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The construction and characterization of a Pro-Thr box deletion of a Cellulomonas fimi endoglucanase… Shen, Hua 1990

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THE CONSTRUCTION AND CHARACTERIZATION OF A PRO-THR BOX DELETION OF A CELLULOMONAS FIMI ENDOGLUCANASE (Cen A) By Shen Hua B.Sc, Nan Kai University, Tianjin, China, 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Microbiology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February 1990 © Shen Hua, 1990 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 scholarly 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 The University of British Columbia Vancouver, Canada DE-6 (2/88) ABSTRACT The catalytic domain is separated from the cellulose-binding domain in Cellulomonas fimi endoglucanase CenA by a proline-threonine rich sequence called the Pro-Thr box. To study the function of the Pro-Thr box region, a deletion mutant, cenAAPT, was made from cenA by an oligonucleotide directed in vitro mutagenesis. The truncated enzyme, CenAAPT, was purified to homogeneity by affinity chromatography on cellulose and characterized. Comparing CenAAPT to CenA, the following characteristics were observed: 1) the Pro-Thr box affected the migration of CenA on SDS-PAGE; 2) the deletion of the Pro-Thr box altered the high affinity interaction with cellulose; 3) the truncated enzyme showed 40-50% reduction in catalytic activity towards both microcrystalline and amorphous cellulose; 4) the truncated enzyme was as sensitive as CenA to a C.fimi protease, and both enzymes were cleaved at the same site adjacent to the binding domain. The Pro-Thr box is not essential for the catalytic activity of CenA or its binding to cellulose, but it does contribute to both functions. ii TABLE OF CONTENTS PAGE ABSTRACT ii TABLE OF CONTENTS iii LIST OF TABLES vi LIST OF FIGURES vii LIST OF ABBREVIATIONS viii ACKNOWLEDGEMENTS ix INTRODUCTION 1 I Cellulose and cellulases 1 II Cen A and Cex of C.fimi 2 IU Structural features of Cen A and Cex 2 IV Objectives 3 MATERIALS AND METHODS 6 I Bacterial strains, phage and plasmid 6 JI Media 6 IE Buffers 7 TV Enzymes, reagents and DNA recombination techniques 7 V Gel electrophoresis of DNA 7 VI Deletion of the Pro-Thr box-encoding sequence from cenA 7 (A) Construction of an M13mpl9cenA clone 8 (B) Preparation of uracil-containing Ml3mpl9ce/iA single-stranded DNA 8 (C) Synthetic oligodeoxyribonucleotides 8 (D) Primer annealing reaction and T7 DNA polymerase extension 9 iii TABLE OF CONTENTS (continued) PAGE (E) Transfection and purification of phage 9 (F) DNA sequence determination 9 VH The construction of pUCl8-1.5cenAAPT 9 Vm Screening colonies for endoglucanase activity 10 IX Purification of CenAAPT 10 (A) Growth of cells 10 (B) Preparation of cell extract 10 (C) Cellulose affinity chromatography 10 (D) Electrophoresis of proteins 11 X Binding affinity for Avicel 11 XI Sensitivity to C.fimi protease 12 (A) Preparation of C.fimi protease 12 (B) Hydrolysis of CenAAPT with C.fimi protease 12 XII Assay of enzyme activity 12 (A) Carboxymethylcellulase assay 12 (B) Activity on Avicel and phosphoric acid swollen Avicel 13 (C) Cellulose azure assay 13 (D) C.fimi protease assay 13 XUI Determination of protein concentration 13 RESULTS 14 I Construction of pen AAFT 14 (A) Subcloning of cenA gene into M13mpl9 14 (B) in vitro mutagenesis 14 iv TABLE OF CONTENTS (continued) PAGE (C) Subcloningof cenAAPTintopUC18 15 TJ Purification of CenAAPT by affinity chromatography on cellulose 15 in Characterization of CenAAPT 15 (A) Sensitivity to C.fimi protease 15 (B) Binding to Avicel 16 (C) Activity 16 DISCUSSION 36 REFERENCES 40 v LIST OF TABLES PAGE Table 1: Bacterial strains, phage and plasmid 6 Table U: Purification of CenAAPT 17 Table III N-terminal amino acid sequence of p30 produced from ngCenAAPT 18 by C.fimi protease Table IV: Extinction coefficient of ngCenAAPT 19 Table V: Enzyme activity against a range of cellulosic substrates 20 Table VI: M r determination for ngCenA and ngCenAAPT 21 vi LIST OF FIGURES PAGE Fig. 1: Structure of CenA and Cex of C.fimi 4 Fig. 2: Scheme for the construction of CenA Pro-Thr box deletion plasmid pUC18-cercAAPT 22 Fig. 3: Screening of M13rnpl9-ce«A clones by BamHL digestion of phage DNA 25 Fig. 4: Screening of potential deletion mutants (M13mpl9-ce«AAPT) by Hpal digestion of RF DNA 26 Fig. 5: Dideoxy nucleotide sequencing gel showing deletion of the Pro-Thr box coding sequence from the cenA gene 27 Fig. 6: Screening of potential deletion mutants (pUC18-ce«AAPT) for CMCase activity 28 Fig. 7: Deletion of ngCenAAPT in cell extracts 29 Fig. 8: Restriction analysis of pUC18-cenAAPT clones 30 Fig. 9: Purification of ngCenAAPT by affinity chromatography on cellulose 31 Fig. 10: SDS-PAGE analysis of the purification of ngCenAAPT 32 Fig. 11: Time course of proteolysis of ngCenA and ngCenAAPT 33 Fig. 12: Adsorption of ngCenAAPT and ngCenA to Avicel 34 Fig. 13: Sequence alignments of CenA and CenAAPT Pro-Thr box and flanking regions and locations of C.fimi protease primary cleavage sites in the nonglucosylated protein 35 vii ABBREVIATIONS AND SYMBOLS Ap ampirillin gene BSA bovine serum albumin CBD cellulose binding domain CMC(ase) carboxymethyl cellulose(ase) dNTP deoxynucleoside triphosphate A deletion in DNA or protein E 2 0 5 / E 2 8 0 extinction coefficient at 205nm or 280nm HB AH hydroxybenzonic acid hydrazine GuHCl guanidine-HCl IPTG isopropyl-6-D-thiogalactoside kb 1000 base pairs kDa kilodalton lacP/O E.coli 6-galactosidase gene promotor and inhibitor Mr relative molecular mass PAGE polyacrylamide gel electrophoresis PMSF phynelmethylsulfonyl fluoride RF DNA replication form DNA SDS sodium dodecyl sulfate TJV ultra violet viii ACKNOWLEDGEMENTS This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada. I would like to thank Drs. R.A.J. Warren, D.G. Kilburn, and R. C. Miller Jr. for their expert guidance and for opening up my eyes to the challenges of scientific research. I would like to thank Dr.N.R. Gilkes for helpful discussions. Also, my thanks to all the members of the cellulase group especially my laboratory fellows in Room 206, H. Smith, E. Kwan, J. Coutinho, E. Ong, and A. Macleod for their friendship and kindness which have made my stay in the laboratory an enjoyable and pleasant one. Special thanks are due to E. Ong for taking the photographs in this thesis, to all my friends in China and elsewhere, and to my family, for their love and encouragement throughout the years. I dedicate this thesis to my parents. ix I N T R O D U C T I O N I Cellulose and cellulases Cellulolytic microorganisms produce a complex of enzymes capable of hydrolysing B-1,4-glucosidic bonds, and degrading cellulose to cellobiose or glucose. Many cellulolytic bacteria and fungi have been identified and characterized ( Beguin, et al., 1987; Coughlan, 1985). Potential applications of cellulase for the production of fuels from biomass and degradation of agricultural products have intensified research in the enzymatic degradation of cellulose. Cellulose is the major component of the cell wall of plants. It is a linear polymer of up to 14,000 glucose residues joined by B-l,4-glucosidic bonds. The linear chains of cellulose are organized in bundles of parallel chains to form fibrils. These fibrils comprise both ordered structure, the crystalline regions, and less ordered structure, the amorphous regions. Cellulase systems are composed of a variety of enzymes with different specificities and modes of action, which act togather to hydrolyze cellulose. Cellulose degradation requires the activities of three basic types of enzyme (Coughlan, 1985; Coughlan and Ljungdahl, 1988; Wood, 1985). Endoglucanases attack the amorphous region of cellulose fibers, cleaving internal B-l,4-glucosidic bonds at random and generating new non-reducing ends. Exoglucanases attack the cellulose molecules preferentially from the non-reducing end, liberating cellobiose subunits and then proceeding into the crystalline regions of the fibers. B-glucosidases hydrolyze cellobiose and low molecular weight cellodextrins into glucose. The rapid development in the study of the molecular biology of cellulase genes has opened new areas of interest: 1) the organization of cellulase genes, 2) their regulation at the molecular level, and 3) the study of structural features required for enzyme activity. This thesis focuses on the influence of the Pro-Thr box on the function of C.fimi CenA. 1 II CenA and Cex of C.fimi The gram-positive cellulolytic bacterium Cellulomonas fimi produces a complex array of cellulases when grown on cellulosic material. Of these, CenA and Cex are the two major components in the culture medium; they bind tightly to Avicel. The genes encoding CenA and Cex have been cloned in E. coli and their nucleotide sequences determined (Gilkes et al, 1984a; O Neill et al., 1986; Whittle et al., 1982; Wong et al., 1986). CenA hydrolyzes Cm-cellulose at random while Cex shows preference for the termini (Gilkes et al., 1984b). The native CenA and Cex from C. fimi are glycoproteins with Mrs of 49.3 and 53 kDa, respectively. The recombinant CenA and Cex from E. coli are non-glycosylated, with Mrs of 48.7 and 47.3 kDa, respectively (Langsford et al., 1987). The glycosylated CenA and Cex are protected from attack by a C. fimi protease when bound to cellulose, but the non-glycosylated recombinant CenA and Cex are both cleaved precisely at the C-terminus of the Pro-Thr box, resulting in the formation of a 30 kDa fragment from CenA and a 39 kDa fragment from Cex. Both of these fragments retain catalytic activity but no longer bind to cellulose (Langsford et al., 1987; Gilkes et al., 1988). Therefore, both CenA and Cex have independently functioning catalytic and cellulose-binding domains. III Structural features of CenA and Cex CenA consists of a polypeptide of 449 amino acids including a leader peptide of 31 amino acids which functions to export CenA to the periplasm of E. coli (Wong et al., 1986). Cex consists of a polypeptide of of 484 amino acids including a 41 amino acid leader peptide which functions to export Cex to the periplasm of E. coli ( O'Neill et al., 1986). Each enzyme contains three distinct regions (Gilkes et al., 1988; Warren et al., 1986) (Fig. la). The binding domain which is a region of low charge, rich in hydroxyamino acids with 112 amino acid residues at the N-terminus of CenA, and 108 amino acid residues at the C-terminus of Cex. These regions are highly conserved, with about 50% sequence homology (Fig. lb). The more highly charged catalytic domains, 2 which are 283 amino acid residues at the C-terminus of CenA, and 315 amino acid residues at the N-tenninus of Cex, both contain sequences which resemble the sequence at the active site of hen egg white lysozyme (Warren et al., 1986). The third region consists of a highly conserved short sequence of about 20 residues containing only proline and threonine, termed the Pro-Thr box [(PT)4T(PT)7 for CenA and (PT)3T(PT)3T(PT)3 for Cex] (Fig. Ic), which separates the binding domain and the catalytic domain. A similar domain structure occurs in many fungal and bacterial cellulases and 8-glucosidases. In most cases, proline and hydroxyamino acid rich segments separate the functional domains (review by Beguin, in press). The most intensively studied cellulases are T. reesei CBH I and CBH II. CBH I and CBH II contain a conserved element of approximately 30 residues, at the C-terminus of CBH I and at the N-terminus of CBH U, which appears to be a binding domain. The core enzymes, obtained by mild papain treatment, lack the conserved region, retain activity towards soluble substrates but have much reduced Avicelase activity. A Pro-Thr-Ser rich sequence connects the binding domain and the core enzymes (Tomme et al., 1988; van Tilbeurgh et al., 1986). Intact and core CBH I and CBH II were studied by small angle X-ray scattering (Schmuck et al., 1986; Abuja et al., 1988a,b). Both intact enzymes are tadpole-shaped, with an isotropic head and a long tail. The core enzymes lack tails. IV Objectives The striking similarities in the organization of CenA and Cex and the extensive sequence homology between their binding domains make it very interesting to understand the relationship between their structural features and their differential substrate specificities. The role(s) of the Pro-Thr box, almost perfectly conserved and sited between functional domains in both enzymes, is not yet clear. Determining the role of the Pro-Thr box in CenA should contribute to an understanding of the mechanism of action of CenA. It should also contribute to the understanding of other cellulase systems because of the common occurrence of the presence of proline-and hydroxyamino acid- rich 3 OOKUUUX UKOU8 UVQUSS mwnnicH WKKO CXttGED 112 134 ; — i OROEKD imauK OWGEO PT ! ! maexn. t in 316 333 300 CMO cn 4 4 iff], tlllllll (Irja iMiiiii « o » JLSJL »« » in ipn- L C O I « 1 lit « l i j i s s * cm cn cm co • 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 jfw]«rno«111 iftfii i oroiifiu trrit s i I U I . or?i»r?tj_s «LijiLklT f i f r jjjjo « iLoJ*|v!s »LtJ« ' o »uJs »wuil« t BO « ? a « 4 4 4 4 4 4 4 4 F c c i i iiv a[(]s[i|r tn»]« sir s il f I f U i l l T LsJ T L«J « «UJI « I F S t l p II • t s i r ' ' t c t i t s i c c 1 « 0 cm co i t i 4 / C I « f 1 1 s » t «3 112 134 DOO p i r i n n i p T n p i u n p i f i EXO f T P I P I I P I H f I M f l f l H 316 335 Fig. 1 Structure of CenA and Cex of C.fimi ( Adapted from Warren et al., 1986.) a. Overall structures. PT denotes Pro-Thr box; AS denotes putative active site. b. Conservation of amino acid sequence in the cellulose-binding domains. The numbers refer to amino acid sequences of the intact proteins, with 1 being the N-terminal amino acid. Conserved residues are boxed. * denotes a gap left in the sequence to improve the alignment. + denotes pairs of nonconserved residues which belong to the same structurally related exchange group, clusters of hydroxyamino acids are underlined. c. Sequence conservation in the Pro-Thr boxes. * denotes a gap left in the sequence to improve the alignment. 4 segments connecting domains in cellulases from various organisms. This work, concerns the function of the Pro-Thr box in CenA. The strategy was to delete precisely from the cenA gene the sequence encoding the Pro-Thr box, using oligonucleotide directed in vitro mutagenesis. The polypeptide produced by the deletion mutant was purified and its affinity for cellulose, its catalytic activity and its sensitivity to C. fimi protease were examined. 5 MATERIALS AND METHODS I Bacterial strains, phage and plasmid A list of the bacterial strains, phage and plasmid used in these studies is given in Table I. Stock cultures of bacteria were maintained at -20°C in 40% glycerol LB medium or -80°C in 10% DMSO LB medium. Table I. Bacterial strains, phage and plasmid Bacterial strain/ phage/plasmid Genetic characters Reference E. coli JM101 supE thi (lac-proAW) [F traD36 pro AB lacPZA Ml 5] Yanisch-Perron et al., 1985 E. coli RZ1032 HfrKL16 PO/45 [fyjA(61-62)], dut\, ungl, Ml, relAl Zbd-279::Tnl0, supEM Kunkel etal, 1987 M13mpl9 pUC18 lac lac ApR pUC18-1.6ce/iA lac ApR Norrander et al., 1983 Norrander et al., 1983 Guo etal., 1988 II Media LB medium contained per liter; 10 g Bacto-tryptone, 5 g Bacto-yeast extract and 5 g NaCl. 2xYT medium contained per liter; 16 g Bacto-tryptone, 10 g Bacto-yeast extract 6 and 5 g NaCl. The pH of all the media was adjusted to 7.2 with NaOH. M9 medium contained per liter; 6 g Na2HP04, 3 g KH2PO4, 0.5 g NaCl, 1 g NH4C1, 5 g Casamino acids, 1 mg thiamine with the addition, after autoclaving, of 10 ml of 0.01M CaCl2, 2 ml of 1M MgS04 and 10 ml of 20% glucose. Agar plates contained 1.5% agar. III Buffers The composition and preparation of buffers were described previously (Maniatis et al., 1982; Messing, 1983). IV Enzymes, reagents and DNA recombination techniques In general, D N A preparation, restriction enzyme reactions and recombinant D N A techniques were as described by Maniatis etal. (1982). Restriction endonucleases, T7 D N A polymerase, T4 DNA ligase, and deoxyribonucleotides were purchased from Pharmacia Inc. (Dorval, Quebec), New England Biolabs Inc. (Beverly, M A , USA), or B R L Inc. (Burlington, Ontario). Al l other chemicals and reagents were from B D H Chemicals (Vancouver, Canada) or Sigma Chemical Company (St. Louis, Mo.). V Gel electrophoresis of DNA Agarose gels (0.7 %) and polyacrylamide gels (6-12%) were used for the analysis and preparation of large (0.5-12 kb) and small (35-450 bp) DNA fragments, respectively. For preparative purposes, large DNA fragments were separated on 0.7% agarose gels running in T A E buffer. The bands of DNA were located by staining with ethidium bromide, then excised. The DNA was recovered with the Geneclean™ kit ( BIO/CAN Scientific Inc. Mississauga, Ontario) as follows: gel slices were dissolved in Nal solution by incubating at 50°C for 5 min; the DNA was bound to glassmilk washed with NEW solution, and eluted with T E buffer. VI Deletion of the Pro-Thr box-encoding sequence from cenA (A) Construction of an M13mpl9ce«A clone The 1588 bp Sstl fragment from pUC18-1.6cenA was cloned into the Sstl site of 7 M13mpl9 to give M13mpl9ce/iA. (B) Preparation of uracil-containing M13mpl9ce/zA single-stranded DNA M13mpl9 phage containing uracil in the DNA was prepared as described by Kunkel (1985) with modifications. E. coli RZ1032 (Kunkel et al., 1987) cells were grown at 37°C in TY medium with vigorous shaking to a density of 108 cells per ml; M13mpl9-cenA phage were then added at a multiplicity of infection of 20. Incubation at 37°C was continued for another 5 hours, then the culture was centrifuged at 5,000 x g for 5 min at 4°C. The clear supernatant containing the phage was used for a second cycle of growth, identical to the first, to obtain a phage titer on the ung host (RZ1032) 6,000 fold greater than that on the ung+ host (JM101). The phage suspension was made 3% in polyethylene glycol (PEG 8000) and 0.5 M in NaCl. After centrifugation at 5000 x g for 15 min, the phage pellet was resuspended in TE (pH 7.6), then extracted once with phenol and once with chloroform/isoamyl alcohol (24:1). The DNA was precipitated with ethanol and resuspended in TE. The DNA concentration was determined by absorbance at 260nm. The purity of the DNA was analysed by agarose gel electrophoresis. (C) Synthetic oligodeoxyribonucleotides A mutagenesis primer 5'-pGC TGG TCG GCT GCG GCG TGA CGT TAA CGC TGG TCG TCG GCA CGG TGC C-3' (48-mer), and a sequencing primer 5'-CGC GGT AGC CCT GCG TCG-3' (17-mer) were synthesized chemically on an Applied Biosystems 380A DNA synthesizer by T. Atkinson using phosphite triester chemistry, essentially as described (Adams et al., 1983; Atkinson and Smith, 1984). The 48-mer was separated from incomplete products by electrophoresis in a 12% polyacrylamide-7M urea sequencing gel, located by UV-shadowing, and extracted from the gel by the crush and soak methed (Atkinson and Smith, 1984). The crudel7-mer oligonucleotide was dissolved in 1.5 ml of 0.5M ammonium acetate solution. The primers were further purified by binding to Sep-Pak C-18 reverse phase cartridges (Millipore/Waters Assoc., Milford, MA) in water, followed by washing with water, and elution with 20% acetonitrile-80% water. The 8 concentrations of the oligodeoxynucleotides were determined by absorbance at 260 nm (D) Primer annealing reaction and 17 DNA polymerase extension One pmole of M13mpl9-ce«A DNA was mixed with one pmole of mutagenesis primer in 18 ul of buffer containing 40mM Tris-Cl (pH 7.5), 20mM MgCl2 and 50mM NaCl. The DNA was denatured by heating the solution in a 1.5 ml microfuge tube at 55°C for 10 min and allowed to anneal by cooling to 23°C over a period of 2 hours and then kept at 4°C overnight. The reaction mixture was then supplemented to give final concentrations of 500uM for each dNTP, ImM ATP, 3mM DTT, 4 units T7 DNA polymerase, 2 units T4 ligase and 2.4 ul of 5x annealing buffer, (final reaction volume of 31ul). The reaction mixture was incubated at 37°C for 2 hours, then 15mM EDTA pH 8.0 was added to terminate the reaction. (E) Transfection and purification of phage The preparation of E. coli JM101 (ung+) competent cells and their transfection with the primer-extended double stranded M13mpl9-cenA DNA were performed as described by Messing (1983). The plaques were screened for the deletion mutation by screening RF DNAs for sensitivity to Hpal. The positive plaques were purified by titering the phage on JM101 cells. (F) DNA sequence determination The cenA APT deletion mutant was sequenced by the dideoxy chain termination method (Sanger et al., 1977). Single stranded templates were prepared from 1.5 ml of phage infected cultures ( Messing 1983). Samples from the enzymatic sequencing reactions were analyzed on 6% denaturing ( 8M urea) polyacrylamide gels. VH The construction of pUC18-1.5ce«AAPT The 1525 bp Sstl fragment from M13mpl9cenAAPT RF DNA was ligated to pUC18 which had been cut with Sstl. E. coli JM101 was transformed with the products. 9 Transformants were screened for endoglucanase activity ( see below) and a positive clone was designated p U C l 8-1.5ce«AAPT. VIII Screening colonies for endoglucanase activity E. coli JM101 clones carrying recombinant plasmids were picked onto both a master LB-agar plate containing 100u.g ampicil l in / m l and a LB-agar plate containing 100u,g ampcil l in / m l , 0.2 m M I P T G and 1% high-viscosity C M C (carboxymethylcellulose). After incubation at 37°C for 12 hours, the plates were exposed to chloroform vapour for 20 min and incubated at 37°C for 30 min. Then the plates were stained with 0.2% Congo-red for 30 min, and destained with I M N a C l . Positive colonies were surrounded by a clear halo against a red background. I X Purification of C e n A A P T (A) Growth of cells A 200 m l overnight culture of JM101 pUC18-1 .5ce«AAPT was added to 60L L B medium, containing 100u,g ampicillin /m l and 0 . I m M I P T G , in a 100L fermenter. The culture was grown for 16 h at 37°C; it was stirred at 200 rpm and aerated with 20 L air / min. The cells were harvested with a Sharpies centrifuge at 45,000rpm. The cell paste was washed with 5 0 m M phosphate buffer p H 7, 0.02% azide. The cells were resuspended in 1 L 5 0 m M phosphate buffer, 3 m M E D T A , 0.02% azide. (B) Preparation of cell extract Cells were ruptured by passage twice through a French Press. Immediately after rupture, the extract was made I m M in P M S F and O.OlmM in pepstatinA, and 1.5% in streptomycin sulfate. The extract was clarified by centrifugation twice at 17,000 rpm in a Beckman JA20 rotor. (C) Cellulose affinity chromatography The clarified cell extract was passed through a jacketed CF1 cellulose column (5cm x 10 30cm) maintained at 4°C at a flow rate of 1 ml/min. The column was washed with 1M NaCl, and then 20mM Tris-Cl, pH 7.4 buffer. The enzyme was eluted with a linear gradient of 0-8M GuHCl in 20 mM Tris-Cl, pH 7.4. The absorbance of the eluate was measured at 280nm. Fractions of 10 ml were collected and assayed for CMCase activity. The peak fractions were pooled and the GuHCl was replaced with 50mM phosphate buffer pH 7 by ultrafiltration with an Amicon PM10 membrane in an Amicon pressure cell. (D) Electrophoresis of proteins SDS-polyacrylamide gels, 0.75 mm thick, were run according to Laemmli (1970). Relative mass (Mr) was estimated by comparison with Sigma SDS-6H Mr standards run on the same gel. Gels were stained with Coomassie Blue (Zacharius et al., 1969), and preserved by drying between layers of cellophane. For sequencing, proteins in gels were transferred electrophoretically to polyvinylidene difluoride membrane, and stained with Coomassie Blue. Protein sequences were determined by automated Edman degradation in an Applied Biosystems 470A gas-phase sequenator. Enzyme activity was detected in gels by a zymogram technique (Beguin, 1983). After electrophoresis, the acrylamide gel was washed twice with 25% isopropanol-50mM citrate pH 6.8 for 30 min , and twice with 50mM citrate buffer pH 6.8 , then laid on a 1% agarose gel in citrate buffer and containing 1% CMC (high viscosity), and incubated at 37°C over night. Then the overlays were stained with Congo Red to observe zones of clearing. X Binding affinity for Avicel Adsorption was determined at 5°C in 1.5 ml Eppendorf tubes. Tubes contained 20 mg Avicel in 1.15 ml phosphate buffer pH 7.0, and 0.05 - 1.6 mg protein ml"1. Tube contents were mixed continuously by rotation. After equilibration for 3 h, the adsorbent and bound protein were removed by centrifugation (twice at 10,000 xg, 10 min) and the unbound protein concentration ([P], mg protein.ml"1) estimated from the absorption of the 11 supernatant at 280 nm using an appropriate blank. The bound protein concentration ([P]ad, mg protein-ml"1) was estimated from the difference between the initial protein concentration ([P]0, mg protein, ml"1) and [P]. Raw data were graphed as an adsorption isotherm plot and as a Scatchard plot XI Sensitivity to C.fimi protease (A) Preparation of C.fimi protease Protease was prepared from the culture supernatant after growth of C.fimi on 0.1% glycerol medium (Gilkes, et al., 1988). (B) Hydrolysis of CenAAPT with C.fimi protease A 35 u.g sample of CenAAPT was incubated with 0.2 unit of C. fimi protease in 200 u,l phosphate buffer pH 7 at 37°C. Samples of 20 |il were removed at intervals and the reaction terminated by the addition of PMSF to a final concentration of 3.6 mg/ml. The samples were analyzed by SDS-PAGE. Protein bands were blotted on polyvinylidene difluoride membranes for sequence determinations. XTI Assay of enzyme activity (A) Carboxymethylcellulase assay CMCase activity was determined by two methods. In the purification process, cell extract was incubated with 25 mg substrate /ml in 0.75 ml of phosphate buffer at 37°C for 30 min, then 50 u.1 of glucose (lmg/ml) and 0.8 ml of DNS reagent (Miller, 1959) were added, and the mixture steamed at 100°C for 15 min, cooled and the absorbance at 550 nm measured. A standard curve was prepared using a 5.56 mM stock solution of glucose. The purified enzyme was assayed by the hydroxybenzoic acid hydrazide (HBAH) method (Langsford et al., 1987). Enzyme was incubated with 4 mg substrate/ml in 0.5 ml citrate buffer at 30°C for 30 min. Then 1 ml of hydroxybenzoic acid hydrazide reagent was added, the reaction mixture was steamed at 100°C for 12 min, allowed to cool, and the absorbance was measured at 420 nm . A standard curve was determined for each assay 12 using a 55.6 nMol stock solution of glucose. Results are expressed as glucose equivalents. (B) Activity on Avicel and phosphoric acid swollen Avicel Enzyme was incubated with 15 mg of Avicel or 10 mg of phosphoric acid swollen Avicel in 1.5 ml of 50mM sodium citrate, pH 7.0, 0.2% BSA, 0.02% Na3N at 37°C for 18 h or 4 h. Reducing sugar release into the reaction supernatant was determined with HBAH reagent. (C) Cellulose azure assay Enzyme was incubated with 40 mg of cellulose azure in 2.0 ml of citrate buffer pH 7.0 for 18 h at 37°C. The A 5 8 5 ^ of the supernatant was measured after an appropriate time. One unit of activity gave a change in A 5 85 ^  of 1.0 / h. (D) C.fimi protease assay C. fimi protease was incubated with 10 mg of Hide Powder Azure in 1.5 ml of phosphate buffer for 1 h at 37°C. The A 5 8 5 „,„ of the supernatant was measured after an appropriate time. One unit of activity gave a change in A 5 8 5 n m of 1.0/ h. XUI Determination of protein concentration Protein concentration was measured routinely by the dye-binding method (Bradford, 1976) using the Bio-Rad Protein Kit (Bio-Rad Laboratories, Canada ). For the binding assay, protein concentration was based on UV absorption at 280 nm. The extinction coefficient (E 1 m g / m l ) for ngCenAAPT was determined by the far-UV 205 method of Scopes (1974). This value was used to estimate the absolute concentrations of solutions of the protein, which was then used to calculate the extinction coefficient at 280 lmg/ml nm (E ). 280 13 RESULTS I Construction of cenAAPT (A) Subcloning of the cenA gene into M13mpl9 The 1588-bp Sstl fragment encoding CenA was cloned into the Sstl site of M13mpl9 (Fig. 2 a). The orientation of the insert was determined by restriction analysis of RF DNA (Fig. 3). A clone with the correct orientation was designated M13mpl9cenA. (B) in vitro mutagenesis Loop-out primer extension was used to delete the sequence encoding the Pro-Thr box of CenA from the cenA gene (Fig. 2 b). If Ml3 is propagated on an E. coli dut ung strain, the progeny DNA has about 1% of its thymine residues replaced with uracil. Such DNA is degraded rapidly when the phage is plated subsequently on a dut+ ung+ host. This phenomenon can be used to enrich for mutants of M13 strains prepared in vitro (Kunkel, 1985). M13mpl9cenA was prepared by two cycles of infection on E. coli RZ1032 dut ung. The relative efficiencies of plating of the phage obtained were 1.0 and 10"6 on the dut ung and dut+ung+ host, respectively. The primer for the loop-out was a 48-mer which was complementary to 21 nucleotides on either side of the sequence encoding the Pro-Thr box of CenA, with a hexamer correspoding to a Hpdl site replacing the Pro-Thr encoding sequence. There are no Hpal sites in M13mpl9, pUC18 (see later) or the Sstl fragment encoding CenA. After primer extension and ligation, the reaction mixture was used to transform a dut+ ung+ E. coli strain. About 30,000 plaques were obtained. Small volume lysates were made from 12 plaques picked at random, and RF DNA prepared from each preparation. The DNA from 5 of the plaques contained a Hpal site (Fig. 4). DNA sequencing confirmed that these phage had the Pro-Thr box encoding sequence replaced by a Hpal recognition site (Fig. 4). 14 (C) Surxloning of cenAAPT into pUC 18 RF DNA was prepared from two individual M13mpl9-cenAAPT clones. The 1525-bp Sstl CenAAPT-coding fragment was cloned into the Sstl site of the pUC18 vector, and transformed into JM101 (Fig. 2 c). The transformants were sreened for CMCase activity (Fig. 6). A positive clone was designated pUC18-1.5ce/zAAPT. Extracts prepared from cells carrying pUC18-1.5ce«AAPT contained two polypeptides with CMCase activity (Fig. 7). The polypeptide of Mr 44 kDa corresponded to intact CenAAPT, that of 30 kDa to an active degradation product. The restriction pattern of pUC18-1.5ce«AAPT was as expected (Fig. 8). II Purification of CenAAPT by affinity chromatography on cellulose CenAAPT retained the cellulose-binding domain (CBD) of CenA. It could be purified by affinity chromatography on CF1 cellulose (Table I, and Fig. 9). CenAAPT was eluted from cellulose with a high concentration of guanidine but could not be eluted with water. The enzyme obtained was virtually homogeneous (Fig. 10). Its Mr was 44.1 kDa, about 4.4 kDa less than that of CenA. HI Characterization of CenAAPT CenA from C. fimi is a glycoprotein, and is referred to as gCenA. CenA produced in E. coli is not glycosylated, and is referred to as ngCenA. (A) Sensitivity to C.fimi protease Both ngCenA and ngCenAAPT were converted into a 30 kDa fragment within 24 hours (Fig. 11, A and B, lane 2-9). The enzymes were hydrolysed at the same rate (Fig. 11, A and B, lane 2-9). A transient 36 kDa fragment was produced from ngCenA but not from ngCenAAPT (Fig. 11 B). The 30 kDa fragment is referred to as p30. The N-terminal amino acid sequence of p30 from both enzymes was Val-Thr-Pro-Glu-Pro-Thr (Table IH). This corresponds to amino acids 135-140 of CenA, and shows that the proteins are cleaved at the same site. 1 5 (B) Binding to Avicel For the binding experiments, protein concentrations were determined by A 2 8 o n m . This was considered to be reliable. Concentrations determined by dye binding were 84% of those determined by adsorbance (Table IV). The adsorption isotherm for ngCenAAPT with Avicel (Fig. 12 insert) indicated that approximately 1.06 nmol ngCenAAPT. mg"1 Avicel was adsorbed at saturation. A Scatchard plot (Fig. 12 main panel) of the same data revealed an almost a linear relationship between [P]ad and [P]ad/[P]\ indicating one class of binding interaction. (C) Activity The molar activities of ngCenAAPT on a range of substrates were compared to those of ngCenA and p30 (Table V). The molar activity of p30 against CMC was 20% higher than that of ngCenA. Higher activities for p30 were also observed against phosphoric acid-treated cellulose and cellulose azure, with 100% and 300% increases, respectively. However, the molar activity of p30 against Avicel was 22% lower than that of ngCenA. These results agreed with those obtained previously (Gilkes et al., 1988). The molar activities of ngCenAAPT against all these substrates were lower than those of ngCenA: 50% lower against CMC and phosphoric acid-treated cellulose, 62% lower against cellulose azure and 40% lower against Avicel. 16 Table II Purification of CenAAPT Material Total Total Specific Recovery Fold protein activitya activity purification (mg) (U) (U/mg) (%) Cell ext. 19,800 11,090 0.56 Strep, sulf. 15,444 11,189 0.725 100.8b 1.3 Eluatepool 43.32 5,465 126.15 48.84c 225 ' CMCase activity is expressed as micromoles of glucose equivalents released per min at 37°C. b- This more than 100% recovery is due to degradation of CenAAPT during purification. The degradation product, p30, has a higher specific activity than CenAAPT. c - This recovery is underestimated due to the activity contributed by p30 in the crude cell extract. 17 T A B L E III N-terminal amino acid sequence of p30 produced from ngCenAAPT by C . fimi protease Cycle Complete digestion (66) 1 V (21.3) b 2 T (13.8) 3 P (13.71) 4 Q (13.39) 5 P (12.40) 6 T (10.18) 7 S (5.53) 8 G (10.33) 9 F (7.08) 10 Y (7.79) 11 V (11.19) 12 D (7.69) 13 P (6.31) 14 T (4.08) 15 T (7.51) 16 Q (6.30) 17 G (9.02) 18 Y (5.36) 19 R (18.49) 20 A (14.88) 21 W (0.51) 22 Q (5.17) 23 A (15.48) a. Sample amount (picomoles) b. Y i e l d (picomoles). 18 Table IV Extinction coefficient of ngCenAAPT lmg/ml lmg/ml lmg/ml Protein concentration Enzyme E 2 o5 E 280 ^ 28o determined by Bradford assay (determined)* (estimated) 1 5 ( p r e d i c t e d ) 0 relative tO UV assay (%)d ngCenA 32.78 2.64 2.47 81 ngCenAAPT 35.785 2.62 2.59 84 p30 35.12 2.38 2.42 89 a" Determined as described by Scopes. ( 1974). b' The absolute concentration of a solution was determined using E ^ and then used to estimate E280. c- Predicted from Tyr and Trp content, accordingly to Cantor and Shimmel (1980). d - The concentration of a solution determined by the Bradford dye-binding assay (relative to BSA) (Bradford, 1976) is expressed as a percentage of that determined from absorbance at 280 nm. 19 T A B L E V . Enzyme activity against a range of cellulosic substrates Sample Enzyme Activity Avicel PASCb CMC Cel. Azure ( m k a t a l / m o l ) a (katal/mol) (katal/mol) ( u n i t s / m m o l ) 0 ngCenA 25.3±0.7 ngCenAAPT 15.03+0.3 p30c 19.77+0.2 3.6±0.2 20.28±0.3 215±41 1.88±0.2 11.17±0.1 79.4+6 7.36±0.6 24.54+0.2 785.8+30 a. katal/mol: activity of 1 mol product/second/mol enzyme. b. PASC, phosphoric acid-swollen cellulose. c. units/mmol: production of OD595 = 1/hour/mmol enzyme. 20 T A B L E V I M r determinations for ngCenA and ngCenAAPT Enzyme Total amino Observed15 Predicted0 Observed less acid residues'1 M r M r predicted (kDa) (kDa) (kDa) ngCenA 418 48.7 43.8 4.9 ngCenAAPT 397 44.1 41.8 2.3 p30 284 30 30.2 0.2 a- From the deduced amino acid sequence of the mature enzyme (Wong et al., 1986). b- Determined by SDS-PAGE. c- Calculated from the deduced amino acid sequence. 21 Fig.2 Scheme for the construction of CenA Pro-Thr box deletion plasmid pUC18-cenAAPT a) subcloning of cenA to M13mp 19 b) oligonucleotide directed mutagenesis c) subcloning of cenAAPT into pUC18 mcs denotes multiple cloning site; BD denotes binding domain sequence CD denotes catalytic domain of CenA PT denotes Pro-Thr box region S, B, H and E denote restriction sites foxSstl, BamHL, Hpal and EcoRl. — denotes single stranded DNA 22 23 T7 DNA polymerase d NTP ATP TA DNA ligase transfection to ung+ host, select Hpal digestion positive clones S 24 12 3 4 5 6 7 8 9 10 11 12 13 14 kb 8.6 7.5 1.4 Figure 3. Screening of M13mpl9-cenA clones by B a m H I digestion of phage DNA Mini-prep DNA from infected cells was digested with BamHI and electrophoresed through a 0.7% agarose gel lane 1 and 14: X DNA digested with HindHSJEcdBl ( standard) lane 2: M13mpl9 DNA digested with BamHI (control) lane3-13: recombinant phage DNA digested with BamHI positive clones: 8.6 kb + 0.23 kb (lane 3,6,8,11,12 ) negative clones: 7.47 kb + 1.37 kb (lane 4,5,7,9,10,13 ) 25 6 7 8 9 10 11 12 13 14 15 16 17 1 2 3 4 5 H H H H H - H - H H H H H H ~ 18 o.c linear ccc. Figure 4. Screening of potential deletion mutants (M13mpl9-ct»nAAPT ) by Hpal digestion of RF DNA Mini-prep DNA from infected cells was digested with Hpal and electrophoresed through a 0.7% agarose gel lanel X DNA digested with HindlU ( standard) lane 2 X DNA digested with Hpal (control) lane3-5 M13mpl9DNA: uncut; cut with Hpal; cut with EcoRI (control) lane 6-17 recombinant phages, H: //maldigestion; - : uncut deletion mutant: Hpal digestion positive (phage clones 3,8,10,11,12) lane 18 single-stranded Ml3mpl9-ct?/iAAPT DNA o.c. denotes open circular form DNA linear denotes linear form DNA ccc. denotes covalently closed circular form DNA 26 c G A c cenA PT N - - Val Pro Thr Thr Ser Val ( Asn Val ) Thr Pro Gin Pro Thr Ser--~C CenAAPT replace N — V a l Ro Thr Thr Ser Val (PronThr12) Thr Pro Gin Pro Thr Ser--C CenA Figure 5. Dideoxy nucleotide sequencing gel showing deletion of the Pro-Thr box coding sequence from the cenA gene The sequence of recombinant phage M 1 3 m p l 9 - c e « A /M13mpl9ce«AAPT was obtained using a chemically synthesized 17-mer oligonucleotide. 27 Figure 6. Screening of potential deletion mutants ( pUC18-ce«AAPT ) for CMCase act iv i ty . T rans formed clones were transferred to pairs o f gr idded LB+ampic i l l in+IPTG+CMC plates. After incubation overnight at 37°C, one plate from each pair was stained with Congo-red and destained with 1 M N a C l . Positive clones were surrounded by haloes. pUC18 was the negative control ( bottom left), pUC18-cenA was the positive control (bottom right). 28 1 2 kDa — 44 - 3 0 Figure 7. Detection of ngCenAAPT in cell extracts Zymogram of polypeptides from JM101/pUC18-1.5ce/tAAPT separated by SDS-PAGE (see Materials and Methods). Lane 1 and 2 denote cen A APT clone 1 and 2. 29 PUC18 1 2 p U C E C 2 - H S B - H S B - H S B - H S B kb 421 2.68 128 29 .1.52 Figure 8. Restriction analysis of pUC18-cenAAPT clones. pUC18: control pUCEC2: control 1 and 2: deletion mutant clones 1 and 2 H, S and B denote Hpal, Sstl and BamUl digestion respectively; - denotes uncut DNA sample. 30 Fractal Nurrtier Figure 9. Purification of ngCenAAPT by affinity chromatography on cellulose. See Materials and Methods for details. After adsorption of the enzyme in 50mM phosphate buffer, pH 7.0, the column was washed with IM NaCl, then 50mM phosphate buffer. The enzyme was eluted with a 0 to 8 M linear gradient of guanidine-HCl. Fractions were 10ml. o A 280nm, • CMCase activity; A Guanidine.HCl concentration. 31 3 4 5 ko* —487 —44.1 Figure 10. SDS-PAGE analysis of the purification of ngCenAAPT lane 1 cell extract lane 2 streptomycin sulfate treated cell extract lane 3 purified ngCenA (control) lane 4 M r standard lane 5 pool of active fractions from the cellulose column 32 Figure 11. Time course of proteolysis of ngCenA and ngCenAAPT React ion mixture contained 35 (ig of ngCenA (A) or ngCenA (B), 200 u l of phosphate buffer and 0.2 unit of crude C. fimi protease. They were incubated at 37°C. Reactions were sampled at 0, 30min, l h , 2h, 4h, 6h, l l h , 24h, (A and B , lane 2-9, respectively), treated with P M S F and analyzed by S D S - P A G E ( 10% acrylamide). Control samples were incubated in the absence of protease, for 24h (A and B , lane 10). A l l lanes were loaded with sample equivalent to 3.4 | ig of initial protein, lane 1, M r marker. 33 E 1.0 - 5 1 0 1 5 2 0 [P](nmol.mL"1) 2 5 0.5 -o • 0.0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 [F^(nrrouTig-1) Figure 12. Adsorption of ngCenAAPT and ngCenA to Avicel The main panel is a Scatchard plot of the adsorption data ( see Materials and Methods for details ). The inset shows the data plotted as adsorption isotherm. • ngCenAAPT; o ngCenA. 34 CenA 112 135 TVPTTS PTPTPTPTTPTFTPTrTTTPTPWTPOPS A A 112 CenAAPT TVPTTS0--114 -gVTPOPS Fig. 13 Sequence alignments of CenA and CenAAPT Pro-Thr box and flanking regions, and locations of C. fimi protease primary cleavage sites in the nonglycosylated proteins. Amino acid sequences were deduced from the nucleotide sequence of the cenA gene. They are numbered with reference to the amino terminus of mature CenA. Underlined sequence was determined by amino acid sequencing (Table III). —denotes a gap to allow alignment of the flanking regions. Boxed amino acids are encoded by the nucleotides constituting the Hpal site of CenAAPT. 3 5 DISCUSSION The CenAAPT polypeptide appeared to be stable in E. coli and could be recovered from cell extracts. The observed and predicted Mrs for the various polypeptides are: ngCenA 48.7 kDa and 43.8 kDa; p30: 30.4 kDa and 29.6 kDa (Gilkes et al., 1988); ngCenAAPT 44.1 kDa and 41.8 kDa. This shows that the discrepancy between the observed and predicted Mrs for ngCenA is caused largely by the conformation of the Pro-Thr box sequence of the protein, but the conformation of CBD may also be a factor. Proteolysis studies of CenA with different proteases under denaturing conditions show that the resistance of the core protein to proteolysis is not due to an absence of cleavage sites within the primary structure of the core peptide, p30 in the case of ngCenA, but to its conformation (N. Gilkes, unpublished result). Proteolysis of ngCenAAPT also results in a core peptide of Mr 30 kDa which has the same N-terminal amino acid sequence as the p30 released from CenA. This suggests that the sequence before the cleavage site is not critical for recognition by C. fimi protease. However, the Pro-Thr box in CenA lies within an extended sequence rich in proline and hydroxyamino acids (N— TTCTGTVPTTS 1 0 1 - 1 1 1 [Pro-Thr box] 1 1 2 1 3 4VTPQQPYSG 1 3 5" 1 4 3—C) so the portion left in ngCenAAPT may mimic the intact Pro-Thr box and give a protease sensitive bridge. Proteolytic processing of the terminal cellulose-binding domains has been proposed as a means of regulating and adjusting the substrate specificities of cellulases during the hydrolysis of complex carbohydrates (Knowles et al, 1987). While there is insoluble substrate to hydrolyse, cellulases bind to it and the glycosylated enzymes are protected from proteolysis. Once the substrate has been solubilized, the enzymes become free in solution and become susceptible to limited proteolysis. Removal of the cellulose-binding regions produces enzymes with improved affinities for shorter, soluble cellodextrins. From the adsorption isotherms, 1.06 and 1.14 nmole. mg"1 of ngCenAAPT and ngCenA, respectively, are required to saturate Avicel PH101 under the stated conditions. 36 This suggests that the overall binding to cellulose is not influenced by the Pro-Thr box. Adsorption of ngCenA to Avicel involves two types of binding : high affinity and low affinity (Fig. 12, main panel, open circles). Scatchard analysis of the adsorption of ngCenAAPT to Avicel (Fig. 12, main panel closed circles) reveals a low affinity interaction but hardly any high affinity interaction. There are two possible explanations for this (N.R. Gilkes, personal communacation). One is that there are two types of site for binding on the cellulose: the crystalline and amorphous regions. The second is that there are two binding sites on the CBD with a single class of binding site on Avicel. The loss of the high affinity component but retention of the low affinity component for binding by ngCenAAPT supports the latter proposal. The removal of the Pro-Thr box may cause some conformational change which eliminates the high affinity site selectively. However, the following evidence favours the first proposal. Avicel has a heterogeneous structure comprising both crystalline and disordered or amorphous regions (Kulshreshtha and Dwetz, 1973). Treatment with H3PO4 converts Avicel to fully amorphous cellulose. The crystalline cellulose structure is changed when it is swollen in concentrated HaPO^ resulting in loss of the parallel chain structure of the original Avicel microcrystals and also a significant reduction in particle size and an increase in surface area. (Blackwell, 1981; Lee et al., 1982; Ooshima et al, 1983; Sarko, 1986). The binding of ngCenA to such amorphous cellulose exhibits only the low affinity component. ngCenA but not ngCenAAPT can be eluted from cellulose with water. This suggests that the Pro-Thr box may also affect the binding interaction between the enzyme and the crystalline regions of cellulose. This further suggests that some conformational changes may result from the deletion. The relative affinities of CenA and CenAAPT for cellulose need to be examined by adsorbing them to cellulose, then determining the conditions for eluting them. ngCenAAPT has significantly lower activity than ngCenA on a range of celluloses, including microcrystalline (Avicel), amorphous ( phosphoric acid-treated cellulose, and cellulose azure) and soluble ( CM-cellulose) substrates (Table V). This indicates that the Pro-Thr box has a crucial role in the normal behavior of this enzyme. The Pro-Thr box may 37 give the enzyme a conformation that makes the catalytic domain more accessible to the substrate. The catalytic activity of p30 against the same range of cellulosic substrates (Table V) agreed with a previous study (Gilkes, et al, 1988). Its decreased activity on microcrystalline cellulose suggests that the binding domain is required for efficient degradation of microcrystalline cellulose. Recent experiments have shown that ngCenA can effect the fragmentation of cotton fibres into small particles, whereas p30 can not (Kilburn et al., 1989 ). This also implies an active role for the CBD of CenA in cellulose degradation. The activity of p30 against soluble and amorphous substrates was significantly increased suggesting that the binding domain somehow inhibits the function of the catalytic domain when the function of the binding domain is not needed. Perhaps the conformation of the entire enzyme makes it less accessible to the substrate than the core protein alone. The Proline and hydroxyamino acid rich segments occur in more than 20 cellulases from various organisms ( personal communication with R.A.J. Warren, ). Of these, T. reesei cellulases display a bifunctional organization that closely resembles that of CenA and Cex (Tomme et al, 1988; van Tilbeurgh et al, 1986). The binding domain of CBH I is at the N-terminus, that of CBH U is at the C-terminus. A Pro-Thr-Ser rich sequence occurs between the two functional domains of CBH I and a repeated Pro-Thr-Ser sequence occurs between the two functional domains of CBH U. CBH II resembles CenA closely. Studies of the truncated proteins obtained by papain hydrolysis of T. reesei CBH I and CBH II showed that their binding to and hydrolysis of Avicel are markedly reduced. The truncated core protein of CBH II has significantly reduced catalytic activity against amorphous cellulose. This reduced activity in the absence of the binding domain is reminiscent of p30 (Tomme et al, 1988; van Tilbeurgh et al, 1988). However, papain hydrolysis removes the Pro-Thr-Ser region as well as the binding domain, so the effect on binding and hydrolysis of cellulose could be all or partly due to the influence of the Pro-Thr-Ser sequence. The Pro-Thr box of C. fimi is very similar in sequence to the hinge region of IgAi 38 (Plaut et al, 1975). The properties of ngCenAAPT suggest that the Pro-Thr box also functions as a hinge. The elimination of high affinity binding and the significant reduction in catalytic activity could reflect a lack of conformational flexibility. The differential responses to C. fimi protease of native CenA free in solution or bound to Avicel (Langsford, et al, 1987) suggest that there is conformational change when CenA binds to Avicel. It would be interesting to compare the sensitivities of ngCenA and ngCenAAPT to C.fimi protease when they are bound to cellulose. Small angle X-ray scattering studies of T. reesei CBHI and CBHII ( Abuja et al., 1988; Abuja et al., 1988; Schmuck et al, 1986) indicate that both CBH I and CBH H have a tadpole-like structure. The core proteins produced by partial papain proteolysis of CBH I and CBH H are the heads in both cases, and the binding domains together with the Pro-Thr-Ser rich regions are the tails . Similar analysis of CenA (N. Gilkes, unpublished result) also reveals a tadpole-like structure. Small angle X-ray scattering studies of CenAAPT and comparison to CenA should offer insights into how the Pro-Thr box is linked to the binding domain. It has been shown that neither CenA nor Cex are cleaved by the Neisseria gonorrhoeae IgAi protease (N.Gilkes, unpublished observations). 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