 5.2 (Figure 4.13A) and 30°C (Figure 4.14A), respectively. The lipase was stable over the pH range of 4 to 8 (Figure 4.13B) and at temperatures lower than 40°C (Figure 4.14B). 126 Table 4.6 Amino acid composition of the extracellular lipase purified from O. piceae compared with that reported for other fungal lipases (mole %). Ophiostoma Geotrichum Candida Penicillium 1 2 3 piceae* candidum cylindracea camembertii Amino acids Lipase A Lipase B Lipase A Lipase B Polar and charged Lys 3.3 4.0 4.3 3.6 4.14 3.79 Arg 2.4 3.7 2.6 2.6 2.18 2.16 Asp/Asn 9.7 12.1 13.1 12.7 12.52 12.39 Glu/Gln 6.6 7.0 6.6 7.3 7.21 6.19 Ser 8.9 8.1 8.2 8.8 5.55 7.02 Thr 7.7 4.8 5.5 6.5 6.87 7.16 Aromatic His 1.7 2.0 1.3 0.8 1.67 1.73 Phe 4.2 5.5 5.5 5.7 4.27 4.04 Tyr 2.9 4.8 3.9 4.7 5.49 5.18 Trp nd 1.3 nd nd 1.11 1.23 Nonpolar Gly 13.1 10.1 11.2 10.8 11.74 12.60 Pro 6.5 6.1 7.2 7.0 4.36 5.14 Cys 1.0 0.9 nd nd 1.39 1.31 Met 0.3 2.2 2.7 . 2.5 0.37 0.34 Aliphatic Ala 11.8 8.1 9.7 9.1 10.81 10.87 Val 7.7 5.7 4.7 4.6 9.14 8.53 Leu 8.8 9.0 8.9 8.9 8.30 7.67 He 3.4 4.6 4.5 4.3 2.88 2.63 * All data points are means of two determinations. 1. Shimadae^ a/. 1989. 2. Raaetal. 1993. 3. Yamaguchi and Mase 1991. nd: not determined. 1 127 Figure 4.13 Effects of pH on the purified O. piceae lipase activity (A) and stability (B). Lipase activity was assayed with olive oil emulsion at 35°C. For stability studies the enzyme was incubated in 0.1 M acetic acid/MES/Tris buffer at different pH's at 4°C for 24 h. After incubation, the remaining activity was detemiined at pH 5.4. The curve in graph A was plotted by fitting the data with Gaussian equation using the software 'Origin' (Microcal Software Inc.). 128 -t—> ;> +-> o CO 0 .> ro 0 B Temperature (°C) Figure 4.14 Effects of temperature on the purified 0. piceae lipase activity (A) and stability (B). Lipase activity was assayed with olive oil emulsion at pH 5.4. For stability studies, the enzyme was incubated in 0.1 M Bis-Tris buffer for 15 min at different temperatures. After incubation, the remaining activity was assayed at 35°C. 129 4.4 Discussion The assay of lipase activity has been commonly carried out with triglycerides from natural (e.g., olive oil) or synthetic (e.g., triolein) sources. It has also been frequently measured by artificial substrates, such as water-insoluble long-chain />-nitrophenyl esters (Winkler and Stuckmann 1979; Kordel et al. 1991; Shabtai and Daya-Mishne 1992; Markweg-Hanke et al. 1995). One of the advantages of using /?-nitrophenyl esters is that the liberated product p-nitrophenol can be directly monitored photometrically. This method is especially useful when pH-stat equipment is not available, and the quantification of fatty acids (in the case of triglycerides as substrates) has to be achieved by tedious manual titration or by using the Duncombe (1963) procedure. In this study, both PNPP and olive oil assays were used in various experiments. Each of the two assays showed consistent results. The PNPP assay proved to be very convenient for monitoring the lipase activity during the laborious purification process. Lipase production may vary widely with the microbial species, the stage of growth and development, and the medium composition (Papaparaskevas et al. 1992; Sztajer and Maliszewska 1989). Since the aim of this study was to understand the production of lipolytic enzymes by 0. piceae in wood, media were designed with carbon sources that are known to be present in wood, e.g., triglycerides and sugars. A l l the plant oils used were composed predominantly of the unsaturated fatty acids, oleic and linoleic acids (Patterson 1989), similar to those found in the wood triglycerides (section 2.3.3). The results indicated that plant oils 130 were better carbon sources than carbohydrates for inducing extracellular lipase production by O. piceae. This conclusion was in agreement with those reported for Candida deformans (Muderhwa et al. 1985), Rhizopus oligosporus (Nahas 1988), Rhodotorula glutinis (Papaparaskevas et al. 1992), and Calvatia gigantea (Christakopoulos et al. 1992). The lipase activity of O. piceae in culture filtrates increased steadily during the exponential growth phase, and rapidly declined at the stationary phase. A similar lipase production pattern has been reported for other microorganisms (Pal et al. 1978; Chander et al. 1980; Samad et al. 1990; Christakopoulos et al. 1992; Ohnishi et al. 1994a). The rapid decrease in lipase activity at stationary phase might be caused either by the degradation of the lipase by proteases secreted into the media (Abraham et al. 1993; Ohnishi et al. 1994a), or by the change of pH in the medium. Nitrogen sources in culture media are also known to play an important role in inducing lipase production (Iwai and Tsujisaka 1984). Some reports indicated that peptone was the preferred nitrogen source for lipase production by microorganisms (Tsujisaka et al. 1973; Nakashima et al. 1988; Salleh et al. 1993). Other reports showed that ammonium phosphate was better than organic nitrogen sources (Papaparaskevas et al. 1992; Christakopoulos et al. 1992). The results from this study showed that for O. piceae cultures, when inorganic nitrogen sources (ammonium salts) were used, the pH of the media decreased rapidly during cultivation, and the low pH was harmful to the lipase (the purified lipase was unstable at pH's below 4). Therefore, it is possible that it is the low medium pH value rather than the nitrogen sources that has a greater influence on lipase production. Peptone is well known to have high 131 buffering capacity when used in a culture medium (Iwai and Tsujisaka 1984; Samad et al. 1990; Baillargeon and McCarthy 1991; Ohnishi et al. 1994a). For O. piceae, when peptone was added to the medium containing 0.5% ammonium sulfate, the pH of the medium increased slightly with the fungal growth, and the lipase activity increased by more than 3 fold. However, when peptone was used as the sole nitrogen source, the extracellular lipase activity was comparatively low, which might be due to inactivation of the lipase by proteases, because the production of proteases was enhanced in the absence of ammonium salts (Abraham et al. 1993; Breuil and Huang 1994). To purify a lipase from lipid-abundant medium, de-lipidation is usually considered to be a crucial factor (Aires-Barros et al. 1994). It was observed in this study that during ammonium sulfate fractionation of the culture filtrate containing olive oil as carbon source, the majority of the lipase was driven to the surface of the solution, forming a brownish sticky aggregate. The brownish color was probably a result of the peptone components used in the culture medium. When the sticky aggregate was re-suspended in buffer, a cloudy solution was obtained, which could not be clarified with treatments such as extended incubation at 30°C, dialysis, or by passing through an Econo-Pac 10 D G column (Bio-Rad). The aggregate was possibly a tight lipid-protein complex such that the lipids were very difficult to separate from the proteins. Similar problems have been encountered during the purification of lipases from lipid-rich tissues such as the pancreas and adipose (Verger et al. 1969; Fredrikson et al. 1981; Aires-Barros and Cabral 1991). Different organic solvents have been used to reduce lipid contamination (Aires-Barros et al. 1994). Acetone was used in this study to precipitate the 132 enzyme following ammonium sulfate fractionation. However, this step could only partially remove the lipids from the proteins. The dark smear appeared on SDS-PAGE gels after silver staining might be attributed to the presence of lipids in the preparation. HIC was shown to be a very powerful step in the purification of the lipase from O. piceae. The column acted like an affinity column due to the high hydrophobicity of the lipase. The enzyme could not be eluted even with 20 m M Tris-HCl buffer. By taking advantage of this property, large amounts of contaminating proteins and brownish peptone components were removed during this step. The elution of the lipase from the column had to be achieved with a non-ionic detergent, Triton X-100. A similar case has been reported with a lipase produced by Pseudomonas fluoresceins (Sztajer et al. 1991). The similar values of the molecular weight obtained for the lipase purified from O. piceae in both denatured (SDS-PAGE) and native (size exclusion chromatography) states indicated that the enzyme was a monomer. Glycosylation is known to be a common feature for fungal lipases. Most fungal lipases have been reported to contain 3% to 15% carbohydrates (Tsujisaka et al. 1973; Huge-Jensen et al. 1987; Baillargeon 1990; Baillargeon and McCarthy 1991; Phillips and Pretorius 1991; Sidebottom et al. 1991; Rua et al. 1993; Ohnishi et al. 1994b). It has been recognized that different degrees of glycosylation of lipases could cause isoforms of an enzyme, which differ in pi value (Baillargeon 1990; Hedrich et al. 1991; Sidebottom et al. 1991). In fact, pi microheterogeneity has been widely reported with fungal lipases (Jacobsen et al. 1989; Baillargeon 1990; Veeraragavan et al. 1990; Hedrich et al. 1991; Baillargeon and 133 McCarthy 1991; Rua et al. 1993; Uyttenbroeck et al. 1993) and mammalian lipases (Verger 1984; Moreau et al. 1988). In this study, the purified O. piceae lipase was resolved as a single band on SDS-PAGE gels, whereas 3 bands were observed on IEF gels. Lipolytic stain demonstrated that all the three bands were lipolytically active. N-terminal sequencing of the first 20 amino acid residues clearly showed that the enzyme preparation had an unambiguous N-terminus. If the isoforms had different N-terminal sequences, multiple signals would appear during the automated sequencing process, which was not observed. The lipase was determined to contain 10.1% carbohydrates. Therefore, it seems that the pi microheterogeneity of the purified O. piceae lipase was caused by the modification of glycosylation. The degree of similarity of the primary amino acid sequence between different lipases is very low except for a few common features, such as the G-X-S-X-G consensus sequence (where X is any residue), which is the substrate-binding site (Svendsen 1994). The N-terminal sequence of the purified O. piceae lipase showed no homology to lipases purified from Aspergillus niger (Torossian and Bell 1991), Candida cylindracea (Rua et al. 1993), Rhizopus niveus (Kohno et al. 1994), and Pseudomonas sp. A T C C 21808 (Kordel et al. 1991). Furthermore, no homology was found in a search of the "non-redundant" protein database (NIH). The optimum pH for the purified O. piceae lipase was about 5.2, which might reflect the natural adaptation of 0. piceae to the wood microniche because most wood tissues have a low pH values of 4 to 6. Similar pH optima were reported for lipases from Rhizopus delemar (Iwai and Tsujisaka 1974). For other microbial lipases, pH optima ranging from 5.5 to 9 have been 134 reported (Seitz 1974; Sugiura et al. 1977; Muraoka et al. 1982; Muderhwa et al. 1985; Christakopoulos et al. 1992; Savitha and Ratledge 1992). The temperature optimum of O. piceae lipase was 30°C, which was consistent with the mesophilic characteristics of this species. In summary, in this chapter lipolytic activities by the sapstaining fungus O. piceae were studied. A medium composition was optimized for a high yield of lipase production by O. piceae. A n extracellular lipase was purified to homogeneity by using ammonium sulfate/acetone precipitation, hydrophobic interaction chromatography, and anion exchange chromatography. The purified lipase was then characterized in terms of its molecular weight, pi, amino acid composition, N-terminal sequence, optimal pH and temperature for activity, pH stability and thermostability. 135 Chapter 5 Substrate Specificities of the Lipase Purified from O. piceae and Effects of Various Chemical Reagents on the Enzyme Activity 5.1 Introduction As presented in chapter 4, an extracellular lipase secreted by O. piceae was purified and characterized. To understand the functions of the lipase in the utilization of lipophilic nutrients by O. piceae colonizing wood, it was important to study the various factors affecting the enzyme activity, and its mode of action on substrates. In chapter 4 the effects of pH and temperature on the lipase activity and stability were examined. Besides pH and temperature, various chemical reagents may also greatly affect the lipase-catalyzed hydrolysis (lipolysis) reactions. Generally, lipases differ from most other enzymes because the natural substrates of lipases are insoluble in water and their activity is maximal only when the enzyme is adsorbed to the oil-water interface (Derewenda and Sharp 1993). Therefore, the velocity of the lipolysis reaction is a function of the substrate surface area, and consequently, factors influencing the interface area wil l affect the reaction rate (Patkar and Bjorkling 1994). Lipase activity is also affected by reagents which react with and modify amino acid residues, particularly those at the active site. Recent studies have shown that the active sites of several lipases contain a catalytic triad similar to that found in serine proteases. The triad of lipases 136 from Rhizomucor miehei, Pseudomonas glumae, and human pancreas contains Ser-His-Asp (Brady et al. 1990; Winkler et al. 1990; Noble et al. 1994), and the triad of the lipase from Geotrichum candidum contains Ser-His-Glu (Schrag etal. 1991). Organophosphate compounds can covalently bind to the active site serine, so they are frequently used as active-site-directed inhibitors of serine proteases (Salvesen and Nagase 1989). Similarly, organophosphates were reported to inhibit the activity of several lipases (Brady et al. 1990; Schrag et al. 1991; Derewenda et al. 1992). Besides organophosphates, many other reagents, such as metal ions, surface-active detergents, organic solvents, and fatty acids have been reported to influence lipolysis rates (Wills 1954; Iwai et al. 1964; Muraoka et al. 1982; Derewenda et al. 1992; Shabtai and Daya-Mishne 1992; Markweg-Hanke et al. 1995). Some reagents may affect the interface area, while others may affect enzyme structure or enzyme-substrate interactions. Since lipases from different sources were reported to have versatile properties and sensitivities to various chemical reagents (Macrae 1983; Iwai and Tsujisaka 1984; Sugiura 1984; Aires-Barros et al. 1994; Jaeger et al. 1994), one objective of this chapter was to investigate the effects of some common chemical reagents on the activity of the newly purified lipase from O. piceae. As discussed in chapter 4, lipase activity could be assayed using either the true substrates triglycerides or the artificial substrates, such as water-insoluble long-chain/?-nitrophenyl esters. When triglycerides are used as substrates, lipase activity is usually measured by quantifying the fatty acids liberated with an automatic titration system (pH-stat). In this study, because pH-stat equipment was not available, PNPL was used as the substrate for examining the effects of chemical reagents on the 137 lipase activity. In this assay, the reaction product />-nitrophenol was measured sp ectrophotometrically. The second objective of this chapter was to examine the fatty acid specificity and positional specificity of the lipase purified from O. piceae. Lipases may show specificity with respect to two parts of their substrates: the fatty acyl or alcohol parts. These are referred to as fatty acid specificity and positional specificity, respectively. Fatty acid specificity refers to the enzyme selectivity on the chain length of fatty acid residues in the substrates. Positional specificity reflects the preference for cleavage on the three positions of the glycerol moiety of a triglyceride. Knowledge of the positional specificity of a lipase is of practical value in oleochemistry (Yokozeki et al. 1982) and organic synthesis (Wang et al. 1988). It would also further enhance our understanding of the mechanism of the lipase functions, and the information might be useful for developing control strategies. Although lipases primarily hydrolyze acyl glycerol ester bonds, some lipases have been reported to hydrolyze the acyl ester linkages of waxes (Misra et al. 1983). In chapter 3 it was shown that when O. piceae grew on the sapwood of lodgepole pine or trembling aspen, the wood triglycerides decreased rapidly, and the amount of wood waxes and steryl esters also dropped. It would be informative to assess the hydrolysis rates of these natural compounds by the lipase purified from O. piceae in vitro. However, it is difficult to separate wood waxes from steryl esters by either column chromatography or the solid phase extraction technique described in chapter 2. Thus, this study compared the hydrolysis rates of the O. piceae lipase on the 138 triglycerides, waxes, and cholesteryl esters from synthetic sources, which have similar fatty acid compositions to those found in wood. Finally, the cleavage of the lipase on triglycerides isolated from trembling aspen wood was examined. 5.2 Materials and Methods 5.2.1 Lipase preparation and activity assay The extracellular lipase of O. piceae 387N was purified as described in section 4.2.3. Lipase activity was assayed either with />-nitrophenyl esters, or with triglycerides, waxes, and cholesteryl esters. Different techniques were used to determine the reaction products generated. When/?-nitrophenyl esters were used as substrates, the activity was measured by quantifying p-nitrophenol liberated by spectrophotometry. When triglycerides, waxes, and steryl esters were used as substrates, the activity was measured by quantifying the released fatty acids by GC as described in section 5.2.6. 5.2.2 Investigation of different chemical reagents on lipase activity Effects of organic solvents, N-ethylmaleimide, reducing agents, metal ions, fatty acids, SDS, EDTA, and several inhibitors on the lipase-catalyzed lipolysis reactions were examined. The substrate PNPL was dissolved in DMSO, which was mixed with 0.05 M acetate buffer (pH 5.4) containing the reagents investigated. The mixture was pre-warmed in a water bath at 30°C 139 before the lipase was added. Immediately after the enzyme was added and mixed, the absorbance changes at 404 nm was recorded continuously in a spectrophotometer. The amount of the hydrolysis product /?-nitrophenol was calibrated with a standard. 5.2.3 Examination of fatty acid specificity and positional specificity The fatty acid specificity of the purified lipase was investigated using />-nitrophenyl esters containing fatty acid residues of various chain-lengths (C2 to C18). A l l the ester substrates were dissolved in DMSO at 15 mM. The reaction mixture comprised 100 ul of the p-nitrophenyl ester/DMSO solution, 1.9 ml of 0.05 M acetate buffer (pH 5.4) containing 0.4% (v/v) Triton X-100 and 0.2% (w/v) gum arabic. The mixture was pre-incubated in a water bath at 30°C before 5 ug purified lipase was added. The reaction product /?-nitrophenol was determined as described in section 5.2.2. The positional specificity of the lipase was examined by T L C analysis of the enzymatic reaction products using triolein (of 99% purity, Sigma Chemical Co.) as substrate. The reaction mixture containing 0.2 ml triolein (150 mg), 1.8 ml of 0.05 M acetate buffer (pH 5.4), and 3.4 ug lipase was incubated at 30°C with shaking at 300 rpm. Samples were taken at 10 min intervals, for 120 min. Immediately after each incubation, 10 ml diethyl ether was added and the solution mixed vigorously. The mixture was rapidly cooled to -70°C, and the diethyl ether layer was then decanted. Aliquots of the diethyl ether extracts were applied to a silica gel plate (20x20 cm, layer thickness 250 um, Whatman International Ltd, Kent, England), and subjected 140 to a two-stage development procedure (slightly modified after Bilyk et al. 1991): (1) toluene : diethyl ether : ethyl acetate : acetic acid (80:10:10:1, v/v); and (2) hexane : diethyl ether : acetic acid (80:20:2, v/v). Spots were visualized by dipping the plate in a solution containing 5% (w/v) ammonium molybdate, 75% (v/v) ethanol, and 1% (v/v) sulfuric acid, then heating the plate in an oven at 105°C. 5.2.4 Hydrolysis of synthetic triglycerides, waxes, and cholesteryl esters The standard substrates, purchased from Sigma Chemical Co., were (1) triglycerides including l,2-dipalmitoyl-3-oleoyl-rac-glycerol (C16:0/C16:0/C18:l, cis-9, abbreviated as PPO) and 1,3-dipalmitoyl-2-oleoyl-glycerol (C16:0/C18:l,cis-9/C16:0, abbreviated as POP); (2) waxes including lauric acid oleyl ester, palmitic acid oleyl ester, and oleic acid oleyl ester; (3) cholesteryl esters including cholesteryl myristate, cholesteryl palmitate, and cholesteryl oleate. Each substrate (150 mg) was dissolved in 0.4 ml acetone, and mixed with 4 ml 0.05 M acetate buffer (pH 5.4) containing 0.2% (w/v) gum arabic and 10 m M CaCl 2 . Each reaction mixture received 6.8 ug lipase, and the samples were shaken (300 rpm) at 30°C for 3 h. Immediately after incubation, the lipids were extracted with 25 ml diethyl ether:hexane (1:1, v/v), and the. analyses of the fatty acids released were performed as described in section 5.2.6. 141 5.2.5 Isolation, saponification, and hydrolysis of wood triglycerides Triglycerides were isolated from the total extractives of trembling aspen sapwood by SPE, and the purity was examined by TLC analysis as described in sections 2.2.3 and 2.2.4. The triglycerides obtained (5.7 mg per treatment) were saponified with K O H as described in section 2.2.7. To study lipase hydrolysis, 5.7 mg wood triglycerides were dissolved in 100 ul acetone, and mixed with 2 ml 0.05 M acetate buffer (pH 5.4) containing 0.2% gum arabic and 10 m M CaCl 2 . After adding 17 ug of the purified lipase, the reaction was incubated with shaking (300 rpm) at 30°C for 2 h. After incubation, the hydrolysis products were immediately extracted with 25 ml diethyl ether:hexane (1:1, v/v) and the analyses of fatty acids released were carried out as described in section 5.2.6. 5.2.6 Isolation, identification and quantification of fatty acids released The diethyl ethenhexane extracts of the above enzyme hydrolysis and saponification treatments (sections 5.2.4 and 5.2.5) were dried with a rotary evaporation apparatus, and re-dissolved in chloroform. Free fatty acids from the chloroform solutions were isolated by SPE as described in section 2.2.3. The free fatty acids obtained were methylated as described in section 2.2.6, and then identified and quantified with GC. The GC temperature programming and operating conditions were the same as described in section 2.2.5, but a DB-17 fused silica capillary column (length 30 m, diameter 0.25 mm, J & W Scientific) was used, instead of a DB-5 142 column. Methyl esters of fatty acids (all of 99% purity, Sigma Chemical Co.) were used to identify and calibrate the various fatty acids. 5.3 Results 5.3.1 Effects of chemical reagents on the lipase activity 5.3.1.1 Organic solvents PNPL was used as a substrate for the O. piceae lipase. Since PNPL is insoluble in aqueous buffers, it was dissolved in organic solvents prior to mixing with the reaction buffer. Triton X -100 and gum arabic were added to increase the dispersion of PNPL in the buffer. When PNPL dissolved in an organic solvent was mixed with the acetate buffer containing Triton X-100 and gum arabic, the mixture was an emulsion-like cloudy solution at room temperature. However, this mixture became clear at temperatures above approximately 28°C, which made it possible to measure the reaction photometrically. This assay system enabled the continuous monitoring of the release of the reaction product/>-nitrophenol using a spectrophotometer. The effect of several organic solvents on the lipase activity was examined. As illustrated in Figure 5.1, the highest lipase activity was obtained when the substrate was prepared in DMSO, followed by acetone and acetonitrile (MeCN), and lowest in isopropanol and tetrahydrofuran (THF). Compared with the enzyme activity in DMSO, the activity in acetone, MeCN, isopropanol, and THF decreased by 40%>, 58%, 74%, and 90%, respectively (Figure 5.1). n — • — r i — 1 — r 2.0. 1.5-1.0. 0.5. 0.0. — • — D M S O • A c e t o n e — A — M e C N — T — Isopropanol _ * _ T H F m/ A 20 ' 40 ' 60 ' 80 ' 100 ' 120 ' 140 ' 160 ' 180 ' 200 -20 Time (second) Figure 5.1 Effects of organic solvents (10%, v/v) on the O. piceae lipase activity. The substrate was prepared by mixing 200 ul 15 mM PNPL dissolved in the different solvents examined, with 1.8 ml 0.05 M acetate buffer (pH 5.4) containing 0.4% (v/v) Triton X-100 and 0.2% (w/v) gum arabic. The mixture was pre-warmed in a water bath at 30°C before 17 ug lipase was added. Since PNPL was insoluble in buffers without organic solvents, a control (without any solvent) could not be performed. A l l data points are means of three determinations with standard deviations less than 10% of the means. PNPL: j9-nitrophenyl laurate, DMSO: dimethyl sulfoxide, MeCN: acetonitrile, THF: tetrahydrofuran. 144 Since the highest activity was recorded with DMSO, the effect of DMSO concentrations on the enzyme activity was further investigated, and the maximum reaction rate was obtained at 20% to 30% (v/v) (Figure 5.2). 5.3.1.2 Sulfhydryl agent and reducing agents A'-ethylmaleimide (NEM) is a sulfhydryl agent which can covalently bind to the sulfhydryl groups in enzymes. Dithiothreitol (DTT) and B M E are two common reducing agents which can be used to break down disulfide bonds in enzyme structures. The results showed that N E M (10 mM), DTT (2 mM) and B M E (2 mM) had no detectable influence on the O. piceae lipase activity (Table 5.1). 5.3.1.3 Metal ions and fatty acids The effects of several metal ions at a concentration of 20 mM on the purified O. piceae lipase activity are shown in Figure 5.3. Ca and Mn enhanced the activity by approximately 30%, Y 2~i~ 2~i~ 2~i~ 3 ] and K , Co and M g appeared to slightly increase the activity, whereas Hg and Fe completely inactivated the enzyme. Butyric acid and caproic acid at a concentration of 20 m M exerted strong inhibition on the lipase activity 4 min after the reaction was started (Figure 5.4). Long chain fatty acids could not be tested because they are not miscible with the reaction mixture employed. 145 T r — - 1 1 • 1 • 1 • 1 • 1 ' 1 ' 1 r D M S O concentrat ion (%, v/v) Figure 5.2 Effects of DMSO concentrations on the 0. piceae lipase activity. The reaction rnixture was prepared by adding 50 ul 15 mM PNPL in DMSO, appropriate volume of DMSO for each treatment, and 0.05 M acetate buffer (pH 5.4) containing 0.4% (v/v) Triton X-100 and 0.2% (w/v) gum arabic to a final volume of 2 ml. The mixture was pre-warmed in a water bath at 30°C before 5 ug lipase was added. The data points shown are the relative activities after 3 min. PNPL: p-nitrophenyl laurate, DMSO: dimethyl sulfoxide. 146 Table 5.1 Effects of N E M , DTT and B M E on the O. piceae lipase activity. j9-Nitrophenol liberated (umol) Time Control N E M DTT B M E (min) (10 mM) (2 mM) (2 mM) 1 0.14 0.13 0.15 0.15 2 0.29 0.27 0.31 0.31 3 0.45 0.43 0.48 0.47 4 0.61 0.58 0.64 0.62 5 0.76 0.74 0.80 0.77 6 0.89 0.87 0.94 0.90 7 1.01 1.00 1.06 1.00 8 1.10 1.10 1.17 1.10 9 1.18 1.19 1.25 1.17 10 1.25 1.27 1.33 1.24 The reaction mixture was prepared by adding 100 ul 15 mM PNPL in DMSO, appropriate amount of the reagent stock solutions, and 0.05 M acetate buffer (pH 5.4) containing 0.4% (v/v) Triton X-100 and 0.2% (w/v) gum arabic to a final volume of 2 ml. The mixture was pre-warmed in a water bath at 30°C before 5 ug lipase was added. A l l data points are means of three determinations with standard deviations less than 10% of the means. PNPL: p-nitrophenyl laurate, DMSO: dimethyl sulfoxide, NEM: N-ethylmaleimide, BME: p-mercaptoethanol, DTT: dithiothreitol. Time (min) Figure 5.3 Effects of metal ions (20 mM) on the 0. piceae lipase activity. The reaction mixture was prepared by adding 100 ul 15 mM PNPL in DMSO, appropriate amount of stock solution of salts, and 0.05 M acetate buffer (pH 5.4) containing 0.4% (v/v) Triton X-100 to a final volume of 2 ml. The mixture was pre-warmed in a water bath at 30°C before 5 ug lipase was added. Al l data points are means of three determinations with standard deviations less than 10% of the means. PNPL: /?-nitrophenyl laurate, DMSO: dimethyl sulfoxide. o E T3 ro CD O c CD .c Q. O 1.4-1.2-1.0-0.8-0.6. 0.4-0.2-0.0--0.2. i • r • ' • • • • • • - • - - Control -v— Butyric acid 20 mM - • — Capro ic acid 20 mM 0 - r -4 —r~ 6 i 8 10 Time (min) Figure 5.4 Effects of fatty acids on the O. piceae lipase activity. The reaction mixture was prepared by adding 100 ul 15 mM PNPL in DMSO, appropriate amount of the fatty acid stock solutions, and 0.05 M acetate buffer (pH 5.4) containing 0.4% (v/v) Triton X-100 and 0.2% (w/v) gum arabic to a final volume of 2 ml. The mixture was pre-warmed in a water bath at 30°C before 5 u.g lipase was added. A l l data points are means of three determinations with standard deviations less than 10% of the means. PNPL: /?-nitrophenyl laurate, DMSO: dimethyl sulfoxide. 149 5.3.1.4 Ionic detergent and chelating agent A n ionic detergent (SDS) and a chelating agent (EDTA) inhibited lipase activity in a time-dependent manner (Figure 5.5). At low concentrations, e.g., 5 m M SDS or 10 m M EDTA, inhibition on the enzyme activity was not observed until 8 min after the enzyme was added. Increases in SDS or E D T A concentrations increased the inhibitory effect. As shown in Figure 5.5, in the reaction mixtures containing 25 m M SDS or 20 mM EDTA, a drop in lipase activity was observed 3 min after the enzyme was added, and the activity appeared to be completely inhibited after 7 min. 5.3.1.5 Enzyme inhibitors Lipase activity was examined in the presence of three inhibitors: PMSF, E600, and diethyl pyrocarbonate (DEPC). The lipase was most sensitive to DEPC as shown in Figure 5.6. The enzyme activity was completely inhibited in 4 min by 2 m M DEPC, while 5 m M DEPC immediately inhibited the enzyme activity. E600 also strongly inhibited the lipase, 100% inhibition was observed at 10 m M in 5 min (Figure 5.6). The lipase was less sensitive to PMSF, 5 m M slightly inhibited the activity after 8 min. Time (min) Figure 5.5 Effects of SDS and E D T A on the 0. piceae lipase activity. The reaction mixture was prepared by adding 100 ul 15 mM PNPL in DMSO, appropriate amount of SDS or EDTA stock solution, and 0.05 M acetate buffer (pH 5.4) containing 0.4% Triton X-100, 0.2% (w/v) gum arabic and 10 mM CaCl 2 to a final volume of 2 ml. The mixture was pre-warmed in a water bath at 30°C before 5 ug lipase was added. Al l data points are means of three determinations with standard deviations less than 10% of the means. PNPL: /7-nitrophenyl laurate, DMSO. dimethyl sulfoxide, SDS: sodium dodecyl sulfate, EDTA: ethylenediamine tetraacetate. Time (min) Figure 5.6 Effects of different inhibitors on the 0. piceae lipase activity. The reaction mixture was prepared by adding 100 ul 15 mM PNPL in DMSO, appropriate amount of stock solutions of inhibitors, and 0.05 M acetate buffer (pH 5.4) containing 0.4% (v/v) Triton X-100 and 0.2% (w/v) gum arabic to a final volume of 2 ml. The mixture was pre-warmed in a water bath at 30°C before 5 ug lipase was added. A l l data points are means of three determinations with standard deviations less than 10%o of the means. PNPL: p-nitrophenyl laurate, DMSO: dimethyl sulfoxide, PMSF: phenylmethylsulfonyl fluoride, E600: diethyl /?-nitrophenyl phosphate, DEPC: diethyl pyrocarbonate. 152 5.3.2 Fatty acid specificity and positional specificity The relative activities of 0. piceae lipase toward the p-nitrophenyl esters with fatty acid residues of various chain lengths are shown in Figure 5.7. The lipase showed very low activity toward fatty acid esters with chain lengths shorter than 6 carbons, i.e., acetate, butyrate, and caproate, whereas high activity was recorded on esters with intermediate or long fatty acid chains, especially caprate, laurate and myristate. When triolein was hydrolyzed by the purified lipase, the thin layer chromatograms obtained showed that 1,2 (2,3)-diolein was detected, while 1,3-diolein was not (Figure 5.8). This suggested that the O. piceae lipase did not hydrolyze the ester bond at position 2 of the triglyceride. 5.3.3 Hydrolysis of the synthetic triglycerides, waxes, and cholesteryl esters The lipase had much higher hydrolysis rates on triglycerides than on waxes and cholesteryl esters (Table 5.2). The cleavage rate for POP was almost double of that for PPO. The ratio of palmitic to oleic acids released in the hydrolysis was 1.1 to 1 for PPO, and 1.8 to 1 for POP (Table 5.2). The relative hydrolysis rate of the lipase for the waxes, i.e., lauric acid oleyl ester, palmitic acid oleyl ester, and oleic acid oleyl ester, was only 3% to 6% of the rate for POP. Similarly the hydrolysis rate was low for the cholesteryl esters, less than 2% of that for POP (Table 5.2). 153 120- i 1 1 1 p 100J Co 80-\ "I 60^ CO I 40-1 CD 20 J .0- 2 ' 4 ' 6 ' 10 12 14 16 18 Carbon number of the constituent fatty acid Figure 5.7 Fatty acid specificity of the O. piceae lipase. The substrate was prepared by mixing 100 u.1 15 mM p-nitrophenyl esters dissolved in DMSO with 1.9 ml 0.05 M acetate buffer (pH 5.4) containing 0.4% (v/v) Triton X -100 and 0.2% (w/v) gum arabic. The mixture was pre-warmed in a water bath at 30°C before 5 ug lipase was added. The relative activity for each substrate is expressed as the percentage of activity on p>-nitrophenyl laurate. Notes: 2 (p-nitrophenyl acetate), 4 (p-nitrophenyl butyrate), 6 (p-nitrophenyl caproate), 10 (p-nitrophenyl caprate), 12 (p-nitrophenyl laurate), 14 (p-nitrophenyl myristate), 16 (p-nitrophenyl palmitate), 18 (/>-nitrophenyl stearate). DMSO: dimethyl sulfoxide. A l l data points are means of three determinations with standard deviations shown by error bars. 154 Figure 5.8 TLC chromatograms of the hydrolysis products obtained through the action of the O. piceae lipase on triolein. Lane 1: standard compounds, a: triolein, b: oleic acid, c: 1,3-diolein, d: 1,2 (2,3)-diolein, e: 2-monoolein; lane 2: triolein before adding lipase; lanes 3, 4, 5, 6 are hydrolysis products of triolein after 10, 30, 50, and 90 min, respectively. 155 Table 5.2 Hydrolysis of synthetic triglycerides, waxes, and cholesteryl esters by 0. piceae lipase. Substrates* Fatty acids released* (umol/h mg enzyme) Relative activity (%)§ Triglycerides PPO 16:0 18:1 105.3 92.2 total 197.5 56.3 POP 16:0 18:1 224.0 126.5 total 350.5 100.0 Waxes W120 12:0 12.3 3.5 W160 16:0 20.1 5.7 W181 18:1 15.7 4.5 Cholesteryl esters CE140 14:0 1.0 0.3 CE160 16:0 5.4 1.5 CE181 18:1 5.9 1.7 t Abbreviations: PPO: l,2-dipalmitoyl-3-oleoyl-rac-glycerol (C16:0/C16:0/C18:l, cis-9), POP: 1,3-dipalmitoyl-2-oleoyl-glycerol (C16:0/C18:l,cis-9/C16:0), W120: lauric acid oleyl ester, W160: palmitic acid oleyl ester, W181: oleic acid oleyl ester, CE140: cholesteryl myristate, CE160: cholesteryl palmitate, CE181: cholesteryl oleate. | A l l data points are means of two determinations with standard deviations less than 10% of the means. § Calculated by taking total fatty acids released from POP as 100%. 5.3.4 Hydrolysis of the triglycerides isolated from trembling aspen wood 156 Alkaline saponification with K O H was used to non-selectively release all fatty acid residues from triglycerides. The triglycerides in trembling aspen sapwood were composed predominantly of linoleic acid (18:2), which accounted for 88.7% of the total fatty acid residues identified (Table 5.3). Other fatty acid residues detected include linolenic, stearic, oleic, palmitic, myristic, eicosenoic and eicosadienoic acids (Figure 5.9, Table 5.3). Treatment of the wood triglycerides with the purified lipase from O. piceae released fatty acid residues in a similar proportion to that obtained by alkaline saponification (Table 5.3). 157 Table 5.3 Hydrolysis of the triglycerides isolated from trembling aspen sapwood by saponification and by the lipase purified from O. piceae. Fatty acids released§ Relative percentage (mole %) ug nmol Saponification treatment* 14:0 4.6 19.1 0.2 16:0 68.2 252.3 2.6 18:0, 18:1* 102.2 344.8 3.5 18:2 2552.0 8665.7 88.7 18:3 110.8 378.7 3.9 20:1 14.5 44.7 0.5 20:2 19.6 60.9 0.6 total 2871.9 9766.2 ceae lipase treatment* 14:0 4.1 16.9 0.5 16:0 38.3 141.7 4.5 18:0, 18:1* 68.8 232.2 7.3 18:2 774.7 2630.6 82.7 18:3 36.7 125.6 3.9 20:1 10.8 33.4 1.1 20:2 nd nd -total 933.4 3180.4 t Wood triglycerides (5.7 mg) were dissolved in 200 ul acetone and mixed with 5 ml 0.5 M KOH in 90% ethanol, which was saponified at 70°C for 2 h. % Wood triglycerides (5.7 mg) were dissolved in 100 ul acetone, mixed with 2 ml 0.05 M acetate buffer (pH 5.4) containing 0.2% (w/v) gum arabic and 10 mM CaCl 2. After adding purified O. piceae lipase 17 ug, the reactions were incubated with shaking (300 rpm) at 30°C for 2 h. * Stearic and oleic acid methyl esters appeared in one peak in GC chromatograms under the analysis conditions used. § A l l data points are means of two determinations, nd: not detected. 158 2.2e4 2.0e4 l.Se4 1.6e4 1.4e4 : 1.2e4: l,Oe4 SOOO SOOO 4000 j 2000 n o ll A 6 7 IO 20 30 40 50 Time (min) 2.2e4 2,Oe4 1.8e4 1.6e4 1.4e4 1.2e4 1 l,Oe4 s o o o i SOOO 4000 i 2000 o B IO 20 30 40 50 Time (min.) Figure 5.9 Gas chromatogram showing fatty acids in the hydrolysis products of the triglycerides isolated from trembling aspen sapwood. A: KOH saponification; B: 0. piceae lipase treatment. A l l acids were detected as methyl ester derivatives. Column: DB-17, conditions: see section 5.2.6. Peaks: 1 myristic, 2 palmitic, 3 stearic and oleic, 4 linoleic, 5 linolenic, 6 eicosenoic, 7 eicosadienoic. Stearic and oleic acid methyl esters appeared in one peak under the analysis conditions used.' 159 5.4 Discussion Studying the structure and function of the O. piceae lipase can enhance our understanding of the mechanism by which this fungus assimilates lipophilic nutrients from wood. The activity of a lipase can be affected by various chemical reagents, which can be used to provide some information about the structure and function of a lipase. N E M is a reagent frequently used to probe essential sulfhydryl groups (-SH) in enzymes. If an enzyme is inactivated by a mild treatment with N E M , then a free sulfhydryl group is probably required for activity of the enzyme. The O. piceae lipase was not affected by 10 m M N E M , indicating that sulfhydryl groups were not essential to this lipase. It was also shown that the lipase was not affected by the disulfide bond reducing agents B M E and DTT, which implied that the enzyme either did not have disulfide bonds, or if present, the disulfide bonds were not essential for activity. DEPC can react with the amino and imidazole (histidine) groups in enzymes. The strong inhibition of the O. piceae lipase by DEPC suggested the importance of a free amino group and/or a histidine residue to this enzyme. Similarly, E600 exerted an inhibitory effect on O. piceae lipase, indicating the crucial importance of a serine residue to the function of this lipase. E600 has been reported to inhibit pancreatic and gastric lipases, as well as lipases from Candida antarctica, Rhizomucor miehei, Pseudomonas cepacia, and Humicola lanuginosa (Rouard et al. 1978; Brzozowski et al. 1991; Moreau et al. 1991; Derewenda et al. 1992; Patkar and Bjorkling 1994). 160 Free fatty acids, one of the lipolysis products, tend to inhibit lipase-catalyzed hydrolysis reactions. It has been suggested that fatty acid molecules may accumulate at the lipid/water interface, thereby blocking the access of enzymes to unreacted substrates (Yamaguchi et al. 1985; Chen 1989; Markweg-Hanke et al. 1995). In this study, the use of PNPL as a substrate allowed the continuous monitoring of the release of hydrolysis product /»-nitrophenol with small amounts of enzyme compared with the amount of enzyme required for detecting the activity by titration method. Unfortunately lauric acid is not miscible with the reaction system used, and was not tested as an end product inhibitor. The short chain fatty acids, butyric (C4) and caproic (C6) acids inhibited the lipolysis reaction. It seems likely that one of the reaction products of PNPL hydrolysis, lauric acid, would have a similar inhibitory effects. The 2+ 2+ enhancement of the lipolysis by Ca and Mn might be caused by the formation of soaps, resulting from the interaction of these ions with lauric acid released. The formation of such soaps would effectively remove fatty acids from the substrate dispersion, thus decreasing the product inhibitory effect (Iwai and Tsujisaka 1984). Some heavy metal ions, such as Fe 3 + and H g 2 + , have been reported to inhibit lipases of Aspergillus niger, Chromobacterium, Pseudomonas, and mammalian lipases (Yamaguchi et al. 1973; Sugiura and Isobe 1974; Sugiura et al. 191 A; Fredrikson et al. 1981; Garcia et al. 1991). In this work it was shown that Fe 3 + and Ffg 2 + completely inactivated the O. piceae lipase. The mechanism of Fe inhibition has yet to be clarified, whereas it has been proposed that Hg forms mercury derivatives with proteins (Patkar and Bjorkling 1994). 161 The lipase purified from O. piceae showed high specificity toward substrates with fatty acid chains of 10 to 18 carbons. This fatty acid specificity pattern is different from that of several other microbial lipases reported. For example, lipases secreted by Penicillium cyclopium (Iwai et al. 1980) and Penicillium roqueforti (Mase et al. 1995b) are highly specific for substrates with short chain lengths of 3 to 8 carbons. A lipase from Rhizopus delemar displays a high specificity for chain lengths of 6 to 14, and a low specificity for 16 and 18 carbons (Mase et al. 1995b). Interestingly, Geotrichum candidum was reported to produce 4 forms of lipase. A l l show high specificity towards a chain length of 20 carbons, followed by 8, and 10 carbons. However, they show very low activity on 2-6 and 12-18 carbons (Sugihara et al. 1994). The diversity of fatty acid specificity of lipases may reflect the diverse substrates present in the growing environment for different microorganisms. The triglycerides are mainly composed of long chain fatty acids in most wood species (chapter 2), thus the high specificity of the O. piceae lipase for long chain fatty acid residues might reflect the natural adaptation of the fungus to its unique microniche. When triglycerides are used as substrates, lipases may show selectivity in terms of cleavage on the 3 ester bonds. Although some lipases, such as those isolated from Penicillium cyclopium (Okumura et al. 1976) and Pichia burtonii (Sugihara et al. 1995) lack positional specificity, other lipases, such as those secreted by Aspergillus niger, Rhizopus delemar (Okumura et al. 1976), Humicola lanuginosa (Liu et al. 1973b; Omar et al. 1987), and Pyihium ultimum (Mozaffar and Weete 1993) have a marked preference for the primary ester bonds (positions 1 or 3) of triglycerides. This study revealed that the 0. piceae lipase only cleaved fatty acids 162 from position 1 or 3 of triolein, since no 1,3-diolein was detected with a reaction carried out for 2 h (Figure 5.8). However, it was also found that when POP was used as substrate, one third of the released fatty acids was oleic acid which is located at the position 2 of POP (Table 5.2). This effect may be ascribed to the phenomenon of acyl migration and the action of the lipase. Chemically, 1,2 (2,3)-diglycerides and especially 2-monoglycerides are unstable and undergo acyl migration to give 1,3-diglycerides and 1-monoglycerides, respectively (Macrae 1983; Sonnet and Gazzillo 1991). In this study, no accumulation of 1,3-diolein was seen in the reaction, while the accumulation of 2-monoolein was observed (Figure 5.8). It appeared that the O. piceae lipase first cleaved one of the two primary ester bonds, followed by the other. The 2-monoolein generated probably underwent acyl migration, forming 1-monoolein, which was then hydrolyzed by the enzyme. The lack of accumulation of 1,3-diolein suggested that either the acyl migration from l,2(2,3)-diolein to 1,3-diolein was minor, or the 1,3-diolein generated through acyl migration was rapidly hydrolyzed by the lipase. We confirmed with 2-monoolein that acyl migration did take place under the reaction conditions used in the absence of the lipase (detected by TLC, developing solvents were chloroform : acetone : methanol, 85:15:1, v/v, Rf values of 1-monoolein and 2-monoolein were 0.34 and 0.30, respectively). The hypothesized sequence of the O. piceae lipase catalyzed lipolysis of triglycerides is illustrated in Figure 5.10. This hypothesis was further supported by comparing the data generated from the lipolysis of PPO and POP by the lipase. With PPO [one palmitic (P) and one oleic (O) in primary ester bonds], the ratio of released palmitic to oleic was 1.1 to 1, while with POP (two Figure 5.10 A proposed reaction sequence of O. piceae lipase on triglyceride lipolysis. -R -R -R -R -R -R triglyceride 1,2 (2,3)-diglyceride 1,3-diglyceride 2-monoglyceride 1-monoglyceride glycerol fatty acid main reaction reaction not likely to occur acyl migration or negligible 164 palmitic in primary ester bonds) the released palmitic to oleic was 1.8 to 1 (Table 5.2). The data also demonstrates the priority of the release of fatty acid residues from positions 1 (3) over position 2. In summary, in this chapter the effects of some commonly used chemical reagents on the purified O. piceae lipase activity were examined, and the action of the enzyme on various substrates investigated. Enzyme activity was not influenced by A^-ethylmaleimide, P-2+ 2+ mercaptoethanol or dithiothreitol, was enhanced by Ca or M n , but could be severely 2+ 3+ inhibited by Hg and Fe , butyric acid, caproic acid, diethyl pyrocarbonate, diethyl p-nitrophenyl phosphate, SDS, or EDTA. The lipase showed high specificity toward substrates with intermediate and long chain fatty acid residues, and it belongs to a group of 1(3) positional specific lipases. The rate of hydrolysis toward the triglyceride POP was 25-50 fold higher than that toward waxes (oleyl esters) and cholesteryl esters. Finally, it was conclusively shown that the purified lipase could effectively release fatty acid residues from the triglycerides isolated from wood. 165 Chapter 6 Concluding Remarks Control of sapstaining fungi and surface moulds on all lumber is of great economic importance. Some existing antisapstain chemicals are toxic to a wide range of organisms, or are not as effective as prior formulations. Alternative methods are needed to minimize the environmental impact and increase efficacy against all fungi causing stain. Some research groups have focused on biological control agents to outcompete staining fungi (Benko 1987; Seifert et al. 1987; Freitag et al. 1991; Kreber and Morrell 1993), others have considered using colorless mutants which do not cause stain (Behrendt et al. 1994; 1995). Another possible approach is to identify and disrupt the key enzymes involved in fungal metabolism (Abraham and Breuil 1995b). Most of these strategies for controlling sapstain would benefit from a better understanding of the physiology of the organisms involved. Ophiostomatoid fungi are among the most aggressive and costly sapstaining microorganisms. The lack of information available on the physiology of this group of fungi was the motivation for this research program. Our overall approach was to examine the physiology and biochemistry of sapstaining fungi, in order to identify the metabolic processes crucial and/or unique to these organisms. Once the important metabolic processes in the nutrition or pigmentation of these fungi are understood, it may be possible to manipulate or block them. 166 Thus, fundamental research is needed to understand how sapstaining fungi assimilate and utilize nutrients from wood, and how they produce pigments. The project described in this thesis, a component of the research program described above, was designed to examine the role of lipid utilization by sapstaining fungi. Specifically, it focused on analyzing the lipid nutrients in natural wood substrates, investigating the ability of sapstaining fungi to degrade and utilize these lipids, and characterizing lipolytic enzymes produced by the sapstaining fungi. Information on the chemical composition of the lipids in the wood of lodgepole pine was sparse. The current work has provided the most detailed and extensive study to date on the identity and quantity of the nonvolatile lipids in the wood of lodgepole pine. In particular, significant differences were observed between the content of lipophilic extractives in heartwood and sapwood. While the high content of resin acids (4.0-5.6%) may be one of the factors preventing sapstaining fungal growth in heartwood, abundant triglycerides in the sapwood (1.1-1.3%) were shown to be nutrient sources for the sapstaining fungi. When colonizing sapwood, Ophiostoma spp. were capable of secreting lipases to hydrolyze triglycerides into assimilable fatty acids and glycerol. In liquid culture, it was confirmed that Ophiostoma species were able to use triglycerides, fatty acids, or glycerol as sole carbon sources for growth. 167 A n extracellular lipase produced by O. piceae was purified and characterized in this study. It was conclusively shown that in vitro this lipase could release fatty acid residues from the triglycerides isolated from wood. Among different lipid substrates, the lipase mainly hydrolyzed triglycerides, although slight activity was measured on waxes and steryl esters. The enzyme belonged to a group of 1 (3) positional specific lipases. It showed little activity for substrates with short chain fatty acids (C2 to C6), but demonstrated high specificity for substrates with intermediate and long chain fatty acid residues (CIO to C18). This was consistent with the natural substrates in wood, where triglycerides are mainly composed of long chain fatty acids (C16 and C18). The temperature optimum of 30°C for lipase activity was consistent with the mesophilic characteristics of O. piceae. The optimum pH at approximately 5.2 reflected the natural adaptation of O. piceae to the wood microniche where low pH values of 4 to 6 are typical. Inhibition of the lipase by diethyl /?-nitrophenyl phosphate suggested that a serine residue was probably a crucial amino acid in the active site of this enzyme. The data and information generated from this project, together with other on-going projects in the group, have enhanced our understanding of the physiology of sapstaining Ophiostoma species. Ophiostoma fungi produce extracellular lipases to hydrolyze triglycerides during the early colonization stage in wood. Other studies in the group have shown that proteolytic enzymes are widely produced by Ophiostoma spp. and their enzyme activities could be inhibited in Vitro (Abraham et al. 1993; Breuil and Huang 1994; Abraham and Breuil 1995a, b; Breuil et al. 1995). It was also found that the black pigment produced by O. piceae strongly 168 resembles other fungal melanins (Brisson et al. 1996). The Ophiostoma spp. examined were shown to have homologous genes for the enzymes of the dihydroxynapthalene pathway of melanin biosynthesis (Eagan et al. 1996). At this point, the results suggest that it may be difficult to completely inhibit fungal growth by targeting just one enzyme or metabolic pathway. Furthermore, testing potential inhibitions on wood is constrained by the lack of specific, cheap, stable, and non-toxic enzyme inhibitors (Abraham and Breuil 1995b). However, the progress to date has indicated what areas should receive attention in on-going characterization of metabolic features and key enzymes of sapstaining fungi. Studies on the interaction between the key enzymes and substrates will provide clues for designing and synthesizing specific, non-toxic enzyme inhibitors. Future formulations containing inhibitors targeting multiple metabolic pathways may lead to a new paradigm of effective and environmentally acceptable agents and methods for sapstain control. Although this thesis has addressed some aspects of the lipids in wood and their degradation by Ophiostoma fungi, it has raised more questions. Though one lipase was purified and characterized, it is necessary to find out whether O. piceae can produce other extracellular and intracellular lipases. For the 0. piceae lipase purified in this work, further research is needed to study the mechanisms of inhibition, the kinetics of lipolysis reactions, and the tertiary structure of the enzyme. These studies will clarify the mode of action of the lipase on substrates, thereby facilitating the design of inhibitors which target fungal lipases. Also, polyclonal and monoclonal antibodies to the purified lipase can be produced. Immunochemical and histochemical approaches with antibodies can provide effective techniques for determining the 169 enzyme secretion and regulation during the fungal colonization in wood. 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