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Gene cloning and characterization of an exocellobiohydrolase (Cbh B) from Cellulomonas fimi Shen, Hua 1995

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GENE CLONING A N D CHARACTERIZATION OF A N EXOCELLOBIOHYDROLASE (Cbh B) F R O M CELLULOMONAS by  H U A SHEN B. Sc., Nan-Kai University, China,  1982  M . Sc., University of British Columbia ,  1990  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Microbiology and Immunology)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA December, 1995 © Hua Shen, 1995  FIMI  In  presenting this  degree at the  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make it  agree that permission for extensive  scholarly purposes may be granted her  for  It  is  by the  understood  that  head of copying  my 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 objective of this study was to characterize a cellulose-binding polypeptide, Cbpl20, from Cellulomonas fimi. The gene cbpllO was cloned, its nucleotide sequence determined and the amino acid sequence it encodes deduced. The gene encodes a polypeptide of 1090 amino acids. Mature protein is 1037 amino acids long with a predicted molecular weight of 109,765. Cbpl20 is non-glycosylated as judged by lack of reaction with the periodic acid-Schiff reagent. The enzyme comprises five domains: an N-terminal catalytic domain of 643 amino acids, followed by three fibronectin type III repeats of 97 amino acids each, and a C-terminal cellulose binding domain of 104 amino acids. The catalytic domain is in family 48 of glycoside hydrolases. Cbpl20 is a cellobiohydrolase (cellobiohydrolase B; CbhB) based on its ability to hydrolyze bacterial microcrystalline cellulose (BMCC), viscometric analysis of carboxymethylcellulose (CMC) hydrolysis, and the release of cellobiose as the major product from insoluble cellulose. CbhB catalyzes hydrolysis with inversion of anomeric carbon configuration. It removes cellobiose units from the reducing end of the cellulose molecule. It has an optimum p H of 7.0 for catalytic activity. CbhB binds to cellulose with similar affinity to other well-characterized C. fimi cellulases such as CenA and Cex. CbhB is the second exo-cellobiohydrolase found in C. fimi. Therefore, the cellulase system of C. fimi resembles those of fungi in comprising multiple endoglucanases and cellobiohydrolases. Furthermore, both the C. fimi and fungal cellulase systems contain cellobiohydrolases that hydrolyze the cellulose molecule from either the reducing end or the non-reducing end. The exo-cellobiohydrolases from C. fimi, CbhA, CbhB and the xylanase/exoglucanase Cex, all have low but detectable endoglucanase activity by the  CMC-Congo red plate assay. It is thus clear that there is not a clear distinction between endo- and exoglucanases.  iv Table of contents  Abstract  ii  Table of Contents  iv  List of Tables  viii  List of Figures  ix  List of Abbreviations  xi  Acknowledgements  1. Introduction  xiii  1  1.1 Importance of cellulase studies  1  1.2 Cellulose  2  1.3 Cellulose hydrolysis by bacteria and fungi  4  1.3.1 The synergistic process of cellulose hydrolysis  4  2.3.2 Cellulase activity — measurement and mode of action  8  1.3.3 The catalytic mechanisms of glycoside hydrolysis  11  1.3.4 Structure and functional relationships ofcellulases  12  1.3.5 Cellulomonas fimi cellulases 1.4 Objectives of study  2. Materials and methods  15 16  18  2.1 Chemicals, buffers and enzymes  18  2.2 Bacterial strains, plasmids and phages  18  2.3 Media and growth conditions  18  2.4 Recombinant D N A techniques  21  2.4.3 Production and isolation of single-stranded DNA 2.4.2 Site-directed in vitro mutagenesis  21 21  V  2.5 Cloning of CbhB gene from C. find 2.6 cbhB D N A sequence determination  22 23  2.7 Detection of cellulose-binding polypeptides from C. fimi  24  2.8 Detection of protein  24  2.8.1 SDS-PAGE  24  2.8.2 N-terminal amino acid sequencing  25  2.8.3 Determination of protein concentrations  25  2.9 Expression of cbhB and production of CbhB  25  2.10 Purification of CbhB  26  2.11 Proteolysis of CbhB  27  2.12 Assays for cellulose and hemicellulose hydrolysis  27  2.12.1 Reducing sugar assay  27  2.22.2 Total sugar assay  28  2.22.3 Hydrolysis of fluorogenic and chromagenic substrates  28  2.22.4 CMC-Congo Red plate assay  29  2.22.5 Viscometry assay  30  2.13 Analysis of products released from various substrates by CbhB  30  2.23.2 Products from oligosaccharides 2.23.2 BMCC and PASC hydrolysis products  30 31  2.14 Amino acid sequence alignments and database searches  31  2.15 Kinetic studies  31  2.16 p H optimum of CbhB  32  2.17 Determination of binding parameters  32  3. Results 3.1 Cloning of the cbhB gene 3.2 Nucleatide sequence of cbhB  34 34 34  vi 3.3 Amino acids sequence of CbhB  34  3.4 Relationship of CbhB to other B-l,4-glycanases  38  3.5 Production and purification of CbhB from E. coli  49  3.5.1 Construction ofpTugSH2 for cbhB expression  49  3.5.2  49  Construction of pTugSH3 for cbhB expression  3.5.3 Production of CbhB by E. coli and its purification 3.6 Hydrolytic activities  55 58  3.6.1 Activity on hemicelluloses  58  3.6.2  Activity on celluloses  58  3.6.3  Kinetics of the hydrolysis ofBMCC  63  3.6.4  Hydrolysis of cellodextrins by CbhB  65  3.6.5  CbhB is an exocellobiohydrolase  3.6.6  The endoglucanase activity of C. fimi exoglycanases  65 71  3.7 Determination of stereochemical course of hydrolysis  76  3.8 pH dependence studies  76  3.9 Binding to BMCC  76  4. Discussion  81  4.1 Structure of CbhB  81  4.2 Catalytic properties of CbhB  85  4.2.1 CbhB is an exocellobiohydrolase 4.2.2  85  The intrinsic endoglucanase activity of exoglucanases  87  4.3 Similarities between fungal and bacterial cellulase systems  90  4.4 Summary  92  5. R E F E R E N C E S  95  vii  6. APPENDIX  109  List o f Tables  Table 1.1 Bacterial exoglucanases  9  Table 1.2 Methods of measuring cellulase activities  10  Table 2.1 Bacterial strains  19  Table 2.2 Plasmids and phages  20  Table 3.1 Family 48 p-l,4-glycanases  48  Table 3.2 Activity of CbhB on hemicellulosic substrates  59  Table 3.3 Comparison of the activities of CbhB, CbhA and CenA on cellulosic substrates  60  Table 3.4 Kinetic parameters for hydrolysis of BMCC by CbhB and CbhA Table 4.1 Processing sites in proenzymes from C.  ftmi  63 82  ix  List of Figures  Figure 1.1  Model of lignocellulose structure  Figure 1.2 Cellulose and its hydrolysis  3 5  Figure 1.3 Mechanism of synergistic action between cellobiohydrolases CBHI and CBHII  7  Figure 1.4 Stereochemical classification of [3-1,4-glycanases  11  Figure 1.5  17  C. fimi cellulases and xylanases  Figure 3.1 Screening of a C. fimi genomic library with a cbhB probe  35  Figure 3.2 Schematic representation of the cloned C. fimi D N A fragment carrying the cbhB gene  37  Figure 3.3 Nucleotide sequence of cbhB and its flanking regions and the deduced amino acid sequence of CbhB  40  Figure 3.4 Leader peptides of C. fimi f3-l,4-glycanases  43  Figure 3.5 Comparison of fibronectin type Ill-like sequences from C. fimi [3-1,4-glucanases  44  Figure 3.6 Amino-acid sequence alignment of family II CBDs from C. fimi (3-1,4-glycanases  44  Figure 3.7 Alignment of the catalytic domains of the [3-1,4-glucanases of family 48  46  Figure 3.8 Construction of plasmid pTugSH2  53  Figure 3.9 Construction of plasmid pTugSH3  55  Figure 3.10 Detection of CbhB from E. coli culture  56  Figure 3.11 Purification of CbhB by cellulose affinity chromatography  57  Figure 3.12 Soluble sugar released from cellulose by CbhB  61  Figure 3.13 Determination of k t and K M values  64  c a  Figure 3.14 HPLC analysis of the products of hydrolysis of cellodextrins  X  by CbhB  66  Figure 3.15 HPLC analysis hydrolysis of cellohexaose by CbhB  68  Figure 3.16 Cleavage of various cello-oligosaccharides by CbhB  70  Figure 3.17 Specific fluidity versus reducing sugar release for the hydrolysis of C M C  72  Figure 3.18 CMC-Congo red plate assay of exoglycanases  75  Figure 3.19 Activity versus p H for the hydrolysis of BMCC  77  Figure 3.20 Isotherm for the adsorption of CbhB to BMCC  79  Figure 3.21 Double-recipocal plot of adsorption data for CbhB  80  Figure 4.1  Hypothetical model of cellulase systems from aerobic bacteria and fungi  94  xi List of Abbreviations  a.a. Ap  amino acid r  ampicillin resistant  BMCC  bacterial microcrystalline cellulose  bp  base pair  CBD  cellulose binding domain  Cbpl20  cellulose binding polypeptide 120  CMC  carboxymethylcellulose  DMSO  dimethyl sulfoxide  dNTP  deoxynucleoside triphosphate  DTT  dithiothreitol  HBAH  p-hydroxybenzoic acid hydrazide  HPLC  high performance liquid chromatography  IPTG  isopropyl-B-D-thiogalactoside  kb  kilobase pairs  kcat  enzyme turnover number  kDa  kilodaltons  KM  Michaelis constant  Km*"  kanamycin resistant  LB  Luria-Bertani medium  M  relative molecular mass  r  PASC  phosphoric acid-swollen cellulose  PFU  plaque-forming units  PMSF  phynelmethylsulfonyl fluoride  pNPC  p-nitrophenyl B-D-cellobioside  pNPG  p-nitrophenyl B-D-glucopyranoside  pNPGla  p-nitrophenyl B-D-galactopyrannoside  pNPX  p-nitrophenyl B-D-xylopyranoside  RBB-xylan  4-O-methyl-D-glucurono-D-xylan-Remazol brilliant Blue  SDS-PAGE  sodium dodecyl sulfate-polyacrylamide gel electrophoresis  TE  tris-EDTA  TYP  tryptone, yeast extract, phosphate  U.V.  ultra violet  <Psp ( l / T | S p )  specific fluidity  Acknowledgments  First of all, I am indebted to my supervisor Dr. Tony Warren for offering me the opportunity to study in Canada in such an interesting field, and take the challenge of a foreign student. I would like to sincerely thank my joint supervisors Drs. Tony Warren, Doug Kilburn and Bob Miller for their encouragement and superior guidance through this work. I am grateful to Dr. Neil Gilkes for his intelligent input into this project. Special thanks go to Dr. Peter Tomme for sharing his enormous knowledge of the cellulase field with me and his help with HPLC operation. I greatly apprietiate my thesis committee Drs. Louis Glass and Jack Saddler for their expert critiques and judgment of this work. Members of the cellulase lab, too numerous to mention, provided a friendly, helpful and enjoyable environment in which to work. Special thanks go to Patti Miller, Helen Smith and Emily Kwan for their warmth and their vital role in running the lab. My thanks also go to Drs. Zahra Assouline, John Coutinho, Edgar Ong and Alasdair MacLeod, the former members in Room 206 for their friendship, the occasional dinner and movie, and for creating a well-organized, efficient lab system. Dr. Alasdair MacLeod deserves special thanks for frequent listening to and discussing of my results and problems, correcting my manuscripts and his sense of humor that melted some bad times into laughs. I also thank the National Sciences and Engineering Research Council of Canada for the grants by which this thesis was supported. I would like to thank my friends, here in UBC, U.S.A. and back in China for their encouragement, which played also a vital part throughout these years. Finally, I sincerely thank my family for their love and support.  1 1 Introduction  1.1 Importance of cellulase studies  Cellulose is the major structural component of plant biomass. Synthesized at about 4 x 10 tonnes annually (Coughlan, 1990), it is the most abundant carbohydrate 9  on the earth. Many microorganisms possess the ability to degrade plant biomass, providing themselves with energy and carbon. In addition to their role in carbon recycling in the environment, cellulases are also involved in plant/parasite interaction (Mateos, 1992; Meletzus et al., 1993; Boccara et al, 1994), spore germination (Ramalingam et ah, 1992) and plant cell wall growth and differentiation (Bonghi et al, 1992; Kemmerer and Tucker, 1994). Enzymatic degradation of cellulose has been of great research interest in biotechnology due to the vast abundance of cellulose. Production of ethanol from plant biomass could improve energy security and reduce air pollution (Wyman, 1994). Other potential applications include the "environmentally friendly" enzymatic treatment of pulp fibre to modify paper properties, or de-inking of recycled paper 0effries et al, 1992 and 1994). The non-hydrolytic cellulose-binding domain (CBD) has promising applications in the textile industry; in biotechnology, it can be used as an affinity tag for protein immobilization or purification (Kilburn et al, 1993). Although extensive study has been done over the years, the mechanism by which microorganisms degrade plant cell wall materials still remains unclear. This can be attributed to the complexity of the physical organization of the substrate as well as the complex polysaccharidases produced by bacteria and fungi. Understanding the structure of cellulose, the structural and functional relationship of individual enzymes, the regulation of these enzymes and the way individual enzymes cooperate could  2 eventually lead to the engineering of enzyme systems with enhanced hydrolytic efficiency, as well as individual enzymes with desired properties.  1.2 Celluloses  Cellulose is a linear polymer composed of up to 10,000 B-l,4-linked D-glucose residues, in which each glucose residue is rotated by 180° relative to the preceding residue. The basic repeating unit is cellobiose (Fig. 1.2) (Beguin and Aubert, 1994). The cellulose chains are associated by inter- and intramolecular hydrogen-bonding to form tightly packed, water insoluble microfibrils (Blackwell, 1982) that contain highly ordered crystalline regions as well as less ordered "amorphous" regions (Coughlan, 1985). In plant cell walls, the major natural source of cellulose, the microfibrils are embedded in an amorphous matrix of hemicelluloses that is further associated with pectin and lignin (Preston, 1974) (Fig. 1.1). In secondary cell walls, the hemicelluloses are mostly xylans (B-l,4-linked xylopyranose) substituted with a variety of side chains, mannans (B-l,4-linked mannose polymer with branches) and glucomannans (Puis, 1993). In primary plant cell walls, xyloglucans form the interface between the microfibrils and the wall matrix (Carpita and Gibeaut, 1993). The enzymes degradading cellulose are called "cellulases". In order to degrade cellulose in such a complex form, cellulolytic microorganisms usually produce an array of polysaccharide hydrolases, such as xylanases and mannanases, in addition to enzymes that hydrolyze B-l,4-glucosidic bonds in cellulose. Due to the complexity of the cell wall materials, purified celluloses are used as substrates in cellulase studies. This requires celluloses of various degrees of crystallinity and polymerization. Bacterial microcrystalline cellulose (BMCC), obtained from the bacterium Acetobacter xylinum, is highly pure and crystalline in structure (Gilkes et al., 1992). CF1™ is a fibrous cellulose from cotton; phosphoric acid swollen  3  CF1 (PASC) is used as amorphous cellulose. Soluble cellulose can be prepared by introducing either carboxymethyl or hydroxyethyl substituents into the cellulose chain. For example, carboxymethylcellulose (CMC) provides a convenient substrate for assaying endoglucanase activity. Cellooligosaccharides are used to determine the modes of action of the cellulases. Other forms of B-glycan are also used in this study to detect |3-glycanase activity other than cellulases. For example, xylan and mannan are used to detect hemicellulase activity. Various chromogenic and fluorogenic cellulosic and hemicellulosic substrates such as p-nitrophenyl-B-cellobiose (pNPC) and azobarley-P-glucan can be used to detect cellulases and hemicellulase activity fluorimetrically or colorimetrically.  Figure 1.1 Model of lignocellulose structure. (A) Cutaway view showing the organization of the cell wall layers composing woody fibres. (B) Probable relationship of lignin and hemicellulose to the cellulose microfibrils in the secondary walls. The diameter of each cell is approximately 25 uM. P.W., primary cell wall; S.W. 1 - S.W. 3, secondary cell walls. (Adapted from Beguin and Aubert, 1992).  4  1.3 Cellulose hydrolysis by bacteria and fungi  Earlier, cellulase research focused on fungi and on Trichoderma in particular. In the last twenty years, however, there has been increasing interest in cellulases produced by bacteria. Cellulolytic fungi produce cellulases in large quantity. The softrot fungus Trichoderma reesei produces about 20 grams of cellulases per liter of culture fluid upon cultivation under appropriate conditions (Wood and McCrae, 1979). Cellulolytic bacteria can produce highly efficient cellulase systems; for example, the thermophilic anaerobe Clostridium sp. produce a cellulosome complex with specific activity about 50-fold higher than the extracellular system produced by T. reesei (Johnson et al, 1982).  2.3.2 The synergistic process of cellulose hydrolysis The degradation of crystalline cellulose involves the synergistic interaction of cellulolytic components having different specificities. Based on the cellulase systems produced by Phanaerochate chrysosporium (Eriksson and Pettersson, 1975; Eriksson, 1978) and Trichoderma koningii (Wood and McCrae, 1978), a synergy model was proposed in the late seventies (Fig. 1.2). Three major classes of enzymes are required for enzymatic cellulolysis. Endoglucanases (l,4-(3-D-glucan gluconohydrolases; EC attack internal pM,4-glucosidic bonds randomly in the amorphous region, greatly increasing the number of free ends for the action of exoglucanases; cellobiohydrolases or exoglucanases (l,4-|3-D-glucan cellobiohydrolases; EC liberate cellobiose units from the ends of cellulose chains; and (3-glucosidases Q3-Dglucoside glucohydrolase; EC hydrolyze cellobiose into glucose. Since then, the model has been extended to other fungal systems (McHale and Coughlan, 1980; Wood et al, 1980; Henrissat et al, 1985).  5  A  B  II  Crystalline region  i  Amorphous region Adsorption of cellulases Endoglucanases  Figure 1.2 Cellulose and its hydrolysis (Adapted from Beguin and Aubert, 1994). A) Molecular structure of cellulose: 0-1,4 linked D-glucopyranose polymer (only 3 residues shown). B) Synergism between endoglucanases, cellobiohydrolases and p"glucosidases in a fungal cellulase system. Shaded glucopyranose residues indicate the reducing ends of the cellulose chains.  6 Although generally accepted, many observations suggest that this model is oversimplified. It does not account, for example, either for the fact that cellobiohydrolase I (CBHI) and cellobiohydrolase II (CBHII) from T. reesei need different endoglucanase ratios to obtain maximum synergy (Henrissat et al, 1985) or that not all endoglucanases appear able to act synergistically with cellobiohydrolases (Wood and McCrae, 1978). In addition, synergy is also observed between pairs of cellobiohydrolases, the socalled "exo-exo" synergy, from T. reesei (Fagerstam and Pettersson, 1980; Henrissat et al, 1985; Kyriacou et al, 1987) and Penicellium pinophilum (Wood and McCrae, 1986). Wood and McCrae (1986) postulated that CBHI and CBHII could exhibit different substrate stereospecificities, each attacking one of the two types of naturally-occuring, nonreducing end group that might be found in the cellulose crystallite (Fig. 1.3). However, the concept that exoglucanases hydrolyse cellulose molecules only from the nonreducing end has been abandoned, as discussed below. Exo-exo synergism can be more apparent than real because of possible contamination with traces of endoglucanase. Wood et al, (1989), for example, demonstrated that the synergy between CBHI and CBHII from P. pinophilum disappeared when the enzymes were further purified; synergy reappeared when endoglucanase was introduced to the system. It was believed for many years that exoglucanases remove cellobiose from the non-reducing ends of cellulose chains. Bronnenmeier et al, (1991) observed that Avicelase II from Clostridium stercorarium removed cellobiose from the reducing end of cellodextrin. Biely et al (1993) showed clearly, using cellodextrin labelled with tritium at the reducing end, that CBHI from T. reesei hydrolyses from the reducing end whereas CBHII hydrolyses from the non-reducing end. Thus exoglucanases can hydrolyze cellulose chains from either end. This may also account for exo-exo synergy. In trying to understand cellulose hydrolysis, cellobiohydrolases have received  7  C«tU/fo«c -G C  •  O  O G \ s N z' o \ O • V G G G-  O  r~G  < CCHI  G-G CeOobtose  CcOoios«  -° <\ xN—o— -G  '  G  G.  GO  CQH  G  ,G-  o  I  G-G OUoblo*€ ( I  G  a  0  G G N / O , G G * G/ O !  Figure 1.3 Mechanism of synergistic action between cellobiohydrolases CBHI and CBHII. (Adapted from Wood et al, 1988). (a) and (b), Since cellobiose is the repeating unit in the cellulose chain, theoretically two types Of reducing end groups (I and II) will exist in the cellulose crystallite; (c), Cellobiohydrolase I attacks the non-reducing chain end of configuration I; (d) cellobiohydrolase II attacks the non-reducing chain end of configuration II exposed by cellobiohydrolase I.  particular attention because of their central role in the degradation of crystalline cellulose (Fagerstam and Pettersson, 1980; Henrissat et al, 1985; Niku-Paavola et al, 1986) and the large proportion of the extracellular protein they account for in the fungal system (Eriksson and Wood, 1985). About 60% of the protein secreted by T. reesei is CBHI; CBHII accounts for a further 20%. The cellulase systems of other soft rot fungi, such as P. pinophilum, and of white rot fungi like P. chrysosporium all contain cellobiohydrolases (CBHs) like CBHI and CBHII.  8 The distinction between exoglucanases and endoglucanases was also clouded by the persistence of endoglucanase activity in highly purified preparations of CBHI and CBHII from T. reesei (Stahlberg et al, 1993). However, the endoglucanase activity could have arisen from as little as 0.2% contaminating endoglucanase. To ensure that a given exoglucanase is free of contaminating endoglucanase, it is necessary to produce the enzyme in a heterologous host devoid of B-l,4-glucanases, such as Escherichia coli. In comparison with the fungal systems, little is known about the mechanism by which bacteria degrade cellulose. In the past, bacteria were thought not to produce exoglucanases. Although cellulolytic bacteria were known to produce numerous endoglucanases, there were relatively few reports of even single exoglucanases in bacterial cellulase systems (MacKenzie et al, 1984; Gardner et al, 1987; Bronnenmeier et al, 1991; Morag et al, 1991; H u et al, 1992) (Table 1.1). However, with the report of several cellobiohydrolases from bacteria in the past year (Meinke et al, 1994; Kruus et al, 1995; Zhang et al, 1995; this work), it is increasingly evident that bacterial cellulolytic system resemble their fungal counterparts.  1.3.2 Cellulase activity — measurement and mode of action The measurement of cellulase activity is difficult for several reasons. Whereas most enzyme theory is based on reactions in solution, the cellulase-cellulose system involves an interface between the enzymes and the insoluble substrate surface. The complexity of the cellulase enzyme system and the synergistic interaction between its various components complicate the assessment of individual enzymes. The situation is further complicated by uncertainty regarding the structures of the various forms of cellulose employed as substrates (Wood and Bhat, 1988). The effectiveness of various microorganisms can be assessed by "total cellulase" activity using standard procedures published by IUPAC (International Union of Pure  9 Table 1.1 Bacterial exoglucanases organisms  enzyme  family  reference  Cellulomonas fimi  CbhA*  6  Cellulomonas fimi  CbhB*  48  this work  Clostridium stercorarium  Avicelase II  48  Bronnenmeier et al, 1991  Clostridium thermocellum YS  S8  48  Morag et al, 1991  48  Kruus et al, 1995  Clostridium thermocellum A T C C 27405 CelS*  Meinke et al, 1994  Microbispora bispora  CbhH  H u et al, 1992  Ruminococcus flavefaciens FD-1  ExoA  Gardner et al, 1987  Streptomyces flavogriseus  45-CD  MacKenzie et al, 1984  Thermomonospora fusca  E3*  6  Zhang et al, 1995  A n sterisk indicates that the enzyme was produced from non-cellulolytic host in which the gene encoding for the enzyme was cloned.  and Applied Chemistry) (Ghose, 1984). However, to develop an understanding of the mechanism of cellulase action, the various enzymes of the cellulase system should be evaluated in as many ways as possible, using a wide variety of cellulosic substrates of differing degrees of polymerization and crystallinity, as described in section 1.2. Glycosyl-hydrolases can be classified according to their roles in cellulose hydrolysis. Various methods used to characterize their activities are shown in Table 1.2. Endoglucanases are characterized by their essentially random hydrolysis of cellulose chains which causes a rapid decrease in degree of polymerization of the substrate. Endoglucanases can be easily identified by their activity on carboxymethylcellulose (CMC) because their random internal-cutting pattern allows them to avoid the negatively charged substituted residues. They can also hydrolyze alkali-swollen (amorphous) cellulose, and crystalline celluloses such as Avicel and cotton to a lesser extent. Exoglucanases are usually recognized by their ability to hydrolyze crystalline cellulose, and by their ability to remove cellobiose units from the  10  Table 1.2 Methods of measuring cellulase activities  1  Enzyme  substrate  Complete cellulase  cotton  Assay solubilization -estimation of cellulose in residue -reducing sugar released weight loss  Cellobiohydrolase  Endoglucanase  filter paper  solubilization  Avicel  -release of reducing sugar  BMCC  -release of reducing sugar  amorphous cellulose  -release of soluble sugar  cellodextrin  analysis by T L C or H P L C  CMC  release of reducing sugar  b  analysis by TLC or HPLC  amorphous cellulose  solubilization: release of soluble sugar  cotton  swelling in alkali  pNPG  d  release of p-nitrophenol  cellobiose  release of glucose  cellodextrin  increase in reducing power  Adapted from Wood and Bhat (1988). bacterial microcrystalline cellulose; b carboxymethylcellulose; hydroxyethylcellulose; c  1  f  cellodextrin  a  e  e  decrease in viscosity  C  1  d  solubilization  Avicel  HEC  B-Glucosidase  3  p-nitrophenyl-B-glucoside; thin-layer chromatography; high performance liquid chromatography.  11 end of a cellulose chain. True exoglucanases do not hydrolyze CMC; they can only remove unsubstituted residues from the ends of C M C and cannot remove further residues once a substituted moiety is encountered.  1.3.3 The catalytic mechanisms of glycoside hydrolysis All glycoside hydrolases (glycosidases) characterized to date catalyze hydrolysis stereoselectively. The configuration at the anomeric carbon of the scissile bond is either retained (a to a, P to P) or is inverted (a to (3, P to a) upon hydrolysis (Fig. 1.3) (Sinnott, 1990). Retaining glycosidases proceed via a double displacement mechanism while *  those that invert do so with a single displacement.  Figure 1.4 Stereochemical classification of P-l,4-glycanases. (Adapted from MacLeod, A . M . , 1994). Only one glucose residue is shown for simplicity. The site of bond cleavage is indicated by A.  C. fimi cellobiohydrolase B (CbhB), studied in this work, is an inverting enzyme. This type of mechanism involves protonation of the glycosidic oxygen of the scissile bond by an acidic amino acid residue (the general acid catalyst). In a concerted process, direct stereospecific attack at the anomeric carbon of the scissile bond by a  12 water molecule also occurs. The nueleophilicity of this water molecule is greatly increased through deprotonation by a basic amino acid residue (the general base catalyst). The resulting hydroxyl group then acts to displace the leaving group resulting in a product with the opposite stereochemical configuration to the original substrate (Sinnott, 1990).  13.4 Structure and functional relationships of cellulases To date, over 200 cellulase and xylanase sequences are available. Most of the Bglycanases are composed of a catalytic domain joined to one or more ancillary domains, frequently by a linker sequence (Gilkes et al., 1991). This modular organization was initially demonstrated by the analysis of fragments released by limited proteolysis (Van Tilbeurgh et al., 1986; Gilkes et ah, 1988; Tomme et al., 1988). Domains in new enzymes can now be recognized by comparison of their primary structures with those of other enzymes whose structural and functional organization has been defined.  Catalytic domains  Twenty one cellulases and xylanases were first classified  into six families by hydrophobic cluster analysis which can detect regions corresponding to highly conserved folds in the catalytic domains (Henrissat et al., 1989). Now based on their amino acid sequence similarities, the catalytic domains of glycoside hydrolases have been classified into 48 families including chitinases, amylases and B1,3-glucanases in addition to cellulases and xylanases (Tomme et al., 1995a). Sequence similarities reflect the fact that the enzymes within the family use a similar mechanism, inverting or retaining, and have similar overall tertiary structures (Claeyssens and Henrissat, 1992; Gebler et al, 1992; Henrissat and Bairoch, 1993). Structure similarities are confirmed with three-dimensional structures in families 6 and 11 (Rouvinen et al, 1990; Arase et al, 1993; Campbell et al, 1993; Spezio et al, 1993; Torronen et al, 1994; Tomme et al, 1995a). For all cellulases and xylanases tested, all enzymes in a family  13 catalyze hydrolysis with the same stereochemistry, supporting this hypothesis (Gebler et al, 1992; Schou et al, 1993). Important amino acid residues are conserved in the same family; thus, sequence comparison of numerous family members can narrow down the candidates for putative catalytic residues. Some families contain enzymes from only bacteria (families 8 and 48) or fungi (family 7), while other families contain both bacterial and fungal enzymes (families 5,6,10,11,12 and 13), indicating that lateral gene transfer occurred (Gilkes et al., 1991). Enzymes of the same family may have different substrate specificities (family 8 contains both (3-1,4-glucanases and (3-1,3(4)glucanases) suggesting divergent evolution from a common ancestor, through a small number of amino acid substitutions.  Cellulose-binding domains Most cellulose-binding domains (CBDs) can be classified into nine families (families I to IX, Tomme, et al., 1995b). T. reesei and C. fimi CBDs are the most extensively studied and classified into families I and II. Family I CBDs are all fungal, about 33-36 amino acids long. Family II CBDs are bacterial, about 100 amino acids long (Fig. 3.6). They all contain four conserved aromatic residues (tyrosines in family I and tryptophans in family II, respectively), believed important for the interaction with cellulose (Claeyssens and Tomme, 1990; Reinikainen, et al., 1992). Mutation of conserved tryptophan residues in the CBDs of C. fimi CenA (Din et al, 1994) and Pseudomonas fluorescens subsp. cellulosa xylanase A (Poole et al, 1993) significantly reduces binding affinity, providing supporting evidence for this hypothesis. High resolution NMR structural analysis of a related CBD from C. fimi Cex shows that the corresponding tryptophan residues are exposed to solvent and accessible to the substrate (Xu et al, 1995). This is also in agreement with a functional role for these tryptophan residues. Removal of the CBD affects enzyme activity on various cellulosic substrates (Gilkes et al, 1988; Tomme et al, 1988; Hefford et al, 1992; Coutinho et al, 1993). It was  14 proposed that the CBD could enhance activity on insoluble substrates by increasing the local enzyme concentration on the substrate, or by disrupting non-covalent associations and thereby increasing substrate accessibility (Knowles et al, 1987; Teeri et al., 1992). The isolated CBD of C. fimi CenA disrupts the structure of ramie fibers and releases small particles of cellulose from both ramie and cotton fibres (Din et al, 1991). In addition to cellulases, CBDs also occur in chitinases (Fujii and Miyashita, 1993) and hemicellulases (Kellet etal, 1990; Ferreira et al, 1993; Millward-Sadler et al, 1994; Irwin et al., 1994). This suggests that the degradation of cellulose and hemicellulose are coordinated processes and the organization of the enzymes involved reflects the association of the carbohydrate polymers which comprise the plant cell wall.  Linker regions and other domains The various domains or modules in multidomain cellulases are typically joined by distinct linker regions (Gilkes et al, 1991). They are usually sequences of 20 - 60 amino acids rich in proline and hydroxyamino acids. Linkers may allow flexibility between domains while maintaining a spatial separation appropriate for domain interaction (Tomme et al, 1995a). Deletion of the linker sequence from C. fimi CenA reduces activity on both insoluble and soluble cellulose substrates (Shen et al, 1991). This is probably a result of steric hindrance due to reorientation of the catalytic domain and the close proximity of the CBD to the catalytic site, since the isolated catalytic domain has greater activity on amorphous and soluble substrates. Linkers are often glycosylated (Tomme et al, 1988; Ong et al, 1994), which contributes to stability in an aqueous environment and protects against proteolysis (Langsford et al, 1987; MacLeod et al, 1992). Fibronectin type III (Fnlll)-like sequences occur in some C. fimi cellulases in duplicate or triplicate (Meinke et al, 1991b; 1993 and 1994; Shen et al, 1995) (Fig. 1.4). This domain can also be found in other enzymes involved in hydrolysis of insoluble  15 polysaccharides, such as chitinases and amylases (Tomme et al, 1995a). Fibronectin is a membrane protein in animal cells, functioning in protein-protein interactions and protein-carbohydrate interactions (Pierschbacher and Ruoslahti, 1987). The roles of Fnin repeats in cellulose degradation remain to be investigated. Deletion of the Fnlll module between the catalytic domain and the chitin-binding domain in Bacillus circulans WL-12 chitinase A l drastically reduces the activity of the enzyme on chitin, but not on the soluble derivative carboxymethylchitin (Watanabe et al, 1993). In this case, it can be argued that the Fnlll sequence acts as a linker for the proper function of both domains on insoluble substrates.  1.3.5 Cellulomonas fimi cellulases C. fimi is a Gram positive, coryneform, mesophilic, soil bacterium capable of growth on cellulose. It can be isolated from forest humus, and waste material with a high cellulose content (Choi et al, 1978; Malekzadeh et al, 1993). Synthesis of cellulases and other related enzymes in C. fimi is controlled by readily metabolized carbon sources: induced in the presence of the polysaccharides and repressed by carbon sources such as glucose (Greenberg et al, 1987a and 1987b). When grown under inducing conditions, it secretes an array of cellulases as well as high levels of B-1,4mannanase and B-l,3-glucanases (Langsford et al, 1984; Gilkes, unpublished result). C. fimi also secrets a low molecular weight serine protease which degrades some of these enzymes (e.g. CenA and Cex) into functional domains, thus adding another layer of regulation and complexity to the system (Langsford et al, 1987; Gilkes et al, 1988). Revealing the mechanism by which C. fimi degrades cellulose requires characterization of each individual enzyme and determination of their structural and functional relationships. The properties of cellulases purified from the native host can be influenced by trace enzyme contaminants (Bronnenmeier and Staudenbauer, 1990; Wood and Garcia-Campayo, 1990). This problem can be surmounted by gene cloning  16 and study of the recombinant protein. The cenA, cenB and cex genes were first isolated as E. coli clones expressing polypeptides which reacted with an antiserum to supernatant proteins from a C. fimi culture grown in the presence of cellulose (Gilkes et al, 1984a; Whittle et al, 1982). The cenC gene was isolated by taking advantage of the capacity of CenC to bind to Sephadex (Moser et al, 1989). The occurrence of CBDs on these enzymes, and the fact that Cex is predominatly a xylanase, suggested that other enzymes important in biomass degradation may contain CBDs as well. Thus C. fimi culture supernatants were screened for other cellulose-binding proteins (Cbps). Several Cbps were thus identified by adsorption to BMCC. Three of them, Cbp75, Cbp95 and Cbpl20, were shown to have unique N-termini (Meinke et al, 1993). Cbp75 and Cbp95 have been characterized as CenD and CbhA, respectively (Meinke et al, 1993 and 1994). Cbpl20 is characterized as CbhB in this work. It is clear now that C. fimi possesses a cellulolytic system resembling that of fungi. Figure 1.4 shows all C.fimi cellulases and xylanases characterized to date.  1.4 Objectives of study  The overall objective of this study was to investigate the role of Cbpl20 in cellulose degradation by C. fimi. The approach was to clone the gene encoding Cbpl20, determine its nucleotide sequence and deduce the amino acid sequence of the encoded polypeptide. Sequence alignment with the other B-glycanases would reveal its functional domains. The second part was to overexpress the gene in and purify the gene product from the heterologous host, E. coli, which is devoid of B-glycanase activity, thereby eliminating B-glycanase contamination. Then, its hydrolytic activities would be screened with various cellulosic and hemicellulosic substrates. Finally, its substrate specificity, mode of hydrolysis and binding parameters would be determined.  17  CenA CenB CenC  I  l\NK\\K\\3  • • • • • • • • • > \ \ \ S I s \ \ \  CenD  mmwrnmrnvm  CbhA CbhB  48  Cex  10  XylD  11  E^giK\\\\\S|ggB  catalytic domain  ^  CBDII g g ]  CBDIII  CBDIV  linker gg"§  Fn3 module  iSS^  other  Figure 1.5 C. fimi cellulases and xylanases. (Adapted from Miller Jr. et al, 1995). Each enzyme is represented as a linear map to show the arrangement of the structural and functional domains (Tomme et al, 1995a; Millward-Sadler et al, 1994). The length of each domain is proportional to the number of amino acid residues it contains. CenA, CenB, CenC and CenD are endo-P-l,4-glucanases. CbhA and CbhB are exoglucanases. Cex is a mixed function (3-1,4-xylanase and |3-l,4-glucanase. XynD is a (3-1,4-xylanase. All the enzymes contain a catalytic domain and one or more cellulose-binding domains. Some contain additional domains such as fibronectin type III (Fnlll) modules. Numbers refer to the glycanase family to which each catalytic domain belongs (Henrissat and Bairoch, 1993). CbhB was originally designated CenE (Shen et al, 1994). Family 48 is a new cellulase family formerly called family L (Shen et al, 1994).  18  2. Materials and methods  2.1 Chemicals, buffers and enzymes  All the chemicals used were of analytical or HPLC grade. The buffers and solutions were prepared according to Sambrook et al, (1989). Restriction endonucleases, polymerases, ligase and nucleotides were from Pharmacia or New England Biolabs (NEB) and buffers and conditions for these enzymes were as recommended by the manufacturers. A v i c e l ™ (type PH101) was from KMC International. Bacterial microcrystalline cellulose (BMCC) was prepared from cultures of Acetobacter xylinutn, (ATCC 23769) as described by Hestrin (1963). CF1 cellulose and carboxymethyl-cellulose (CMC) were from Sigma. A l l media components were obtained from Difco.  2.2 Bacterial strains, plasmids and phages  The bacterial strains, plasmids and phages used in this study are described in Tables 2.1 and 2.2. Bacterial stocks were maintained at -70°C in LB medium containing 10% DMSO. Plasmid D N A was stored in TE buffer or water at -20°C. Phages were stored in TYP medium at 4°C.  2.3 Media and growth conditions  C. fimi A T C C 484 was grown in modified (low salt) LB medium (Miller, 1972) (10 g tryptone, 5 g yeast extract, 0.5 g NaCl per liter) supplemented with 0.1% glucose for D N A preparation. For the isolation of cellulose-binding polypeptides, C. fimi was grown in basal salts medium (Stewart and Leatherwood, 1976)  19 supplemented with 0.1% cellulose powder CC41 (Whatman) and 0.09% Torula yeast extract. Cultures were grown in shake flasks at 200 rpm, 30°C, for 2-5 days. E. coli strains were grown in LB or TYP broth (16 g tryptone, 16 g yeast extract, 5 g NaCl and 2.5 g K2HPO4 per liter). Small-scale cultures of E. coli containing pTZ or pTug-based vectors were grown in TYP supplemented with antibiotics (ampicillin at 100 ug/mL or kanamycin at 50 ug/mL) in shake flasks at 200 rpm, 30°C. Flask volumes were limited to 25% of capacity. Solid medium contained 1.5% (w/v) agar.  Table 2.1 Bacterial strains Bacterial strain  Genotype  Reference or source  C. fimi  A T C C 484  E. coli strains JM101  supE thi 1 A(lac-proAB) F[tra D36  Yanisch-Perron  proAB lad* lacZ AMI5]  et al., 1985  HfrKL160/45[/ysA(61-62)] dutl  Kunkel et al.,  ungl thil relAl Zbd279::Tnl0 supE44  1987  +  RZ1032  XLl-Blue  rec Al end A l gyr A96 thil hsdR17 supE44 rel AlHuynh et al, lac [F proAB lacl* lacZ AM15 TnlO (ter )l r  XLl-Blue MRF' A(mcrA)182 A(racrCB-/isdSMR-mrr)172  1985 Jerpseth et al,  rec A l end Al gyr A96 thil hsdR17 supE4A relAl1992 lac [F proAB lacl* lacZ AM15 TnlO (tet )] T  SOLR  el4" (mcrA) A(mcrCB-/zsdSMR-mrr)171 sbcC recB rec] wmuC::Tn5(kan ), uvrC lac gyrA96 r  relAl thil endAl X [ F proAB lacfi AM15] Su' R  StrataGene  20  Table 2.2 Plasmids and phages  Plasmid or phage  Characteristics  Reference or source  lambda-C. fimi  C. fimi genomic D N A fragments  Custom made by  genomic library  carried on A.ZAPII phagemid  StrataGene  ExAssist  helper phage to excise  StrataGene  pBluescript from XZAPII M13K07  pBluescript#5  helper phage for preparation  Vieira and Messing,  of single-stranded DNA: Knv  1987  pBluescript carrying a 3776 bp  This study  fragment of C. fimi D N A containing the CbhB gene pTZ18U/R  p/flc; F l ori, Apr  Mead et al, 1986  pTug  ptac; K m  Graham et al, 1995  pTZSHl  pTZ19U carrying a 3776 bp  r  This study  fragment of C. fimi D N A containing the CbhB gene pTugSH2  pTugS3N8 carrying the CbhB gene  This study  pTugSH3  pTugE07K3 carrying the mature  This study  CbhB coding sequence  21 2.4 Recombinant D N A techniques  Recombinant D N A work was generally carried out as described by Sambrook et al., (1989). D N A fragments were isolated from agarose gels and then purified using G e n e C l e a n ™ kits (Bio 101, La Jolla, CA). Competent cells were prepared and transformed as described by Miller (1987). Oligonucleotides were synthesized on an Applied Biosystems automated D N A synthesizer, model 380B (Oligonucleotide Synthesis Lab, U.B.C.), and purified by butanol extraction (Sawadogo et al., 1991).  2.4.1 Production and isolation of single-stranded DNA Single stranded D N A for sequencing was prepared as follows: single, overnight colonies of E. coli JM101 containing pTZ18-based constructs were inoculated in 2 mL TYP medium containing 100 n g / m L ampicillin and 10  9  P F U / m L M13K07 helper phage. Following 1 hour incubation at 37°C, kanamycin was added to 70 ug/mL. Cultures were grown overnight at 37°C and 200 rpm. Cells were removed by centrifugation in a microfuge at room temperature for 5 min. The phagemid was precipitated at 4°C with 1.7 M ammonium acetate and 12% (w/v) PEG-6000. Single-stranded D N A was isolated from the phagemid as described by Sambrook et al, (1989). The yield of D N A was estimated from agarose gels.  2.4.2  Site-directed in vitro mutagenesis Single-stranded uracil-containing D N A was prepared from E. coli RZ1032  (Kunkel et al., 1987) as described in section 2.4.1. Site-directed mutagenesis was carried out as described below (Zhou et al., 1990). Template D N A (0.15 pmoles) was mixed with mutagenic primer (1.4 pmoles) in 10 uL sequencing buffer (40  22 m M Tris-HCl, p H 7.5; 10 m M MgCl^ 50 m M NaCl). Annealing was carried out by heating the mixture at 68°C for 10 min, then allow cooling to room temperature over 30 min. For the synthesis of the complementary strand, 10 x T7 D N A polymerase buffer (500 m M Tris-HCl, p H 7.5; 100 m M DTT; 70 m M MgCLj 10 m M ATP; 5 m M each dATP, dCTP, dGTP, dTTP), 1.5 units of T4 ligase and 7 units of T7 D N A polymerase was added to the annealing mixture (final volume of 30 uL). The mixture was then incubated on ice for 3 min, at room temperature for 3 min, and at 37°C for 1-2 hours. E. coli JM101 was then transformed with 2-5 uL of this mixture. Single-stranded D N A was prepared from the transformants and screened for the mutations by dideoxy sequencing (see section 2.6).  2.5 Cloning of CbhB gene from C. fimi genomic library  C. fimi genomic D N A was isolated using the SDS-proteinase K procedure described by Ausubel et al, (1987). D N A fragments were generated by random shearing; After size-fractionation, fragments of 2-6 kb were ligated to an EcoRl linker and inserted into the vector lambda ZAPII to form a genomic library (StrataGene). A n oligonucleotide based on the N-terminal sequence of CbhB was synthesized and labeled with 32p using [oc-32p]-ATP and T4 polynucleotide kinase (Sambrook et al, 1989) as a probe to screen the genomic library. E. coli XLl-Blue grown in LB medium to A600 = 0.85 was infected with the lambda ZAPII C. fimi genomic library (1.4 x 10^ PFU/mL) to give 5 x 10^ plaques on each of five 132 mm Petri plates. The plaques were blotted in duplicate onto Hybond-N+ nylon membranes (Amersham). Probe hybridization was as described by the manufacturer, in 6 x SSC (90 m M sodium citrate, 900 m M sodium chloride), 5 x Denhardt's solution (Sambrook et al, 1989), 0.5% SDS, 0.2 m g / m L salmon sperm D N A at 48°C for 21 hours. Filters were rinsed in 6 x SSC buffer at 48°C for 20 min,  23 then washed in 6 x SSC buffer at 51 °C for 20 min, and finally in 4 x SSC buffer at 54°C for 30 min. pBluescript DNAs with potential inserts were excised as described by the manufacturer (StrataGene) from the positive plaques. Positive clones were confirmed by D N A sequencing using the probe as the primer. The lengths and relative positions of the inserts were determined through restriction analysis with Ncol (at the 5' end of cbhB ) and Hindlll (after the 3' end of cbhB, on the vector). Finally by D N A sequencing from the 3' end of each insert, a clone carrying a 3.8 kb D N A fragment (pBluescript#5) was found to contain the entire cbhB gene.  2.6 cbhB D N A sequence determination  Overlapping subclones of cbhB were made as follows to determine the D N A sequence. The 3.8kb D N A fragment from pBluescript*5 (see Table 2.2) was digested with Pstl and SstI, giving 3 and 4 fragments (with sizes of 0.8-1.7 kb) respectively, on 0.7% agarose gel. These fragments were inserted into both pTZ18U and pTZ18R through the appropriate restriction sites. Single stranded DNAs of both strands for each subclone were then prepared as described in section 2.4.1. M13-based universal and reverse primers were used to determine the sequence of each subclone. Sequencing gaps were filled in using synthetic oligonucleotides based on the determined sequence. Single-stranded D N A sequencing by the modified dideoxy chain termination method was described previously (Sanger et al., 1977; Tabor and Richardson, 1987) with the following modifications: in the primer extension reaction, T7 D N A polymerase (Sequenase 2.0™ kit, United States Biochemical) was used, the reaction temperature was increased to 43°C and 7-deazaGTP was substituted for dGTP. The nucleotide mixes were adjusted for the high G+C content of C. fimi D N A .  2.7 Detection of cellulose-binding polypeptides from C. fimi  A sample (1.5 mL) was taken each day for 5 days from a C. fimi culture. The cells were removed by centrifugation and the supernatants were adsorbed with 2 mg of Avicel by incubation on ice for 30 min with occasional shaking. The Avicel was recovered by centrifugation and then washed with 1 mL of 1 M NaCl, followed by 1 mL of 50 m M phosphate-azide buffer (pH 7.0). The washed Avicel was boiled for 2.5 min with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer, and the extracted polypeptides were analyzed by SDS-PAGE (see section 2.8). The N-terminal amino acid sequence of the polypeptide band corresponding to CbhB was determined (see section 2.8).  2.8 Detection of protein  2.8.1 SDS-PAGE Proteins were resolved by 0.1% sodium dodecyl sulfate-7.5% polyacrylamide gel electrophoresis (SDS-PAGE) as described previously (Laemmli, 1970; Schagger and von Jagow, 1987) using a Bio-Rad MiniPROTEIN™ apparatus. Molecular weight standards (BIO-RAD) were as follows (kDa): myosin, 205; (3-galactosidase, 116.25; phosphorylase B, 97.4; bovine serum albumin, 66.2; ovalbumin, 45; carbonic anhydrase, 31; trypsin inhibitor, 21.5; lysozyme, 14.4; aprotimin, 6.4. Protein bands were visualized by staining with Coomassie Brilliant Blue R250.  25 2.8.2  N-terminal amino acid sequencing Polypeptides were separated by SDS-PAGE and then transferred  electrophoretically to a polyvinylidene difluoride (PVDF) membrane (Millipore). The transferred polypeptides were visualized with Coomassie Blue (Matsudaira, 1990) and the desired bands excised from the membrane. The excised bands were sequenced by automated Edman degradation using an Applied Biosystems 470A gas sequenator (Protein Microchemistry Facility, University of Victoria, Victoria, B.C.)  2.8.3  Determination of protein concentrations Protein concentrations were determined by dye binding (Bradford, 1976)  using the Bio-Rad protein kit, with bovine serum albumin as the standard. Where appropriate, the concentrations of purified protein solutions were determined by absorbance measurements at 280nm (A280nm) using the extinction coefficients for the polypeptides. The extinction coefficients (280nm; 1 mg/mL; 1cm) were calculated from the Trp and Tyr content and the theoretical molecular masses of the polypeptides (Cantor and Schimmel, 1980). The molecular masses were calculated from the primary structure of the proteins, deduced from D N A sequence analysis. The calculated extinction coefficients of the proteins were: CbhBn, 2.46; CbhB, 2.5. The predicted values were in good agreement with the extinction coefficients, CbhBn, 2.56; CbhB, 2.5, determined experimentally by the far-U.V. method of Scopes (Scopes, 1974).  2.9 Expression of cbhB and production of CbhB  E. coli JM101 containing the plasmids pTugSH2 and pTugSH3 (see Table 2.2) encoding CbhB with different N-termini were grown in 6 L TYP  26 supplemented with 50 ug/mL kanamycin at 30°C, 200 rpm in 2 L shake flasks. At A600 = 5, IFTG was added to 0.1 m M and incubation continued for 3 hours. Cells were collected by centrifugation (10,000 x g) at 4°C for 10 min. The cell pellet was resuspended in 600 mL osmotic shock buffer (0.1 M Tris-HCl, p H 8.0,1 m M EDTA, 0.02% NaN3). A n equal volume 40% (w/v) sucrose in the same buffer was then added to the suspension, to give 20% sucrose in a final volume of 1.2 L (1/5 of the culture volume). The mixture was kept on ice for 5 min. Cells were recovered by centrifugation again as above for 20 min, then resuspended in 1.15 L 5 m M MgSC>4. Concentrated phosphate-azide buffer, p H 7.0, and PMSF were added to the cell resuspension to 50 mM, and 0.5 m M respectively, making a final volume of 1.2 L. Cells were removed by centrifugation (10,000 x g) at 4°C for 10 min. CbhB was purified from the osmotic shock fluid as described below.  2.10 Purification of CbhB  Osmotic shock fluid from E. coli was stirred with 160 g of CF-1 cellulose (Sigma) in phosphate-azide buffer, p H 7.0, for 1 hour at 4°C. The cellulose was recovered by vacuum filtration on a glass fibre filter disk (GF/C, Whatman), then washed twice with 500 mL 1 M NaCl in the same buffer. The cellulose was packed into a column (5 cm x 60 cm) and attached to an FPLC system (Pharmacia). Washing was continued by passing 1.2 L of 1 M NaCl in phosphate-azide buffer, followed by 1.2 L of phosphate-azide buffer through the column at a flow rate of 1 mL/min. CbhB was then eluted with distilled water at a flow rate of 1 mL/min. Fractions were collected in test tubes contained concentrated phosphate-azide buffer p H 7.0, to give a final concentration of 50 mM. The absorbance of the elute was measured continuously at 280 nm and peak fractions were selectively checked for purity by SDS-PAGE. The appropriate fractions were pooled and  27 centrifuged (14,000 x g) for 30 min at 4°C to remove any fines. The supernatant was concentrated to greater than 15 mg protein/mL by diafiltration through an Amicon PM30 membrane. The purified polypeptide was analyzed again by SDSPAGE. The final protein concentration was determined by A280- Purified protein solutions were stored at 4°C.  2.11 Proteolysis of CbhB  CbhB (4 nmol) was digested with 8 m U papain in 150 uL proteolysis buffer (100 m M Tris-HCl, p H 8.0, 2 m M EDTA, 0.02% N a N , 5 m M cysteine) at 37°C for 1 3  hour. Samples (15 uL) were removed at 0, 6,16, 30 min and 1 hour. PMSF was added to each sample at 5 m M to stop proteolysis. The samples were analyzed by SDS-PAGE.  2.12 Assays of cellulose and hemicellulose hydrolysis  2.12.1 Reducing sugar assay Hydrolysis of barley B-glucan (Megazyme) and oat-B-glucan (Megazyme) was measured by reducing group production using the hydroxybenzoic acid hydrazide (HBAH) reagent as described previously (Langsford et al., 1987). Substrate was prepared as recommended by the manufacturer. CbhB (2 nmol) was incubated with 1% (w/v) substrate in 1.25 mL buffer (50 m M Na citrate-azide buffer, p H 7.0) at 37°C for 24 hours. Samples (20 uL) were removed from the reaction and added to 480 uL buffer, then 1 mL hydroxybenzoic acid hydrazide (HBAH) reagent was added. The reaction mixture was steamed at 100°C for 12 min, allowed to cool, and the A420 nm was measured. A standard curve was determined using a 55.6 n M stock solution of glucose.  28 Hydrolysis of Roth-xylan (Carl Roth KG), |3-glucomannan (Sigma), (3galactomannan (Sigma), and [3-lichenan (Sigma) was also measured by reducing group production, using the dinitrosalicylic acid (DNS) reagent (Miller, 1959). Substrate concentrations were 0.66% (w/v) lichenan, 0.13% (w/v) (3glucomannan, 0.13% (w/v) (3-galactomannan, and 0.66% (w/v) Roth-xylan respectively. CbhB (2 nmol) was incubated with the substrates in 0.75 mL phosphate-azide buffer, p H 7.0, at 37°C (50°C for Roth-xylan) for 24 hours. Then 50 uL of glucose (lmg/mL), and 0.8 mL dinitrosalicylic acid reagent (DNS) were added. The mixture was steamed at 100°C for 15 min, cooled and the A550nm measured. A standard curve was prepared using a 5.56 m M stock solution of glucose.  2.12.2 Total sugar assay Hydrolysis of the insoluble celluloses BMCC and PASC was determined by measuring the release of soluble sugar using phenol-sulfuric acid (Chaplin, 1986). BMCC (1.5 mg) or PASC (15 mg) was incubated with 1.2 nmol CbhB in 1.5 mL phosphate-azide buffer, p H 7.0, at 37°C and 2 rpm for 1 hour. Insoluble substrate was removed by centrifugation; 200 |iL of the supernatant were removed and mixed with 200 uL of 0.5% phenol, and 1 mL concentrated H 2 S O 4 on a vortex mixer. After standing at room temperature for 30 min, the A550nm was measured. A standard curve was prepared using a 5.56 m M stock solution of glucose.  2.12.3 Hydrolysis of fluorogenic and chromogenic substrates The fluorogenic substrates used were p-nitrophenol-(3-D-cellobiose (pNPC) (Sigma), 6.25 mM; p-nitrophenyl-fJ-D-glucopyranoside (pNPG) (Sigma), 6.25 mM; p-nitrophenyl-(3-D-xylopyranoside (pNPX) (Sigma), 1 mM; p-  29 nitrophenyl-(3-D-galactopyranoside (pNPGla) (Sigma), 6.25 mM. The substrates (0.5 mL) were incubated with 2 nmol CbhB (diluted in 0.5 mL 50 m M phosphateazide buffer, p H 7.0), at 37°C for 20 hour. The reactions were stopped with 0.5 mL Na2CC>3. p-nitrophenyl release was calculated from the absorbance at 410nm (Gilkes et al, 1984b). Azo-barley-glucan (Megazyme), 0.5% (w/v), in 50 m M Na citrate buffer, p H 7.0, was prepared as recommended by the manufacturer. CbhB (2 nmol) was incubated with 0.5 mL substrate at 37°C for 4 hours. Unhydrolyzed substrate was precipitated by addition of 0.9 mL of precipitation solution (4 g Na acetate, 0.4 g Zn acetate, dissolved in 20 mL distilled water, add 80 mL methyl cellosolve to this solution). The release of dyed barley (3-glucan fragments was measured from the A590nm as described by the manufacturer. 4-O-methyl-D-glucurono-D-xylan-Remazol brilliant Blue (RBB xylan) (Sigma) of 0.575% (w/v) was incubated with 2 nmol CbhB in 0.5 mL phosphateazide buffer, p H 7.0, at 37°C for 2 hours. The reaction was stopped by the addition of 1 mL anhydrous ethanol; insoluble substrate was then removed by centrifugation. Dye release was measured from the A595nm (Biely et al, 1988).  2.22.4 C M C - Congo Red plate assay The plates contained 0.1% C M cellulose (Na salt, low viscosity grade, Sigma; degree of substitution 0.7; degree of polymerization 400) and 1.5% agar in 50 m M phosphate -azide buffer, p H 7.0. The enzymes were diluted in the same buffer to concentrations of 1,10,100 pmol/|iL, and 20 uL of each dilution were deposited on a C M cellulose-agar plate. The plate was incubated overnight at 30°C, then flooded with Congo Red solution (2 mg/mL). The Congo Red was rinsed off with distilled water and the agar washed with 1 M NaCl (Teather and Wood, 1982).  30  2.12.5 Viscometry assay Hydrolysis of C M C was monitored for the increase in specific fluidity and the generation of reducing groups, as described previously (Gilkes et al., 1984). In the 37°C reaction water bath, 8 mL 4% (w/v) C M C in 50 m M Na citrate-azide buffer, p H 7.0, was prewarmed with appropriate amount of buffer for 10 min. Then 40 nmol CbhB was added to the prewarmed C M C solution, made final volume 10 mL, and mixed quickly. A 5.0 mL portion was immediately transferred to a Cannon-Fensky viscometer (nomical constant = 0.1 c St/s) for viscometric assay for 26 hours. The retention time (to) of CbhB solution containing no C M C was 8.4 seconds at 37°C. The real incubation times (t) were calculated as the time at the start of viscometric measured (tx) plus half of the measured retention time (tr). The specific fluidities, (psp> (= 1/T|sp) were calculated by the equation: (p p = l / [ ( t r / t o ) - 1] (Thomas, 1956). 0.2 mL aliquots were S  removed from the remaining mixture for reducing groups determination using H B A H reagent (see section 2.12.1).  2.13 Analysis of products released from various substrates by CbhB  2.13.1 Productsfromoligosaccharides Oligosaccharides were from Seikagaku America. Cleavage products were separated on a Waters, Dextro-pak column (Millipore) using an HPLC (Waters). The flow rate was 1 mL/min at room temperature with water as the solvent. The refractive index of the eluate was measured with a Waters 410 differential refractometer. The column was capable of separating the a- from the B- anomer for oligosaccharides G3 and G4, with the a anomer having a slightly longer retention time. Reaction mixtures contained substrate concentrations in the  31 range 0.57 - 4 mM and were incubated with 0.35 - 0.7 nmol CbhB at 37°C in a final volume of 70 uL for 1 hour. 50 uL samples were injected into the column for analysis.  2.23.2 BMCC and PASC hydrolysis products BMCC and PASC at 1 mg/mL were incubated with 1 and 10 nmol of CbhB, respectively, in 1.5 mL 5 m M phosphate-azide buffer, p H 7, at 37°C and 2 rpm for 24 hours. Insoluble substrate was then removed by centrifugation, and 100 uL of the supernatant was analyzed by HPLC as above.  2.14 Amino acid sequence alignments and database searches  Polypeptides sequences homologous to CbhB were searched through electronic mail ( from the GenBank and Swiss-Prot databases (Altschul, 1990) at the National Center of Biotechnology Information (NCBI), National Library of Medicine, NIH, in Bethesda, MD. Amino acid sequence information for family 48 cellulases was also obtained through electronic mail ( from the above source.  Nucleotide  and amino acid sequences were aligned with the PCGENE program from Intelligenetics.  2.15 Kinetic studies  Michaelis-Menten parameters for the hydrolysis of BMCC by CbhB were determined as described below. Activity was measured by soluble sugar release as described in section 2.12.2. Initial rates of enzyme-catalyzed hydrolysis were measured by incubating 1 nmol CbhB with different concentrations of substrate  32 (0.2 to 5 mg/mL BMCC in 1.0 mL 50 m M phosphate buffer, p H 7.0), at 37°C for 1 hour. Insoluble substrate was removed by centrifugation, and 200 uL of the supernatant taken to determine soluble sugar release. Values for K  m  and kc  at  were determined from the initial rate of hydrolysis (v ) versus, substrate 0  concentration, by non-linear regression analysis using the computer program GraFit 3.0 (Erithacus Software Ltd., Staines, U.K.).  2.16 p H optimum of CbhB  BMCC hydrolysis was measured at p H values from 3.5 to 9.2 by the release of soluble sugar (see section 2.12.2). Buffers used were citrate at p H 3.5,4.65, 5.65; phosphate at p H 6.5, 7.08, 7.7, 7.8, 8.2 and Tris-HCl at pH 8.9 and 9.2, all at 25 mM. A glucose standard curve was determined at pH 3.5, 7 and 9.1. The assays were carried out by incubating 1 nmol CbhB with 1 mg/mL BMCC in 1 mL of the above buffers at 37°C at 2 rpm for 1 hour. Insoluble substrate was removed by centrifugation twice, 200 uL supernatant was taken to determine soluble sugar release. The optimum p H was determined from activity versus p H value, by non-linear regression analysis using the computer program GraFit 3.0 (Erithacus Software Ltd., Staines, U.K.).  2.17 Determination of binding parameters  The absorption of CbhB to BMCC was determined using the procedure described by Gilkes et al. (1992). Samples of purified CbhB (2.7 to 26 nmol) were incubated with 1.0 mg BMCC in 1.0 mL phosphate-azide buffer in 2.0 mL Eppendorf tubes (Island Scientific) at 4°C. The tube contents were mixed by rotation at 2 rpm for 1 hour. The BMCC was then pelleted by centrifugation  33 (14,800 x g) for 10 min and the supernatant removed. The supernatant was centrifuged once more in order to remove all BMCC particles. The concentration of the unbound protein [F] was determined from the A280nm  a n  d the extinction  coefficient of the protein (see section 2.8). The concentration of bound protein, [B], was determined from the difference between the initial protein concentration and [F].  34  3. Results  3.1 Cloning of the cbhB gene  CbhB was identified from C. fimi culture supernatant as a 120 kDa cellulosebinding polypeptide (Cbpl20) by cellulose adsorption and SDS-PAGE analysis. Its N terminal amino acid sequence was determined (Meinke et al., 1993). In this study, CbhB from C. fimi was re-sequenced (Shen et al., 1994). The sequence, AVDGEYAQRFLAQYDKIKDPANGYF (Fig. 3.3), agreed with that determined previously, except for D3, R9 and D19 which were originally reported as T3 X9 and R19 (Meinke et al, 1993). To isolate the cbhB gene, an oligonucleotide (5CAGTACGACAAGATCAAGGACCC-3') was synthesized based on the amino acid sequence of Q13 - P20, and the codon bias of C. fimi genes (O'Neil et al, 1986; Wong et al., 1986; Coutinho et al, 1991; Meinke et al, 1991a, 1993 and 1994). The synthetic oligonucleotide was labeled with P and used to screen a C. fimi genomic library. 3 2  The cbhB gene was cloned as described in Materials and Methods section 2.5. Briefly, chromosomal D N A was sheared and ligated to an EcoRI linker and inserted into the EcoRI site of lambda ZAPII to form a genomic library (StrataGene). The P 3 2  labeled probe was hybridized with the C. fimi genomic library (Fig. 3.1). Nine positive clones released inserts of 2 - 6 kb upon EcoRI digestion. The orientations of the inserts were determined by single-stranded D N A sequencing, using the probe as the primer. A n Ncol site was found downstream of the priming site and unique to the cbhB gene. The relative positions of the inserts were determined through restriction analysis with Ncol (at the 5* end of cbhB) and Hindlll (downstream of cbhB, on the vector) (Fig. 3.2). Finally by D N A sequencing from the 3' end of each insert, a clone carrying a 3.8 kb D N A fragment (pBluescript 5) was found to contain the entire cbhB gene. #  Figure 3.1 Screening of a C. fimi genomic library with a cbhB probe. Hybridization of the C. fimi lambda ZAPII genomic library with the synthetic oligonucleotide labeled with [a- P]-ATP to detect clones carrying the cbhB gene. 32  A and B are the duplicate blots from the plate.  36 3.2 Nucleotide sequence of cbhB Overlapping subclones of pBluescript#5 were made for D N A sequencing. Pstl and Sstl digestion of the 3.8 kb D N A fragment resulted in 3 and 4 fragments of 0.8 -1.7 kb, respectively (Fig. 3.2). These fragments were inserted into both the pTZ18R and pTZ18U vectors, so that both stands could be sequenced. Single-stranded D N A was sequenced using M13-based universal and reverse primers and synthetic oligonucleotides. Figure 3.3 shows the nucleotide sequence of cbhB and its flanking regions. Codon usage in cbhB is very similar to that in cex, cenA, cenB, cenC, cenD and cbhA from C.fimi (O'Neil et al, 1986; Wong et al, 1986; Coutinho etal, 1991; Meinke et al, 1991a; 1993 and 1994). Twenty-one codons are not used in cbhB, and of these, fourteen are not used in any of these genes (appendix). cbhB has a mole% G+C content of 70.5 close to the 71 mole% G+C of total C. fimi DNA. cbhB encodes a polypeptide of 1,090 amino acids (Fig. 3.3). The A T G translation initiation codon is preceded by a putative ribosome-binding site. The coding sequence of cbhB terminates 32 nucleotides before the end of the 3.8 kb fragment of C. fimi DNA. There are no repeats within the 32 nucleotides; presumably, the transcription termination signals are located further downstream. There are two overlapping inverted repeats of 11 and 17 nucleotides, 261 to 322 nucleotides upstream of the translation initiation codon for cbhB, and 3 to 64 nucleotides downstream of an open reading frame upstream of cbhB.  3.3 Amino acid sequence of CbhB  Residues 54-76 of the deduced amino acid sequence of CbhB (Fig. 3.3) match the N-terminal amino acid sequence determined for Cbpl20 (Shen et al, 1994). The N terminal sequence of a peptide released from recombinant CbhB by papain, MGYTEA, corresponded exactly to residues 456 - 461 of the deduced amino acid sequence (Fig.  37  Ncol  (398) a.  \  cbhB  h  1  3270  Pstl  . b.  1  Sstl  , 1  (2128) 1  .  cbhB  (559) C.  Pstl  (1270) 1  Sstl  h  Sstl  (1453) (1564)  1  Sstl  (2415) J  cbhB  . >  500bp  Figure 3.2 Schematic representation of the cloned C. fimi D N A fragment carrying the cbhB gene. The major restriction sites used for subcloning are shown: a, Ncol site; b, Pstl sites; c, Sstl sites. Nucleotide s are numbered from the first nucleotide of the translational initiation codon.  38 3.3). The experimentally determined amino acid composition of recombinant CbhB agrees with that calculated from the deduced sequence (data not shown). Mature CbhB is 1037 amino acids long with a calculated molecular weight of 109,765. CbhB from C. fimi does not react with the periodic acid-schiff reagent, so it is not glycosylated (Emily Kwan, unpublished observation). CbhB has a pi of 4.67, predicted from its amino acid sequence with the PCGENE program (Intelligenetics). CbhB has a relatively long leader peptide of 53 amino acids (Fig. 3.3, Fig. 3.4). It is a modular protein, similar to CbhA, CenB and CenD from C. fimi. The N-terminal catalytic domain is 643 amino acid residues, the longest among C.fimi B-l,4-glycanases. The catalytic domain is followed by three fibronectin type III repeats of ~ 97 amino acids each, similar to those present in CenB, CenD and CbhA (Meinke et al, 1991b, 1993 and 1994) (Fig. 3.5). Amino acid residues 934 -1,034 at the C-terminus are 25% identical to the family II cellulose-binding domains (CBDs) present in other  0-1,4-  glycanases from C. fimi (Fig. 3.6), with all the aromatic residues and cystine residues conserved, it is a typical family IICBD.  3.4 Relationship of CbhB to other B-l,4-glycanases  The amino acid sequence of the CbhB catalytic domain is 47% identical to that of CelS from Clostridium thermocellum A T C C 27405 (Wang et al, 1993) and the C-terminal domain of CelA from Caldocellum saccharolyticum (Bergquist et al, 1993) (Fig. 3.7). S8 from Clostridium thermocellum YS and CelS have identical N-terminal sequences (Morag et al, 1993). P70 from C. cellulovorans is also related to CelS (Doi et al, 1993). The N-terminal sequence of Avicelase II from C. stercorarium (Bronnenmeier et al, 1991) is related to the N-terminal sequence of CelS. The partial sequences of ORF1 from C. josui (Fujino et al, 1993) and CelCCF from C. cellulolyticum (Belaich et al, 1993) share identity with the catalytic domains of CbhB, CelS and CelA (Fig. 3.5). Thus, we  39 proposed that the catalytic domains of these enzymes comprise a new family of (3-1,4glycanases called family L (Shen et al, 1994), now re-classified as family 48 (Henrissat, 1991, Henrissat and Bairoch, 1993, and B. Henrissat, personal communication). To date family 48 contains exclusively bacterial (3-1,4-glycanases, mostly from Clostridium spp. Of these. CelS, S8, CbhB and Avicelase II were reported to be exoglucanases (Table 3.1) (Bronnenmeier et al, 1991; Morag et al, 1991; Kruus et al, 1995; Shen et al, 1995).  Figure 3.3 Nucleotide sequence of cbhB and its flanking regions and the deduced amino acid sequence of CbhB. The numbering (+1) of the nucleotide sequence starts from the first nucleotide of the translational initiation codon. The stop codon is indicated by an asterisk. A putative ribosome-binding site is shown in bold italics. Horizontal arrows indicate inverted repeats. Amino acid residues are numbered from the first residue of mature CbhB. The N-terminal sequence determined for native CbhB produced in C. fimi is shown in bold type. The N-terminal sequence of a papain fragment from recombinant CbhB is underlined. The fibronectin type III repeats are boxed. • is the leader peptide processing site in E. coli.  GCGCAGTACACCCCGCTCGGCGCCGCGTCGCAGGCGCTGGCCGCGGCGTGGTTCGGGCAGCCGTTCCCCGCGACCGAGCTGCTCGTGCTGGCCGGCTGGGGTCTGGTCGGC A Q Y T P L G A A S Q . A L A A A W F G Q P F P A T E L L V L A . G W G L V G  >  <  - 3 61  ><  ATCCCGCTCGCGGCCCGGCTGTTCCGCTGGTCGTGAGCCCCGGCACGCTCCAGGAGGGGGAGCGCGACGGCCCGTCGGGAGGTCTCCCGGCGGGCCGTCGTCGTGGGAGGGGCGTGGGAA I P L A A R L F R W S *  -2  CGGACCAGGGTGTGGTCACGCTCCCACCCGAAAGGTTTAGGAGTCTGGACGCGACCTGTGCGAACTCGGAGGGTGGGCCCGACCTGCCCGACGCCCACCGGCGGACCCGCACCGACGCGA  41  -121  GGCCCGGCGGACGGCGACGGACAGGGTCTCGTGCACCTCCACCACCCCGCCCGCGGGGTCCGGTGGGTTCGCCCCCGCGACGACGCGAGGGCACGCCCGCACACCAGAAAOOTACGGACG  -1  ATGTCGTCAACGACCCGCCGGCGATCCGCCTGGGTGGCAGCAGCCACCGTAGGCGTCTCGTCGTTCTTGGCGGTCGCCGGGATCACCCCCGCGATCGCCGCGGCCGGAGCGGGACAACCC M S S T T R R R S A W V A A A T V G V S S F L A V A G I T P A I A A A G A G Q P  12 0 -14  GCCACGGTCACCGTCCCCGCGGCCTCCCCGGTCCGCGCCGCCGTGGACGGCGAGTACGCGCAGCGGTTCCTCGCGCAGTACGACAAGATCAAGGACCCCGCCAACGGGTACTTCAGCGCG A T V T V P A A S P V R A A V D O B Y A Q R P L A Q Y D K I K D P A N O Y F S A  240 27  CAGGGCATCCCGTACCACGCCGTCGAGACCCTCATGGTGGAGGCGCCCGACTACGGCCACGAGACGACGTCCGAGGCGTACTCCTACTGGCTGTGGCTCGAGGCGCTCTACGGCCAGGTC Q G I P Y H A V E T L M V E A P D Y G H E T T S E A Y S Y W L W L E A L Y G Q V  3 60 67  ACCCAGGACTGGGCACCGCTCAACCACGCCTGGGACACCATGGAGAAGTACATGATCCCCCAGTCGGTGGATCAGCCCACGAACTCCTTCTACAACCCCAACTCGCCGGCCACCTACGCG T Q D W A P L N H A W D T M E K Y M I P Q S V D Q P T N S F Y N P N S P A T Y A  4 80 107  CCCGAGTTCAACCACCCGAGCAGCTACCCGTCGCAGCTCAACAGCGGCATCAGCGGCGGCACCGACCCGATCGGCGCCGAGCTCAAGGCGACGTACGGCAACGCGGACGTCTACCAGATG P E F N H P S S Y P S Q L N S G I S G G T D P I G A E L K A T Y G N A D V Y Q M  600 147  CACTGGCTGGCCGACGTCGACAACATCTACGGCTTCGGCGCGACGCCGGGCGCGGGCTGCACGCTCGGCCCGACCGCGACCGGCACGTCGTTCATCAACACCTTCCAGCGCGGCCCGCAG H W L A D V D N I Y G F G A T P G A G C T L G P T A T G T S F I N T F Q R G P Q  72 0 187  GAGTCGGTCTGGGAGACCGTCCCGCAGCCGTCCTGCGAGGAGTTCAAGTACGGCGGCAAGAACGGGTACCTCGACCTCTTCACGAAGGACGCGTCGTACGCGAAGCAGTGGAAGTACACC E S V W E T V P Q P S C E E F K Y G G K N G Y L D L F T K D A S Y A K Q W K Y T  84 0 227  TCGGCGTCCGACGCCGACGCCCGCGCTGTCGAGGCCGTGTACTGGGCCAACCAGTGGGCGACCGAGCAGGGCAAGGCCGCCGACGTCGCCGCGACCGTGGCCAAGGCCGCCAAGATGGGC S A S D A D A R A V E A V Y W A N Q W A T E Q G K A A D V A A T V A K A A K M G  9 60 267  GACTACCTGCGGTACACGCTGTTCGACAAGTACTTCAAGAAGATCGGCTGCACCTCGCCCACCTGCGCCGCAGGCCAGGGCCGTGAGGCCGCGCACTACCTGCTCTCCTGGTACATGGCG D Y L R Y T L F D K ' Y F K K I G C T S P T C A A G Q G R E A A H Y L L S W Y M A  1080 307  ;  TGGGGCGGCGCGACCGACACCAGCTCCGGCTGGGCGTGGCGCATCGGCTCGTCGCACGCGCACTTCGGCTACCAGAACCCGCTCGCCGCGTGGGCGCTGTCGACGGACCCGAAGCTGACG W G G A T D T S S G W A W R I G S S H A H F G Y Q N P L A A W A L S T D P K L T  12  CCGAAGTCGCCGACCGCCAAGGCGGACTGGGCGGCCTCGATGCAGCGCCAGCTCGAGTTCTACACGTGGCTGCAGGCGTCCAACGGCGGCATCGCCGGCGGCGCGACCAACAGCTGGGAC P K S P T A K A D W A A S M Q R Q L E F Y T W L Q A S N G G I A G G A T N S W D  132 0 387  GGCGCCTACGCGCAGCCCCCGGCCGGCACGCCCACGTTCTACGGCATGGGCTACACCGAGGCGCCCGTCTACGTCGACCCGCCGTCGAACCGCTGGTTCGGCATGCAGGCGTGGGGCGTG G A Y A Q P P A G T P T F Y G M G Y T E A P V Y V D P P S N R W F G M Q A W G V  144 0 427  CAGCGCGTCGCCGAGCTCTACTACGCGTCGGGCAACGCGCAGGCCAAGAAGATCCTCGACAAGTGGGTCCCGTGGGTCGTCGCGAACATCTCGACCGACGGTGCGAGCTGGAAGGTCCCG Q R V A E L Y Y A S G N A Q A K K I L D K W V P W V V A N I S T D G A S W K V P  1560 467  AGCGAGCTCAAGTGGACCGGCAAGCCCGACACGTGGAACGCCGCCGCACCGACCGGCAACCCCGGCCTCACGGTCGAGGTGACGAGCTACGGCCAGGACGTCGGCGTCGCCGCCGACACC S E L K W T G K P D T W N A A A P T G N P G L T V E V T S Y G Q D V G V A A D T  16  00 347  80 507  GCCCGCGCCCTGCTCTTCTACGCCGCCAAGTCCGGCGAC ACCGCGTCCCGCGACAAGGCGAAGGCGCTGCTCGACGCCATCTGGGCCAACAACCAGGACCCGCTGGGCGTGTCCGCGGTC 1800 A R A L L F Y A A K S G D T A S R D K A K A L L D A I W A N N Q D P L G V S A V 547 GAGACCCGCGGCGACTACAAGCGGTTCGACGACACCTACGTCGCCAACGGTGACGGCATCTACATCCCGTCCGGCTGGACCGGCACGATGCCCAACGGCGACGTC ATCAAGCCGGGTGTC 192 0 E T R G D Y K R F D D T Y V A N G D G I Y I P S G W T G T M P N G D V I K P G V 587 TCGTTCCTCGACATCCGCAGCTTCTACAAGAAGGACCCGAACTGGTCCAAGGTGCAGACGTTCCTCGACGGCGGCGCGGAGCCGCAGTTCCGGTACCACCGGTTCTGGGCCCAGACGGCC S F L D I R S F Y K K D P N W S K V Q T F L D G G A E P Q F R Y H R F W A Q T A  2040 627  GTCGCGGGTGCGCTCGCGGACTACGCGCGGCTCTTCGACGACGGCACCACGACGCCTGACACGACGGCGCCGACGGTCCCGACGGGCCTGCAGGCGGGCGTCGTCACCTCGACCGAGGCG 2160 667 V A G A D Y A R T T P D T L Q ACGATCTCCTGGACGGCCTCGACGGACGACACCCGCGTCACGGGCTACGACGTGTACCGCGGCGCCACGAAGGTCGGCACCGCCACCACGACGTCGTTCACCGACACGGGCCTGACCGCC W T A V T R A T K T T L T A V  2280 707  TCGAGGGCGTACGCGTACACCGTCCGGGCGTTCGACGCGGCCGGCAACGTCTCGGCACCGTCCGCGGCGCTCACCGTCACGACCAAGGCCACGCCGTCCGACACGACCGCTCCGAGCGTC L T V D T T N K  2400 747  CCGGCGATCACGTCGAGCTCGAGCACGGCGAACAGCGTGACCATCGGCTGGTCGGCCTCGACCGACAACGCCGGCGGCTCGGGCCTCGCCGGGTACGACGTGTACCGCGGTGCGACCCGC T A N A G  2520 787  GTGGCGCAGACGACCGCGCTCACGTTCACCGACACGGGCCTGACCGCCTCGACCGCGTACGAGTACACGGTCCGGGCGCGGGACGTCGCCGGCAACGTGTCGGCCCCGTCGACGGCCGTC 2640 827 A G N A Q T F T D T T A Y R D TCGGTCACGACGAAGTCCGACACCACGCCGGACACGACGGCGCCGAGCGTGCCCGCGGGCCTCGCCGCGATGACCGTCACGGAGACGAGCGTCGCGCTCACCTGGAACGCGTCGACCGAC W N V T T K T T P D T T A P A G L A T E T  2760 867  ACAGGCGGCTCGGGCCTCAAGGGCTACGACGTCTACCGCGGCGCCACCAGGGTCGGCTCGACGACCACCGCGTCGTACACCGACACCGGCCTCACGGCGGCGACGGCGTACCAGTACACG 2880 907 S G L K A T R T T T A T D T G L Q Y GTCCGGGCCACGGACAACGCGGGCAACGTCTCCGCCGCGTCGGCCGCCCTGTCCGTCACCACGAAGACGCCGCAGACCGGCGGCTCGTGCTCGGTGGCCTACAACGCCAGCAGCTGGAAC K T P Q T G S C S V A Y N A S W N D N A  3000 947  AGCGGCTTCACGGCGTCGGTCCGGATCACCAACACCGGCACGACGACGATCAACGGCTGGAGCCTCGGCTTCGACCTGACGGCGGGCCAGAAGGTGCAGCAGGGCTGGAGCGCGACCTGG S G F T A S V R I T N T G T T T I N G W S L G F D L T A G Q K V Q Q G W S A T W  3120 987  ACGCAGTCCGGGTCCACCGTGACGGCGACCAACGCACCCTGGAACGGGACCCTGGCCCCGGGCCAGACCGTCGACGTCGGCTTCAACGGCTCCCACACGGGCCAGAACCCGAACCCGGCG T Q S G S T V T A T N A P W N G T L A P G Q T V D V G F N G S H T G Q N P N P A  3240 1027  TCGTTCACGCTCAACGGCGCGTCCTGCACGTGACGCGCTGAGCGAGGACGCACCACCCGCAGGAG S F T L N G A S C T *  3305  43  (3-1,4-glycanases  leader peptide sequences  number of a.a. residues  CenA  MSTRRTAAALLAAAAVAVGGLTALTTTA^AQA^APG  31  CenB  MLRQVPRTLVAGGSALAVAVGVLVAPLATGAAA^APT  33  CenC  MVSRRS SQARGALTAWATLALALAGSGTALA^AS P  32  CenD  MHSASRTRARTRVRTAVSGLLAATVLAAPLTLVAAGAQA^ATG  39  MPRTTPAPGH PARGARTALRTTL A A A A A T L W G A T W L PAQA^AIA  42  CbhA  MSTLGKRAGVRRRVRAVATAATATALVAVPLTTLATSASA^APV  40  CbhB  MS STTRRRSAWVAAATVGVS SFLAVAGITPAIA^AAG  33  Cex  AGQPATVTVPAASPVRA^ AVD XylD  a  53  MSDSFEATRTTRRRRPLQALTGLLAAGALVAGALAAASPAAA^AVT  42  Figure 3.4 Leader peptides of C. fimi (3-1,4-glycanase. Arrows indicate sites of processing. Cleavage is at the same sites in C. fimi and E. coli for most of the (3-1,4-glycanases as indicated by  For CbhB, ^ is the site giving rise  to native CbhB in C. fimi, ^ is the site used only in E. coli. For CenA, cleavage in E. coli  occured at two sites at equal ratio as indicated by ^ and -k There were errors in the reported sequence of the Cex leader peptide: amino acids 22 to 26 were thought to be TRRRA (O'Neill et al, 1986); the correct sequence is T L A A A A , and the length of the leader peptide is 42, not 41, amino acids. The corrected sequence has GeneBank accession number LI 1080.  44  CenB-l CenB-2 CenB-3 CenD-1 CenD-2 CbhA-1 CbhA-2 CbhA-3 CbhB-1 CbhB-2 CbhB-3  TTTDTTPPTTP GTPVATGVTTVGASLSWAASTD-AG-SGVAGYELYRVQ TTGETE P PTTP GTPVASAVTSTGATLAWAPST GDPAVSGYDVLRVQ PPVDTVAPTVP GTPVASNVATTGATLTWTASTD-TGGSGVAGYEVYR-GTPDTTAPTAPTGLRAGTPTASTVP LTWSASTD-TGGSGVAGYEVYR— GGGDVTAPSVPTGLTAGTPTATSVP LTWTASTD-TGGSGVTGYEVYR— PVEDLVAPTVPTGLTAGTTTATSVP LSWTASTDNV AVTGYDVYR— TVTDTTAPSVPAGLTAGTTTTTTVPLSWTASTDNAGGSGVAGYEVLR— TWDTTAPSVPTGLTAGTTTTSSVP LTWTASTDNAGGSGVAGYEVFN— TTPDTTAPTVPTGLQAGWTSTEAT ISWTASTDD TRVTGYDVYR— TPSDTTAPSVPA-ITSSSSTANSVT—• IGWSASTDNAGGSGLAGYDVYR— TTPDTTAPSVPAGLAAMTVTETSVA LTWNASTDT-GGSGLKGYDVYR— W  consensus  ST  GY  CenB-l CenB-2 CenB-3 CenD-1 CenD-2 CbhA-1 CbhA-2 CbhA-3 CbhB-1 CbhB-2 CbhB-3  GTTQTLVGTTTAAAYILRDLTPGTAYSYWKAKDVAGNVSAASAAVTFTTD GTTTTWAQTTVPTVTLSGLTPSTAYTYAVRAKNVAGDVSALSAPVTFTTAA GTTQTLVASPTTATVALAGLTPATAYSYWRAKDGAGNVSAVAAPVTFTTL GT--TLVGTTTATSYTVTGLAADSAYTFSVRSKDGAGNTSAASAAVTARTAA GS--TLVARPTGTSHTVTGLSAATAYTFTVRAVDAAGNVSAASAPVGVTTAP GT—TLVGTTAATSYTVTGLTPATAYSFTVRAKDAAGNVSAASAAAAATTQSG GT - - TWGTTTATS YTVTGLTAGTTYS FS VRAKDVAGNTS AAS AAVS ATTQTG GT--TRVATVTSTSYTVTGLAADTAYSFTVKAKDVAGNVSAASAAVSARTQ GA--TKVGTATTTSFTDTGLTASTAYAYTVRAFDAAGNVSAPSAALTVTTKA GA--TRVAQTTALTFTDTGLTASTAYEYTVRARDVAGNVSAPSTAVSVTTKSD GA--TRVGSTTTASYTDTGLTAATAYQYTVRATDNAGNVSAASAALSVTTKTPQT  consensus  G  TV  L  Y  V  AG SA  657 754 854 453 449 478 57 6 674 687 783 880  7 08 806 905 503 599 529 627 723 737 834 93 0  T  Figure 3.5 Comparison of fibronectin type Ill-like sequences from C. fimi (3-1,4glucanases. The fibronectin type Ill-like sequences are from CenB (Meinke et al., 1991b), CenD (Meinke et al, 1993), CbhA (Meinke et al, 1994) and CbhB (Shen et al, 1995). The number following the acronym for the enzyme indicates the number of the repeat in that enzyme. All sequences are numbered from the first amino-acid residues of the mature enzymes. The consensus shows amino acid residues that are identical in all sequences.  45  CenA Cex CenB CenD CbhA CbhB consensus  CenA Cex CenB CenD CbhA CbhB consensus  APGCRVDYAVTNQWPGGFGANVTITNLGD-PVSSWKLDWTYTAGQRIQQLWNGT PAGCQVLWGV-NQWNTGFTAWTVKNTSSAPVDGWTLTFSFPSGQQVTQAWSST TPSCTWYS-TNSWNVGFTGSVKITNTGTTPLT-WTLGFAFPSGQQVTQGWSAT TGSCWTYT-ANGWSGGFTAAVTLTNTGTTALSGWTLGFAFPSGQTLTQGWSAR SGGCTWYS-ASSWNTGFTGTVEVKNNGTAALNGWTLGFSFADGQKVSQGWSAE GGSCSVAYN-ASSWNSGFTASVRITNTGTTTINGWSLGFDLTAGQKVQQGWSAT CV  W G F V N  WL  GQ  Q W  ASTNGGQVSVTSLPWNGSIPTGGTASFGFNGSWAGSNPTPASFSLNGTTCTGT VTQSGSAVTVRNAPWNGSIPAGGTAQFGFNGSHTGTNAAPTAFSLNGTPCTVG * WSQTGTTVTATGLSWNATLQPGQSTDIGFNGSHPGTNTNPASFTVNGEVCG* WAQSGSSVTATNEAWNAVLAPGASVEIGFSGTHTGTNTAPATFTVGGATCTTR* WSQSGTAVTAKNAPWNGTLAAGSSVSIGFNGTHNGTNTAPTAFTLNGVACTLG* WTQSGSTVTATNAPWNGTLAPGQTVDVGFNGSHTGQNPNPASFTLNGASCT* G  V  WN  G  GFG  G N P F  53 390 961 651 779 986  106 443 1012 704 832 1037  G C  Figure 3.6 Amino-acid sequence alignment of family II CBDs from C. fimi (3-1,4glycanases. The CBDs (or putative CBDs) are from CenA (Wong et al, 1986); Cex (O'Neill et al, 1986); CenB (Meinke et al, 1991a); CenD (Meinke et al, 1993); CbhA (Meinke et al, 1994) and CbhB (this work). All sequences are numbered from the first amino-acid residues of the mature enzymes. Positions of the C-termini, deduced from the occurence of a stop codon in the corresponding gene, are indicated by asterisks. The consensus shows amino acid residues that are identical in all sequences.  46  Figure 3.7 Alignment of the catalytic domains of the B-l,4-glucanases of family 48. Numbers at the ends of sequences refer to the position of the last amino acid in the native polypeptides. Bold type represents amino acid residues conserved in all sequences.  , gaps introduced to improve the alignment;  , additional C-terminal  amino acids. CfiCbhB is CbhB (GenBank accession number L38827) from C. fimi; CthCelS is CelS (GenBank accession number L06942) from Clostridium thermocellum; CsaCelA is CelA CD2 (catalytic domain 2) (GenBank accession number L32742) from Caldocellum sacchrolyticum; CsaORFl (GenBank accession number L01257) is from Caldocellum saccharolyticum; CceCelCCF is CelCCF (GenBank accession number M87018) from Clostridium cellulolyticum; CjoORFl (GenBank accession number D16670) is from Clostridium josui; CstAvill is Avicelase II from Clostridium stercorarium NCIB11745, determined by amino acid sequencing (Bronnenmeier et ah, 1991).  47  C s t A v i l l XSDDPYKQRFLDLWDDLHDPSNGYFSXH-GIPYHAV 35 C f i C b h B AVDGEYAQRPLAQYDKIKDPANOYPSA_QGIPYHAVETLMVEAPDYGHETTSEAYSYWLWLEALYOQVTQDWA 72 CsaCelA SGLGKYGQRFMWLWNKIHDPASGYPNQD_GIPYHSVETLICEAPDYGHLTTSEAFSYYVWLEAVYGKLTGDWS 72 CthCelS KDGTSYKDLPLELYGKIKDPKNGYPSPDEGIPYHSIETLIVEAPDYGHVTTSEAFSYYVWLEAMYGNLTGNWS 80  CfiCbhB CsaCelA CthCelS  PLNHAWDTMEKYMIPQSVDQPTNSFYNPNSPATYAPEFNHPSSYPSQLN_SGISGGTDPIGAELKATYGNADV 144 KFKTAWDTLEKYMIPSAEDQP_MRSYDPNKPATYAGEWETPDKYPSPLEFN_VPVGKDPLHNELVSTYGSTLM 143 GVETAWKVMEDWIIPDSTEQPGMSSYNPNSPATYADEYEDPSYYPSELKFDTVRVGSDPVHNDLVSAYGPN_M 152  CfiCbhB CsaCelA CthCelS  YQMHWLADVDNIYGFGATPGAGCTLGPTATGTSPINTPQRGPQESVWETVPQPSCEEPKYGGKNGYLDLPTKD 217 YGMHWLMDVDNWYGYG KRGDGVSRASPINTPQRGPEESVWETVPHPSWEEPKWGGPNGFLDLPIKD 209 YLMHWLMDVDNWYGFG TG TRATPINTPQRGEQESTWETIPHPSIEEPKYGGPNGFLDLPTKD 214  Cf iCbhB CsaCelA CthCelS  ASYAKQWKYTSASDADARAVEAVYWANQWATEQGKAADVAATVAKAAKMGDYLRYTLPDKYPKKIGCTSPTCA QNYSKQWRYTNAPDADARAIQATYWAKVWAKEQGKFNEISSYVGKAAKMGDYLRYAMPDKYPKPLGCQDKNAA RSYAKQWRYTNAPDAEGRAIQAVYWANKWAKEQGKGSAVASVVSKAAKMGDFLRNDMPDKYPMKIGAQDKTPA  CfiCbhB CsaCelA CthCelS  AGQGREAAHYLLSWYMAWGGATDTSSGWAWRIGSSHAHPGYQNPLAAWALSTDPKLTPKSPTAKADWAASMQR 363 GGTGYDSAHYLLSWYYAWGGALDGA WSWKIGCSHAHPGYQNPMAAWALANDSDMKPKSPNGASDWAKSLKR 353 TGYDSAHYLMAWYTAWGGGIGAS_WAWKIGCSHAHFGYQNPFQGWVSATQSDFAPKSSNGKRDWTTSYKR 356  290 282 285  CfiCbhB QLEPYTWLQASNGGIAGGATNSWDGAYAQPPAGTPTPYGMGYTEAPVYVDPPSNRWPGMQAWGVQRVAELYYA CsaCelA QIEPYRWLQSAEGAIAGGATNSWNGRYEKYPAGTATPYGMAYEPNPVYRDPGSNTWPGFQAWSMQRVAEYYYV CthCelS QLEPYQWLQSAEGGIAGGATNSWNGRYEKYPAGTSTPYGMAYVPHPVYADPGSNCWPGFQAWSMQRVMEYYLE CcelCCF EPYQWLQSAEGAIAGGATNSWNGRYEAVPSGTSTPYGMGYVENPVYADPGSNTWPGMQVWSMQRVAELYYK Cj OSORF1 EPYQWLQSSEOAIAGGATNSJWNGRYESIPSGTSTPYGMGYVENPVYADPGSNTWPGMQVWSMQRVAELYYK  43 6 42 6 429 71 71  Cf iCbhB CsaCelA CthCelS CcelCCF Cj OSORF1  SGNAQAKKILDKWVPWWANI STDGASWKVPSELKWTGKPDTWNAAAP_TGNPGLTVEVTSYGQDVGVAAD TGDKDAGTLLEKWVSWIKSWKLNSDG_TFAIPSTLDWSGQPDTWNGT YTGNPNLHVKWDYGTDLGITAS TGDSSVKNLIKKWVDWVMSEIKLYDDG_TFAIPSDLEWSGQPDTWTGT YTGNPNLHVRVTSYGTDLGVAGS TGDARAKKLLDKWAKWINGEIKFNADG_TFQIPSTIDWEGQPDTWNPTQGYTGNANLHVKVVNYGTDLGCASS TGDTRAKNLLDKWAKWNSEIKFNADG_TFQIPGTLDWEGQPDTWDPTQGYTGNPNLHVKWNYNTDLGCASS  506 496 499 143 143  Cf iCbhB CsaCelA CthCelS CcelCCF CjosORFl  TARALLFYAAKSG DTASRDKAKALLDAIWAN NQDPLGVSAVETRGDYKRFDDTYVANGDGIYIPS LANALLYYSAGTKKY_GVFDEEAKNLAKELLDRMWKLY RDEKGLSAPEKRADYKRPFEQEV YIPA IJINALATYAAATERWEGKLDTKARDKAAELWRAWYNFYCSEGKGVVTEEARADYKRFFEQEV YVPA LANTLTYYAAKSG DETSRQNAQKLLDAMWNNY SDSKGISTVEQRGDYHRPLDQEV FVPA LANTLTYYAAKSG DTTSKENAKKLLDAMWNNY SDSKGISTIEQRGDYHRPLDQEV YVPA  571 560 560 212 212  CfiCbhB CsaCelA CthCelS CcelCCF CjosORFl  GWTGTMPNGDVIKPGVSPLDIRSFYKKDPNWSKVQTFLDGGAEPQFRYHRPWAQTAVAGALADYARLP... GWTGKMPNGDVIKSGVKPIDIRSKYKQDPDWPKLEAAYKSGQVPEFRYHRFWAQCDIAIVNATYEILP... GWSGTMPNGDKIQPGIKPIDIRTKYRQDPYYDIVYQAYLRGEAPVLNYHRPWHEVD1AVAMGVLATYP... GWTGKMPNGDVIKSGVKPIDIRSKYKQDPEWQTMVAALQAGQVPTQRLHRPWAQSEFAVANGVYAILP... GWTGKMPNGDVIKSGVKPIDIRSKYKQDPEWQTMVAALQAGQVPTQRLHRFWAQSEFAVANGVYAILP...  639 62 8 629 270 27 0  Table 3.1 Family 48 (3-1,4- glycanases  organism  enzyme  accession No  Caldocellum saccharolyticum  CelA«  L32742  Cellulomonas fimi (ATCC 484)  CbhB''  L38827  Clostridium cellulolyticum  CelCCi*  M87018  Clostridium cellulovorans  P70  (Doi, et al, 1993)  Clostridium josui  ORFl^  D16670  Clostridium thermocellum (ATCC 27405)  CelS*  L06942  exocellobiohydrolasef  Clostridium thermocellum (YS)  S8*  (Morag, et al, 1993)  cellobiohydrolases  Clostridium stercorarium (NCIB11745)  Avicelase II  (Bronnenmeier, et al, 1991)  exoglucanases  a  C-terminal catalytic domain.  b  N-terminal catalytic domain.  c  Partial sequence data only.  d  Translated open reading frame.  e  N-terminal amino acid sequence.  / study of recombinant protein. 8 study of purified native protein  e  hydrolytic activity  exocellobiohydrolasef  49 3.5 Production and purification of CbhB from E. coli  3.5.1 Construction ofpTugSH2 for cbhB expression pTug3SN8, a pTug (Graham et al, 1995) plasmid derivative, was used for cbhB expression. cbhB was under the regulation of the tac promoter and gene 10 leader sequence on p3SN8. To subclone cbhB to pTug3SN8, a Stul site was first introduced next to the translational initiation codon of cbhB on pTZSHl (see Table 2.2) through in vitro sitedirected mutation. The oligonucleotide 5 ' - A C C A G A A A G G T A A G G C C T A T G T C G T CAACG-3' was used to generate the mutation. The bold faced residues in the oligonucleotide mark the Stul site. The mutation was detected by Stul restriction analysis. D N A sequencing of a 400 bp fragment (from upstream of the translational initiation codon of cbhB to the Ncol site) detected only the mutation of interest. A 870 bp Hindlll - Ncol fragment containing the mutated sequence was subcloned to wildtype pTZSHl, replacing*the equivalent wild-type fragment. As outlined in Figure 3.8, cbhB was inserted at the blunt-ended Ncol site (Ncol') (situated 10 bp downstream of the ribosome binding site on pTug3SN8) on the vector. The resulting plasmid was designated pTugSH2. E. coli JM101 was transformed with pTugSH2 for the expression of CbhB. The presence of pTugSH2 was confirmed by analyzing plasmid D N A by agarose gel electrophoresis.  3.5.2 Construction ofpTugSH3 for cbhB expression The N-terminal amino acid sequence of CbhB obtained from E. coli JM101/pTugSH2 was AAGAGQPATVTVPAASPVRAAVDGEY,  corresponding to  residues -20 to +6, indicating processing between Ala -21 and Ala -20 (Fig. 3.3). The different N-terminal sequence could have arisen by either processing of CbhB at a different site in E. coli, or degradation of Cbpl20 by C. fimi protease (Gilkes et al, 1988)  50 prior to its N-terminal sequence was determined. Nevertheless, in the function studies, it was thought necessary to compare both forms of CbhB. For this purpose, pTugSH3, was engineered to produce in, E. coli, CbhB with the same N-terminus as the enzyme from C. fimi. pTugE07K3 (Xu et al., 1995), another pTug derivative, has the Cex leader peptide coding sequence under the control of the tac promoter and gene 10 sequence. The Cex leader peptide promotes the successful secretion of C. fimi endoglucanase C (CenC) from E. coli (P. Tomme personal communication). Thus, the mature CbhB coding sequence was fused to and in frame with the Cex leader peptide coding sequence on pTugE07K3. A n Nhel site was introduced at the beginning of the mature CbhB coding sequence on plasmid pTZSHl by site-directed mutation. The oligonucleotide 5'C C G G T T C C G C G C C G C T A G C G A C G G C G A G T A C G C - 3 ' was used to generate the mutation. The bold faced residues in the oligonucleotide mark the Nhel site. This mutation also changed the second amino acid of CbhB from valine to serine (codon underlined). The mutation was detected by Nhel restriction analysis. A 400 bp fragment (from the translational initiation codon of cbhB to the Ncol site) was sequenced to ensure that no changes other than the desired mutation had occurred within this region. The 870 bp HindlU-Ncol fragment containing the mutated sequence was subcloned into wild-type pTZSHl, replacing the equivalent wild-type fragment. The expression plasmid pTugSH3 was constructed as outlined in Figure 3.9. The mature CbhB coding sequence was inserted after and in-frame with the Cex leader coding sequence on the pTugE07K3. E. coli JM101 was transformed with pTugSH3 for the expression of CbhB. The presence of pTugSH3 was confirmed by analyzing plasmid D N A by agarose gel electrophoresis. The N-terminal amino acid sequence of CbhB produced with pTugSH3 was ASDGEY, exactly as designed.  51  Figure 3.8 Construction of plasmid pTugSH2. A StuI site was introduced before the translational initiation codon of cbhB by sitedirected mutation (underlined Stul site) on pTZSHl. Plasmid pTZSHl (see Table 2.2) was then digested with Stul and BamHI. The pTug3SN8 was digested with Ncol, bluntended by mung bean nuclease {Ncol'), then digested with BamHL. The 3.3 kb SfulBarriHI fragment containing the cbhB gene was isolated and ligated with the 4.7 kb Ncol' -BamHI fragment of pTug3SN8 to give plasmid pTugSH2 (8 kb).  52 in vitro mutation  Ncol  Ncol BamHl  HindUI  BamHl  1) digest with Ncol 2) mung bean nuclease digest with Stul/BamHl  3) digest with BamHl  Isolate 4.7 kb vector fragment  Isolate 3.3 kb DNA fragment containing the cbhB gene  H  b Ligate  Ncol  BamHl  53  Figure 3.9 Construction of plasmid pTugSH3. A n Nhel site was introduced at the first codon of the mature CbhB coding sequence by site-directed mutation (underlined Nhel site) on pTZSHl. Plasmid pTZSHl (see Table 2.2) was then digested with BamHl, blunt-ended with the Klenow fragment of Poll and dNTP (BamUY), then digested with N M . pTugE07K3 was digested with HmdIII, blunt-ended with the Klenow fragment and dNTP (HindllV), then digested with M i d . The 3.1 kb BamHl'-Nhel fragment containing the mature CbhB gene was isolated and ligated to the 4.8 kb Hindlll'-NheR fragment of pTugE07K3 to give plasmid pTugSH3 (7.9 kb).  in vitro mutation  Ncol  BamHl ,  /  N h e I  Hindffl  BamHl  1) digest with BamHl  1) digest with HindUl  2) Klenow frag., dNTP  2) Klenow frag., dNTP  3) digest with Nhel  3) digest with Nhel  Isolate 3.1 kb DNA fragment containing mature CbhB coding sequence  h  I  Isolate 4.8 kb vector fragment  I  Ligate  BamHl , |NheI  55 3.5.3 Production of CbhB by E. coli and its purification CbhB was detected in cells of E. coli JM101/pTugSH2 and E. coli JM101/pTugSH3 by SDS-PAGE following release by osmotic shock and binding to Avicel. Most of the CbhB was in the periplasmic fraction of the cells (Fig. 3.10). The polypeptides produced by E. coli JM101/pTugSPI2 and E. coli TM101/pTugSH3 were designated CbhBn and CbhB, respectively. CbhB and CbhBn were purified on a small scale by affinity chromatography on CF1™ cellulose, using osmotic shock fluid from a 6 L culture (see Materials and Methods, section 2.9). The purified polypeptides were homologous as judged by SDSPAGE (Fig. 3.11). Typical yields of the polypeptides from 6 L cultures were about 200 mg. The molecular weights of CbhB and CbhBn, calculated from their deduced amino acid sequences (Cantor and Schimmel, 1980) were 109,765, and 111,757, respectively. The extinction coefficients (280nm; lmg/mL; 1cm) determined experimentally for CbhB and CbhBn were 2.5 and 2.56. These values were in agreement with the predicted extinction coefficients of 2.5 and 2.46.  56  1  2  3  4  5  6  7  8  9  10  Figure 3.10 Detection of CbhB from E. coli culture. An E. coli JM101/pTugSH3 culture was fractionated as described in Materials and Methods, section 2.10. Culture supernatant (500 mL) and the osmotic shock fluid (corresponding to 500 mL culture) were incubated with 2.5 mg of washed Avicel (see section 2.7). The Avicel was recovered by centrifugation and the supernatant removed. The Avicel pellet was washed, resuspended in 20 uL of SDS-PAGE loading buffer, boiled, then analyzed by SDS-PAGE. After osmotic shock, the cells corresponding to 50 uL culture were recovered by centrifugation and analyzed by SDS-PAGE. Lanes 2-4 and lanes 5-7 were cultures grown in incubator of 30°C and 32°C, respectively. Lane 1- molecular weight standards as indicated (kDa). Lanes 2 and 5- culture supernatant (500 uL). Lanes 3 and 6- osmotic shock fluid (corresponding to 500 uL culture). Lanes 4 and 7- cell pellet after osmotic shock. Lanes 8,9 and 10- osmotic shock fluid corresponding to 250,500 and 1,000 uL culture.  57  1  2  3  Figure 3.11 Purification of CbhB by cellulose affinity chromatography. The cells from a 6 L culture of E. coli JM101/pTugSH3 were collected by centrifugation and the periplasmic proteins released by osmotic shock as described in Materals and Methods section 2.9. The osmotic shock fluid was incubated with CF1™ at room tempreture for 30 min. The cellulose was recovered and washed with high salt buffer (1M NaCl in phosphate-azide buffer), and low salt buffer (phosphate-azide buffer) and packed into a column. Distilled water was used to elute bound proteins. The eluate was concentrated by ultrafiltration (see Materials and Methods section 2.10). The purity of the polypeptide was analyzed on a 10% SDS polyacrylamide gel, stained with Coomassie blue. Lane 1- osmotic shock fluid. Lane 2- total of 10 ug of C F 1 ™ purified CbhB. Lane 3- molecular weight standards with sizes as indicated.  58 3.6 Hydrolytic activities  Both CbhB and CbhBn were assayed for their hydrolytic activities against various cellulosic and hemicellulosic substrates. No significant differences were apparent between CbhBn and CbhB. Furthermore, CbhB aligns with the other family 48 P-glucanases, therefore, only the results for CbhB are presented in the following sections.  3.6.1 Activity on hemicelluloses CbhB did not hydrolyze P-glucan, xylan, mannan or P-lichenan (Table 3.2), not even when 2 nmol enzyme was used. The control enzymes exhibited activity when 25 to 10 - fold less enzyme was assayed. 4  3.6.2 Activity on celluloses The activities of CbhB on cellulosic substrates were compared with those of CenA, an endoglucanase (Gilkes et al., 1984), and CbhA, an exocellobiohydrolase (Meinke et al, 1994) from C. fimi (Table 3.3). CbhB was more active than CenA but less active than CbhA on bacterial microcrystalline cellulose (BMCC). In 1 hour, the enzymes solubilized 4%, 2% and 9% of the BMCC, respectively. As for the hydrolysis of amorphous cellulose, phosphoric acid-swollen cellulose (PASC) and the soluble substituted cellulose, carboxymethylcellulose (CMC) CbhB was comparable with CbhA. CenA was 9 and 25 times more active on PASC than CbhA and CbhB, respectively. The activity for CenA on C M C was approximately 2 x 10 and 3  1.2 x 10 times greater than those for CbhB and CbhA, respectively. 4  When the activities were converted to mole product/min/mole enzyme, the activities of CbhB on BMCC, PASC and C M C are 1.9 mole cellobiose/min/mole, 1.2  59 mole cellobiose/min/mole and 0.7 mole RS/min/mole. Thus CbhB is most active on the microcrystalline cellulose. Table 3. 2 Activity of CbhB on hemicellulosic substrates.  substrates  assay methods  CbhB (nmol)  activity  positive controls enzymes (pmol)  3  B-Glucan barley-B-glucan  R.S. (HBAH)  2  ND  CenA (0.2)  oat-B-glucan  R.S. (HBAH)  2  ND  CenA (0.2)  azo-barley-glucan  A590  2  ND  CenA (1)  RBB-xylan  A595  2  ND  Cex  (10)  Roth-xylan  R.S. (DNS)  2  ND  Cex  (2)  B-glucomannan  R.S. (DNS)  2  ND  CenB  (0.5)  B-galactomannan  R.S. (DNS)  2  ND  CenB  (0.5)  pNPC  A420  2  ND  Cex  (20)  pNPG  A420  2  ND  Abg  (20)  pNPX  A420  2  ND  Cex  (80)  pNPGla  A420  2  ND  Cex  (1)  B-lichenan  R.S. (DNS)  ND  CenA (0.5)  Xylan  Mannan  chromogenic substrates  Abbreviations: A420, A590 or A595: Activity was expressed as AA420, AA 590 or AA595/h/nmol enzyme; R.S. (HBAH) or R.S. (DNS): Hydrolysis was determined by reducing sugar group assay using the H B A H reagent or the DNS reagent; ND: activity not-detectable; Abg: B-glycosidase from Agrobacterium sp. a :  All controls gave detectable activity with the amounts of enzyme used under the  conditions described in Materials and Methods section 2.12.  60  The major soluble product released from BMCC by CbhB was cellobiose, with traces of glucose and cellotriose (Fig. 3.12A). Cellobiose was also the major soluble product released from PASC, but there was more cellotriose than from BMCC (Fig. 3.12B). Cellotetraose did not accumulated in either case. The product is characteristic of exocellobiohydrolase activity.  Table 3.3 Comparison of the activities of CbhB, CbhA and CenA on cellulosic substrates.  CMC moleRS/min/mol  BMCC a  |igSS/min/nmol  D  PASC ugSS / min / nmol^  CbhB  0.7 ±0.035  0.7 ±0.096  0.4 ± 0 . 0 3  CbhAC  3.7±0.3  1.4 ± 0 . 0 7  1.2 ± 0 . 0 8  0.27 ±0.008  10.6 ± 0 . 9  CenA  a  8129 ± 1 2  Activity expressed as mol reducing sugar groups (RS) generated per min per mol  enzyme. D  c  Activity expressed as ug soluble sugar (SS) released per min per nmol of enzyme. Data are from Meinke et al, 1994.  61  Figure 3.12 Soluble sugar released from cellulose by CbhB. Soluble sugars produced by hydrolysis of BMCC (A) or PASC (B) for 20h were resolved by H.P.L.C.. Glucose (1), cellobiose (2) and cellotriose (3) were resolved as single peaks.  Relative Refractive Index  63 3.6.3 Kinetics of the hydrolysis of BMCC The apparent K and V m  m a x  were determined for the hydrolysis of BMCC (see  Materials and Methods section 2.16). The rate of hydrolysis declined after 1 h, presumably due to product inhibition by cellobiose. Therefore, initial reaction velocity (v ) was determined for 1 hour reaction by measuring soluble sugar release (see Q  Materials and Methods section 2.12.2) and converting it to jig glucose equivalent based on a standard curve. Vmax  d K at steady state were determined from the initial rate of hydrolysis  a n  m  (Vo) versus substrate concentration, by non-linear regression analysis (Fig. 3.13) using the computer program GraFit 3.0 (Erithacus Software Ltd., Staines, U.K.); the values were 1.068 ug/min.nmol and 0.5454 mg/mL, respectively. However, it should be noted that the catalytic mechanism involved in this case is much more complex than that described by classical enzyme kinetics because the enzyme is concentrated on the BMCC through binding by its CBD, which could influence the K value. Nevertheless, the kinetic parameters are valuable for m  comparison with the other cellobiohydrolase, CbhA, from C. fimi (Table 3.4).  Table 3.4 Kinetics parameters for hydrolysis of BMCC by CbhB and CbhA  Vmax  K  g/min.mmol  mgBMCC/mL  m  CbhA*  1.82  0.32  CbhB  1.07  0.54  a  Data are from Meinke et al. 1994.  0  1  2  3  BMCC (mg/mL)  Enzyme Kinetics Simple weighting Reduced Chi squared = 0.0002273 Value  Variable V max Km  1.0680 0.5454  Std. Err. 0.0171 0.0320  Figure 3.13 Determination of k t and K M values. c a  4  5  65 3.6.4 Hydrolysis of cellodextrins by CbhB The products of hydrolysis of soluble cellodextrine by CbhB were separated using an HPLC equipped with a Dextropak column (Waters) and a refractometric detector (Fig. 3.14, Fig. 3.15), allowing resolution of the a- and B-anomers of cellotriose and cellotetraose. CbhB hydrolyzes B-l,4-glucosyl bonds with inversion of anomeric carbon configuration (see section 3.5.5). Determination of the ratio of the a- and Banomers of product released initially, before significant mutarotation can occur, will show the initial site of hydrolysis. Figure 3.16 shows the sites of cleavage of cellohexose, cellopentaose, cellotetraose and cellotriose by CbhB. CbhB removed cellobiose from the reducing ends of cellohexose and cellopentose. CbhB also removed cellotriose from cellohexose. Hydrolysis of cellotetraose by CbhB produced only cellobiose. Cellotriose was not hydrolyzed by CbhB.  3.6.5. CbhB is an exocellobiohydrolase As shown in section 3.6.2, CbhB was more active than CenA on BMCC but 10 4  fold less active than CenA on C M C , implying exoglucanolytic activity of CbhB. The plot of specific fluidity ((p ) versus reducing group production during the sp  hydrolysis of C M C is an indicator of exoglucanase or endoglucanase activity. This is because the random action of an endoglucanase results in a greater increase in specific fluidity than the restricted action of an exoglucanase (Gilkes et al, 1984; Meinke et al, 1994). The C M C used in this study had a nominal degree of polymerization of 400 and a nominal degree of substitution of 0.7. The calculated average molecular weight was 87,400. Incubation of 4 nmol CbhB with 3.2% (360 uM) C M C for 26 hours released approximately 200 nmol reducing sugar per mL (Fig. 3.17 B), equivalent to 0.55 scissions per C M C molecule on average. The slopes for CbhB, CbhA and CenA were 0.02,0.02 and 0.096 (p per mL per nmol, respectively (Fig. 3.17 A). Therefore CbhB  66  Figure 3.14 HPLC analysis of the products of hydrolysis of cellodextrins by CbhB. Panels A , B and C show the products released from cellopentaose, cellotetraose and cellotriose, respectively. The a- and 0- anomers of cellotriose and cellotetraose were resolved, but not those of cellobiose.  A  G2  G-3  0  J  I  I  5  10  15  Retention time (min)  20  68  Figure 3.15 HPLC analysis hydrolysis of cellohexaose by CbhB. Panels A and B show hydrolysis products before and after mutarotation, respectively. The a- and p*- anomers of cellotriose and cellotetraose were resolved, but not those of cellobiose. The inset in panel B represents a cellohexaose molecule; the reducing end is shaked black. Production of cellotriose indicates cleavage at site 1. CbhB is an inverting enzyme. Therefore, cellotetraose is produced by hydrolysis at site 2 because the ratio of a to p cellotetraose before mutarotation (1: 0.9) changed to the equilibrium ratio of 1: 2 after mutarotation..  Figure 3.16 Cleavage of various cello-oligosaccharides by CbhB. Points of hydrolysis are indicated by an arrow.  71 behaved like an exoglucanase, not an endoglucanase. The major soluble product released from BMCC and PASC was cellobiose (see section 3.6.2. and Fig. 3.12) consistent with exo-cellobiohydrolase activity.  3.6.6 The endoglucanase activity of C. fimi exoglycanases The CMC-Congo red plate assay is a convenient way to screen endoglucanase activity. Congo red forms a red complex with oligosaccharides containing a minimum of five monosaccharides linked by 0-1,3- or 0-1,4- bonds (Wood, 1980). When C M C is incorporated into agar, hydrolysis of the C M C to products with fewer than five glucose units is revealed by staining with Congo red. Endoglucanases produce a yellowish "clear" zone against a red background. Exoglucanases appear to lack hydrolytic activity because they cannot reduce the sizes of the glucan chains sufficiently to prevent interaction with Congo red (Wood, 1980; Teather et al., 1982, Gilkes et al, 1984). CbhB was first thought to be an endoglucanase because it produced a zone of clearing (Shen et al, 1994), but its activity in the viscosity assay clearly showed it is an exocellobiohydrolase. This raised the possibility that it has some intrinsic endoglucanase activity. Two other enzymes with exoglucanase activity from C. fimi are the exoglycanase Cex, which is most active on xylan but also hydrolyzes C M C slowly (Gilkes et al, 1984, Gilkes et al, 1991), and CbhA (Meinke et al, 1994). CbhB, CbhA and Cex hydrolyze C M C in the manner characteristic of exoglucanases (Meinke et al, 1994 and Fig. 3.16), but only CbhB appeared to give clearing on C M C plates (Shen et al, 1994). The assays for CbhA and Cex, however, used relatively low concentrations of enzyme. When higher concentrations of enzyme were tested, all three produced zones of clearing in proportion to the amount of enzyme deposited on the plate (Fig. 3.18). The three exoglycanases clearly had weak endoglucanase activity. Although the catalytic activities of these exoglycanases on C M C is low under the conditions of the viscometric  72  Figure 3.17 Specific fluidity versus reducing sugar release for the hydrolysis of CMC. A, relationship between specific fluidity ((p) and production of reducing sugar groups during hydrolysis of C M C by CbhB, CbhA and CenA. B, release of reducing sugar by CbhB, CbhA and CenA with time. Reaction mixtures contained 4 uM CbhB ( • ) , 3.6 nM CenA ( A ) , 500nM CbhA (•) and 3.2% (w/v) C M C in 50 m M Na citrate, p H 7. Data for CenA and CbhA are from Meinke et al., 1994.  Incubation time (hours)  74 assay (see above section), the conditions used in the CMC-Congo red plate assay are different. In order to see clearing, it was necessary to apply a 20 uL aliquot of 100 uM CbhA, CbhB or Cex to the plate. There is very little penetration of the agar by the dye during staining so the assay monitors only the hydrolysis of a small volume of 0.1% C M C near the plate surface. Presumably, it is these extreme conditions that allow detection of the endoglucanase activities of these enzymes. Under the same assay conditions, Cex E127A, an inactive mutant of Cex in which the acid/base catalyst in the active site is mutated to alanine (MacLeod et ah, 1994) did not cause clearing. The endoglucanase CenA was at least 100 times more active than the exoglycanases in the clearing of CMC-Congo red plate. Abg is a Bglucosidase from an Agrobacterium sp. that removes glucosyl residues from the nonreducing end of cellotriose and longer cellooligosaccharides (Day and Withers, 1986). It is also active on C M C and gives a low slope (0.013 cp per mL per nmol) characteristic of an exoglucanase in the viscometric assay (Meinke et al., 1995). Abg did not cause clearing in the CMC-Congo red plate assay showing that this exoglucanase does not have endoglucanase activity. Thus the clearing in CMC-Congo red assay caused by Cex, CbhA and CbhB was clearly due to their intrinsic endoglucanase activities.  75  2000  200  20 pmol  Figure 3.18 CMC-Congo red plate assay of exoglycanases. The enzymes were deposited on a plate containing 0.1% C M C and agar, (see Materials and Methods section 2.12.4). After incubation at 30°C overnight, the plate was stained with 0.2% Congo Red for 15 min, then washed with 1 M NaCl for 30 min. The enzymes from C. fimi were endoglucanase A (CenA), cellobiohydrolases A and B (CbhA and CbhB), the mixed function xylanase-glucanase (Cex) and an inactive mutant of Cex (E127A). The B-glucosidase (Abg) was from an Agrobacterium sp. Except for CenA, 20, 200 and 2000 pmol of each enzyme were used, and each was held in 20 uL of volume. Only 20 pmol of CenA were tested; with more, the zone of clearing was too big and obscured hydrolysis by the other enzymes.  76 3.7 Determination of stereochemical course of hydrolysis  The stereochemical outcome of the hydrolysis reaction can be obtained from the anomeric carbon configuration of the products. Immediately after the hydrolysis of (51,4-glycan, if the ratio of the a- and fj-anomers of cellotriose or cellotetraose is > 1.0, it is an inverting enzyme; if the ratio is < 0.5, it is a retaining enzyme. In this study, CbhB was incubated with cellohexose at 37°C. After 5 min, the ratio of a- to P-cellotriose was 2:1 (Fig. 3.14). Further incubation for 60 min at 37°C resulted in mutarotation to the normal ratio of 1:1.8. Thus, clearly CbhB proceeds with inversion of anomeric configuration, characteristic of a single displacement reaction.  3.8 p H dependence studies.  The optimum p H for BMCC hydrolysis was determined by assaying soluble sugar release in buffers of varying p H (3.5 - 9.3). A plot of catalytic activity versus p H is shown in Figure 3.19. The p H profile curve was bell shaped; the optimum p H was 7.1.  3.9 Binding to BMCC  Gilkes et al, (1992) suggested that the cellulose surface can be considered as an array of overlapping potential binding sites. Therefore, the concentration of binding sites must be determined by a probability function, where the probability of finding a free binding site is dependent on both the concentration and configuration of bound ligand on the cellulose surface. However, if adsorption at only very low concentrations of bound protein is considered, then this complication is avoided. This is because at  Figure 3.19 Activity versus p H for the hydrolysis of BMCC. The p H profile curve was fitted to the data points using GraFit 3.0.  78 these lower concentrations there is a decreased probability of any two neighboring bound protein molecules excluding the binding of a third protein molecule. The modified Langmuir equation then becomes [No] Ka [F] [B] =  [3] 1 + flKa [F]  where [B] is the concentration of bound ligand (mol /g. cellulose), [F] is the concentration of free ligand (molar), [No] is the concentration of binding sites in the absence of ligand (mol/g cellulose), a is the number of lattice units (i.e. cellulosyl residues) occupied by a single ligand molecule and Ka is the equilibrium association constant (litres/mol). If equation (3) is re-arranged in a double reciprocal form we obtain the following; 1 _ [B]  l  =  l  . _ + Ka[No] [F]  a  [4] [No]  This form of the equation emphasizes data for the lower range of ligand concentrations. The slope of a plot of 1/[B] versus 1/[F] would give a value for l/Ka[No], where [No]Ka is defined as the relative equilibrium constant (Kr). This value of Kr allows us to compare the affinities of various related ligands for a given preparation of cellulose, as [No] will be constant. The absolute value of Ka cannot be determined unless [No] is known. The equilibrium adsorption isotherm for CbhB is shown in Figure 3. 20. Saturation of BMCC by CbhB was approached but not attained at the highest protein concentration used (10.77 uM). Adsorption parameters for CbhB were obtained using the model described above. The relative equilibrium constant (K ) was determined r  from the slope of a plot of 1/[B] versus 1/[F] (Fig. 3.21). The K value for CbhB was 48.5 r  L / g cellulose, comparable to those of CenA and Cex: 38 and 40 (Gilkes et al, 1992).  79  a) CO  O CU  u of.  .a  cu  o  u 0co cu  pa  [F] (LIM)  Figure 3.20 Isotherm for the adsorption of CbhB to BMCC. Free and bound polypeptide concentrations [F] and [B], respectively were determined after equilibration at 4°C for 1 hour (see Materials and Methods, section 2.17). The cellulose concentration was 1 g/L; initial CbhB concentration was in the range of 2.7 26 nmol. Data points are means of three replicates.  80  1/[F] (mM" ) 1  Figure 3.21 Double-reciprocal plot of adsorption data for CbhB. The adsorption data for CbhB are plotted in double-reciprocal form (1/[B]) versus 1/[F]). The K value was estimated from the slope of this plot. r  81  4. Discussion  4.1 Structure of CbhB  CbhB is produced as a periplasmic protein in E. coli JM101/pTugSH3 at a level of approximately 33 mg per liter of culture. The recombinant CbhB has the same M by r  SDS-PAGE as Cbpl20 from C. fimi. This is to be expected because Cbpl20 is nonglycosylated as judged by lack of reaction with the periodic acid-Schiff reagent (Kwan, E. unpublished observation). When produced in E. coli JM101/pTugSH2, the protein, CbhBn, has 20 more amino acid residues at its N terminus than Cbpl20 from C. fimi, indicating that the leader peptide is processed between Ala -21 and Ala -20 (Fig. 3.4). CbhB produced in E. coli JM101/pTugSH3 has the same N-terminal sequence as Cbpl20. Signal peptides from Gram-positive bacteria are usually longer than those from Gram-negative bacteria (von Heijne and Abrahmsen, 1989). When genes from Gram-positive bacteria are expressed in E. coli, aberrant cleavages could occur if more than one cleavage site compatible with the (-3, -l)-rule (von Heijne, 1983) exists in the vicinity of the normal processing site. For example, when the genes encoding an a-amylase from Bacillus stearothermophilus and endoglucanase A (CenA) from C. fimi, are expressed in E. coli, 40% or 50% of the polypeptides are cleaved three residues upstream of the normal processing site (Suominen et al, 1987; Din, 1994). CbhB has a rather long leader peptide of 53 residues. Both cleavage sites at -21/-20 (PAIA/AAG) and -1/+1 (PVRA/AVD) are compatible with the (-3, -l)-rule. Also, the prolines at the -24 and -4 positions (bold type) agree with the concept that prolines in positions -6 to -4 having a positive effect on the rate of processing (Yamamoto et al, 1989; Nothwehr and Gordon, 1989). In addition, the -21/-20 site is preceded by a hydrophobic "h" region. Therefore, it is highly likely that the extra N-terminal sequence in CbhBn is the consequence of the  82 leader peptidase of E. coli recognizing the -21/-20 site but not the -1/+1 site in proCbhB. On the other hand, it is also possible that C. fimi processes proCbhB at two sites. The most probable leader peptide processing site predicted by the algorithm of van Heijne (1986) is the -21/-20 site recognized in E. coli. The sequence P V R A / A V D is unusual amongst the processing sites of proenzymes in C. fimi identified to date (Table 4.1). The processing site in all of the enzymes except CbhB is A X A / A , where X is an uncharged amino acid. This contrasts with the V R A / A site in CbhB. The -21/-20 site in CbhB recognized in E. coli, A L A / A , resembles the processing sites of the other enzymes. Initial processing at the -21/-20 site would remove a leader peptide of 33 amino acids, comparable in length to those of CenA, CenB and CenC (Table 4.1). The sequence of amino acids between the -21/-20 and -1/+1 sites in proCbhB includes three proline residues, which is unusual for the leader peptides from C. fimi (Table 3.4). This sequence could be removed by a secondary processing of the proCbhB.  Table 4.1 Processing sites in proenzymes from C. fimi. Enzyme  Leader peptide length  Proline residues  Processing site  CenA  31  0  TTAAQA/APG  CenB  33  2  ATGAAA/APT  CenC  32  0  SGTALA/ASP  CenD  39  2  AAPAQA/ATG  CbhA  40  1  ATSASA/APH  CbhB  53  4  ASPVRA/AVD  Cex  41  2  VLPAQA/ATT  XylD  43  2  SPAAAA/AVT  83 Nevertheless, similar amounts of the enzymes were obtained when CbhB and CbhBn were recovered from E. coli cultures by adsorption to cellulose. The catalytic activities against various cellulosic and hemicellulosic substrates of the two forms of CbhB were not significantly different. Clearly the extra N-terminal sequence did not affect either the catalytic or the cellulose-binding properties of CbhB. Comparison of its amino acid sequence with those of known |3-glycanases reveals the modular structure of CbhB. Like CenB, CenD and CbhA from C. fimi, CbhB has an N-terminal catalytic domain, followed by three fibronectin type III (Fnlll) repeats and a typical family II cellulose-binding domain (CBD). Based on its amino acid sequence, the catalytic domain is classified in family 48 of glycoside hydrolases (Table 3.1), which have the largest catalytic domains of known (3-glycanases (Fig. 3.7). Currently, only three entire and two partial sequences of the eight enzymes in family 48 are available for sequence comparison, and they are almost 50% identical. This precludes using sequence alignments to identify potential catalytic residues at this stage. The catalytic domains of the C. fimi cellulases CenA, Cex, CenB and CenD are all very resistant to proteolysis. These enzymes, when subjected to proteolysis by C. fimi protease (a non-specific serine protease, Gilkes et al, 1989) or papain, all yield fragments corresponding to their catalytic domains, even after prolonged digestion (Gilkes et al, 1988; Meinke et al, 1992; Gilkes, N . unpublished result). The overall conformation of CenA resembles a tadpole (Pilz et al, 1990), that of CenB "beads on a string" (Meinke et al, 1992). In each case, the large catalytic domain accounts for only a minor portion of these extended structures, suggesting a tightly folded, compact structure. The catalytic domain of CbhB, however, is very prone to proteolysis. Under much milder digestion conditions, transient products of various sizes are detectable by SDS-PAGE, including two products with M s of 40 kDa and 70 kDa (data not shown). r  Their N-terminal amino acid sequences, ASDGE and MGYTE, respectively, indicate  84 cleavage between residues 402/403 (Fig. 3.3). Prolonged digestion causes complete degradation without accumulation of any fragments detectable by SDS-PAGE. The susceptibility of CbhB to proteolysis implies a looser folding of its catalytic domain than the catalytic domains of the enzymes which are resistant to proteolysis. The modular structures of CbhB and CenB differ only in their catalytic domains; thus their conformations should differ only in the catalytic domains. CbhB has yet to be analyzed by SAXS (small angle X-ray scattering). Interestingly, Fnlll repeats occur between the two independent functional domains of C. fimi cellulases CenB, CenD, CbhA and CbhB, all capable of hydrolyzing crystalline cellulose (Kleman-Leyer et ah, 1994; Meinke et ah, 1994; Shen et ah, 1995). CenB without the Fnlll repeats still hydrolyses C M C (Meinke et ah, 1992), so the Fnlll repeats are not essential for the hydrolytic function. One possible role of the Fnlll repeats is as a linker. The sequences of four endoglucanase genes, cenA, cenB, cenC and cenD, two exoglucanase genes, cbhA and cbhB, and two xylanase genes, xynD and cex (also called xynB) from C. fimi are known (Gilkes et ah, 1984a; Whittle et ah, 1982; Wong et ah, 1986 Moser et ah, 1989; Meinke et ah, 1993 and 1994; Millward-Sadler et ah, 1994; Shen et ah, 199). The catalytic domains of the enzymes they encode come from six different families of glycoside hydrolases: family 5 (CenD); family 6 (CenA and CbhA); family 9 (CenB and CenC); family 10 (Cex); family H(XynD); and family 48 (CbhB). All of these enzymes have modular structures (Tomme et ah, 1995a) and can be classified into four groups in terms of module composition: CenA and Cex comprise a catalytic domain and a type IICBD separated by a short FT linker; CenB, CenD, CbhA and CbhB comprise a catalytic domain, a typical type II CBD and two or three fibronectin type III repeats; CenC comprises a catalytic domain, two tandem type IV CBDs, and two tandem sequence-related modules of unknown nature; and XynD comprises a catalytic domain, a type II CBD, a second sequence related to type II CBDs, and a sequence with  85 substantial sequence similarity to Rhizobium NodB proteins (Millward-Sadler et al, 1994). Finally, CenA, CenB, CenC, CbhA and CbhB hydrolyse glucosidic bonds with inversion of anomeric configuration, whereas CenD, XynD and Cex are retaining enzymes. Overall, the cellulase system of C. fimi is complex on several levels: the numbers of enzymes, which may be more than the eight characterized to date, the different modular arrangements, the different substrate specificities, the widely different specific activities, and the different families of catalytic domains represented.  4.2 Catalytic properties of CbhB  4.2.1 CbhB is an exocellobiohydrolase CbhB was initially described as an endoglucanase (called CenE) because of its activity in the Congo red assay (Shen et al., 1994) largely due to the vast excess enzyme used (2nmol); however, more detailed analysis shows that it has the characteristics of an exocellobiohydrolase. These include its preference for crystalline and amorphous cellulose over carboxymethylcellulose and its much lower ability to reduce the viscosity of a carboxymethylcellulose solution than CenA per hydrolytic event (i.e. per reducing sugar group generated). Also, CbhB produces mainly cellobiose from BMCC and PASC, consistent with cellobiohydrolase activity. In contrast, C. fimi endoglucanases CenA, CenB and CenD produce significantly higher levels of glucose and cellotriose from these substrates (Meinke et al, 1993). The absolute specific activity values for CbhB on BMCC and PASC are rather low, being 2 and 1.2 mole cellobiose/min/mole protein respectively, but are comparable to the activities of other family 48 exoglucanases, such as Avicelase II of Clostridium stercorarium and CelS of C. thermocellum (Bronnenmeier et al, 1991; Kruus et al, 1995). However, comparison will be more meaningful when assays in different laboratories are more defined with  86 respect to substrates and assay conditions (e.g. method of determining protein concentration). CbhB releases cellotriose as well as cellobiose as the major products from cellohexaose, which is unexpected for a cellobiohydrolase. The same products were released by C B H I and CBHII of T. reesei and P. pinophilum (Wood and McCrae, 1986; Claeyssens et al., 1989) and Avicelase II from C. stercorarium (Bronnenmeier et al., 1991). The difference in the products released from insoluble cellulose and cellohexaose is probably due to the different physical organization of these substrates. In crystalline cellulose, an individual cellulose molecule is rigidly held in position in the crystalline array by intra- and intermolecular hydrogen bonds, restricting its accessibility to the active site of the enzyme, whereas, lacking such physical constraints, the soluble oligosaccharide may access the active site more easily, compromising the normal specificity of the enzyme. Furthermore, this argument would also support the intrinsic endoglucanase activity observed in C. fimi exoglucanases (see section 3.6.6) which is discussed in section 4.2.2. Wood (1989) pointed out that the steric limitations imposed by insoluble celluloses might have an important effect on enzyme-substrate interaction, saying that although oligosaccharides and their derivatives as substrates "have provided valuable information on the active site and binding site requirements of cellobiohydrolases in particular,  there is a regrettable tendency to extrapolate and  make assumptions on the mode of action on insoluble substrates from observations made using soluble derivatives". Avicelase II from Bacillus circulans basically behaves as an exoglucanase but does not release exclusively cellobiose units from oligosaccharides. Thus it was classified as a specific endoglucanase which resembles an exoglucanase (Kim and Kim, 1995). For the reason argued above, the products released from insoluble substrates should be emphasized because they reflect the intrinsic nature of cellulose hydrolysis. Thus, the Avicelase II would be more properly characterized as an exoglucanase.  87 CbhB initially produces more cc-cellotriose than fi-cellotriose from cellohexaose. After mutarotation, the a- and P-cellotriose are present in the equilibrium ratio of 1:1.8 (Stoddart, 1971). Therefore, hydrolysis by CbhB proceeds with inversion of anomeric configuration. Inversion of anomeric configuration is expected to be characteristic of hydrolysis by all family 48 enzymes. Like other family 48 enzymes such as CelS from C. thermocellum, Avicelase II from C. stercorarium, and S8 fromC. thermocellum YS, CbhB is unable to hydrolyze pNPC (Kruus et al., 1995; Bronnenmeier et al., 1991; Morag et al., 1991; this work). Taking into account the fact that cellotriose is not hydrolyzed by CbhB or CelS of C. thermocellum (Kruus et al., 1995), it may be that four glucosyl units must bind in the catalytic site for effective hydrolysis to occur. Nevertheless, as a chromogenic substrate, pNPC provides a convenient and sensitive assay for enzyme purification and screening of gene banks. However, exoglucanases lacking this activity are difficult to detect, consequently few of them have been cloned. It is clear now that hydrolysis of pNPC is not diagnostic of exoglucanase activity, as has been claimed (Deshpande et al., 1984).  4.2.2 The intrinsic endoglucanase activity of exoglucanases The CMC-Congo red assay shows that Cex, CbhA and CbhB have detectable endoglucanase activity, although it is very much less than that of CenA. Therefore, Cex, CbhA and CbhB are predominantly, but not exclusively, exohydrolytic. T. reesei CBHI and CBHII were reported to have endoglucanase activity based on their ability to produce new reducing end groups on phosphoric acid swollen Avicel or filter paper (Stahlberg et al., 1993). These enzymes were purified from culture supernatants that also contained endoglucanases. Contamination of CBHI and CBHII with <0.2% endoglucanase could account for the level of endoglucanase activity observed. The C. fimi enzymes were produced from cloned genes expressed in E. coli, which does not  88 encode 3-1,4-glycanases. Therefore, the endoglucanase activities of Cex, CbhA and CbhB could not result from contaminating endoglucanases. Their properties support the claim that CBHI and CBHII also have endoglucanase activity. Cellulase E3 from Thermomonospora fusca, is an exoglucanase. E3 produced in E. coli also appears to have endoglucanase activity when assayed by zymogram (Zhang et ah, 1995). Exoglucanases are thought to have limited action on C M C because of the carboxymethyl substituents. Exoglucanases can remove the few unsubstituted cellobiosyl units from the ends of C M C chains but soon encounter a glucosyl unit with a substituent, which then blocks further progress of the enzyme along the chain. This means that the effective substrate concentration in a C M C solution at the start of the reaction is much lower for an exoglucanase than for an endoglucanase and quickly approaches zero. Indirect support for this hypothesis comes from the structures of two cellulases that belong to family 6 of related amino acid sequences: endoglucanase E2 from T. fusca (Spezio et al., 1993) and exo-cellobiohydrolase II (CBHII) from T. reesei (Rouvinen et al., 1990). Three-dimensional structures of the two enzymes are virtually superimposable, but there is a striking difference. In E2, the active site is a cleft on the surface of the molecule; in CBHII it is in the corresponding location but it is tunnel-shaped because two surface loops, which are not present in E2, fold over the active-site cleft. CenA and CbhA from C. fimi are also in family 6; CbhA but not CenA has stretches of amino acids corresponding to the surface loops of CBHII (Meinke etah, 1994). Another family 6 exoglucanase, E3 from T. fusca, also has the stretches of sequence corresponding to the surface loops. The active site of exo-cellobiohydrolase I (CBHI) from T. reesei, which belongs to family 7 of B-l,4-glucanases, is also tunnel-shaped (Divne et al., 1994). The intrinsic endoglucanase activity would require that the loop enclosing its tunnelshaped active site can move to allow entry of glucan chains and hydrolysis of internal glucosidic bonds. Again as discussed in section 4.2.1, with their freedom in solution,  89 soluble substrates like C M C or oligosaccharides, may compromise the active site topology and thererby gain access to the active site. It cannot be assumed, however, that all exoglucanases have tunnel-shaped active sites into which cellulose molecules thread from one end. The endoglucanase activity exhibited by C. fimi CbhA, T. reesei CBHI and CBHII and T. fusca E3 would require that the loops occluding their active sites have enough flexibility to allow a cellulose chain to occasionally enter the active sites through the open roof rather than from one end of the tunnel. Enzymes isolated from their native hosts may be contaminated with traces of other (3-glucanases which can distort their activity profiles (Wood and GarciaCampayo, 1990). For example, CelS from C. thermocellum, now characterized as an exoglucanase by use of recombinant enzyme that is devoid of (3-glucanase activity (Kruus et al., 1995), was thought initially to be an endoglucanase (Fauth et al., 1991), very likely because of contamination with endoglucanase activity. Therefore, it is proposed that where possible, assays of specificity should be done with recombinant enzymes. It seems that there is not a clear distinction between the endo- and the exo- mode of action. The apparent difference between so-called exo- and endoglycanases probably reflects the preference of an enzyme for a certain substrate or its cleavage pattern on the cellulose chain. There is no single criterion available for the unambiguous identification of an exoglucanase; however, it can be characterized by comparison of its activities on crystalline and C M C , including reduction of the viscosity of C M C relative to reducing sugar release, the ratio of reducing groups produced from soluble and insoluble cellulose, analysis of the hydrolysis products, and inhibition by cellobiose.  90 4.3 Similarities between fungal and bacterial cellulase systems  The cellulase system of C. fimi contains multiple endoglucanases (Gilkes et al, 1984a; Wong et al, 1986; Moser et al, 1989; Meinke et al, 1993) and exocellobiohydrolases (Meinke et al, 1994 and this work). This is the first time that more than one cellobiohydrolase has been identified in a bacterial cellulase system. In this respect, the C. fimi cellulase system resembles the systems from fungi such as T. reesei. The cellulosome of the bacterium C. thermocellum was thought initially to be devoid of exoglucanases; simultaneous "multicutting" of the cellulose chain by endoglucanases was thus proposed to explain cellulose hydrolysis by this organism (Mayer et al, 1987). However, exoglucanases are present in cellulolytic bacteria, including Clostridia, indicating that bacteria and fungi probably use a similar strategy to hydrolyse cellulose. The cellulase systems of both groups of organisms are proving to be complex. For example, the T. reesei system was thought to comprise two endoglucanases and two exo-cellobiohydrolases, but at least two other endoglucanases are now known to be present (Ward et al, 1993; Saloheimo et al.,1994). In P. chrysosporium, a white-rot fungus, typical endoglucanases appear to be absent and it is suggested that cellobiohydrolase variants provide endoglucanase activity in this organism (Sims et al, 1994). The interactions between the individual components of cellulase systems are not yet well understood in any of the bacteria or fungi. All known fungal exo-cellobiohydrolases belong to either family 6 Or family 7 of glycosyl hydrolases (Henrissat, 1991; Henrissat and Bairoch, 1993). CbhA from C.fimi and E3 from T. fusca belong to family 6 (Meinke et al, 1994; Zhang et al, 1995), but there are no known bacterial enzymes in family 7. All of the known enzymes in family 48 are bacterial. At least four of these are exo-cellobiohydrolases: S8, and its analog CelS, from C. thermocellum (Morag et al, 1991 and 1993; Kruus et al, 1995), Avicelase II from C. stercorarium (Bronnenmeier et al, 1991), and CbhB. Perhaps family 48 exo-  91 cellobiohydrolases are the functional equivalents in bacteria of the fungal enzymes in family 7. Fungal exoglucanases contain both inverting and retaining enzymes (Family 6 and 7), whereas the bacterial counterparts include only inverting enzymes (families 6 and 48). It has proved difficult to detect activity in some family 48 enzymes, such as the C-terminal catalytic domain of CelA from C. saccharolyticum (Bergquist et al., 1993), and P70 from C. cellulovorans (Doi et al., 1993), perhaps because of the lack of a convenient and sensitive assay for exo-cellobioh'ydrolase activity. pNPC is not hydrolyzed by five of the family 48 enzymes (see Discussion 4.2.1). Synergism is detectable between pairs of fungal cellobiohydrolases (Fagerstam and Pettersson, 1980; Henrissat et al, 1985; Kyriacou et al, 1987). Wood and McCrae (1986) explained the synergy by different stereo-specificities of these enzymes, but this model lacks experimental evidence. Since some cellobiohydrolases can behave as endoglucanases, at least on C M C , the synergism seen between pairs of cellobiohydrolases may be real, and not a consequence of endoglucanase contamination. However, synergy between CBHI and CBHII from P. pinophilum disappeared when a trace endoglucanase contaminatant was removed (Wood et al, 1989). Synergism was only restored when a specific endoglucanase was added back to the mixture. In this case at least, it appears that all three enzymes are required for efficient hydrolysis. The analysis of hydrolysis products, using a chromatographic column that resolves the a and (3 anomers of cellotriose and cellotetraose, revealed that CbhB attacks cellohexaose and cellopentose from the reducing end. Similar data have been reported for C. stercorarium Avicelase II, also in family 48 (Bronnenmeier et al, 1991). Several fungal cellobiohydrolases in family 7 (e.g. T. reesei CBHI), like CbhB, appear to attack from the reducing end (Biely et al, 1993). In contrast to the family 7 and family 48 cellobiohydrolases, attack on cellulose by T. reesei CBHII (and presumably other cellobiohydrolases in family 6) appears to be from the non-reducing end (Biely et al,  92 1993). On the basis of these observations it can be speculated that a common feature of both fungal and bacterial cellulase systems is the synergistic interaction of an endoglucanase with two types of cellobiohydrolase (Fig. 4.1). A comparative approach could provide insight into both bacterial and fungal systems. The model presented in Figure 4.1 provides a basis for further experimentation. Analyses of other organisms will determine whether the cellulase systems of all aerobic bacteria and fungi contain multiple cellobiohydrolases that attack cellulose molecules in opposite directions, as suggested in the model. Only two bacterial family 6 cellobiohydrolases are known at present: C. fimi CbhA and a recently described enzyme, E3, from T. fusca (Zhang et al, 1995). It is noted that most of the known family 48 enzymes are from anaerobic Clostridium spp., but family 6 cellobiohydrolases have not been described in this genus. It remains to be seen whether these and other cellulolytic anaerobes that form cellulase complexes use the same general strategy as C. fimi and T. reesei or whether they have had evolved a different mechanisms of attack.  4.4 Summary  Cbpl20 from C. fimi was characterized as cellobiohydrolase B (CbhB) based on its ability to hydrolyze crystalline cellulose, viscometric analysis of carboxymethylcellulose (CMC) hydrolysis, and the fact that cellobiose is the major product released from insoluble cellulose. The identification of a second cellobiohydrolase from C. fimi indicates, for the first time, that the cellulase system of an aerobic bacterium, is parallel to those of aerobic fungi in containing multiple endoglucanases and cellobiohydrolases. Furthermore, cellobiohydrolases from both systems hydrolyze cellulose chains in both directions, one from the reducing and the other from the non-reducing end. The recognition of a bacterial cellulase system  resembling those of fungi opens the way to a more meaningful comparison of the systems. This will certainly facilitate the understanding of the mechanism by which microorganisms degrade cellulose. Using recombinant enzyme, it was shown unambiguously that exoglucanases from C. fimi do have intrinsic endoglucanase activity, eliminating the possibility that this could be due to contamination with endoglucanases possible when enzymes are purified from their native hosts. Cellobiohydrolases do not remove cellobiose units from the end of cellulose chain exclusively, they also cleave internal p-l,4-glucosidic bonds at least in soluble substrates.  94  family 6 cellobiohydrolase  family 7 or family 48 cellobiohydrolase endoglucanase  M  A  .  _  Q.  cellulose microfibril  0" non-reducing end  reducing end  Figure 4.1 Hypothetical model of cellulase systems from aerobic bacteria and fungi. (Adapted from Miller Jr. et al, 1995). Two cellobiohydrolases that attack cellulose molecules at opposite ends are shown as a common feature of the bacterial and fungal systems. Preliminary evidence suggests that family 7 (fungi) or family 48 (bacteria) cellobiohydrolases attack from the reducing end while family 6 cellobiohydrolases (bacteria and fungi) attack from the non-reducing end. Nicks created by endoglucanases activity at disordered regions on the cellulose surface are expanded in both directions by the two cellobiohydrolases. 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Appendix Codon usage of C. fimi B-l,4-glycanases genes number of codons amino acid codon  cenA  cenB  cenC  cenD  cbhA  cbhB  Ala  1 21 2 33 0 14 0 2 1 1 0 22 0 19 0 6 0 21 1 10 4 48 0 3 0 3 0 12 0 0 0 0 17 0 12 0 13 3 0 9 0 10 0 22 0 10 0 15 0 5 0 29 0 21 16 0 13 0 24  l 63 3 73 0 14 0 16 0 0 0 32 0 54 0 6 1 35 0 27 7 79 2 17 0 13 0 13 0 0 0 0 44 0 23 0 31 10 0 29 0 21 1 40 1 22 0 41 0 23 1 61 0 76 26 0 50 0 65  l 58 5 97 2 25 0 23 0 1 0 23 0 71 0 9 1 40 1 59 5 82 3 27 0 17 0 11 0 0 0 0 54 0 30 0 6 5 0 30 1 28 0 48 1 13 2 31 0 18 0 46 1 51 27 0 42 2 59  l 27 4 50 4 15 0 7 0 0 0 26 0 41 0 3 0 21 0 25 3 62 4 17 0 15 0 19 0 0 0 0 29 0 24 0 24 4 0 21 0 9 0 26 0 13 0 26 0 12 0 48 0 56 31 0 20 0 37  3 60 3 67 3 11 1 13 0 1 0 45 0 43 0 6 0 18 0 20 5 72 1 4 0 9 0 23 0 0 0 0 28 0 23 0 21 5 0 25 0 14 0 23 0 24 0 35 0 14 0 42 1 80 17 0 27 0 46  2 70 7 82  Arg  Asn Asp Cys Gin Glu Gly  His De  Leu  Lys Met Phe Pro  Ser  Thr  Trp Tyr Val  GCT GCC GCA GCG CGT CGC CGA CGG AGA AGG AAT AAC GAT GAC TGT TGC CAA CAG GAA GAG GGT GGC GGA GGG CAT CAC ATT ATC ATA TTA TTG CTT CTC CAT CTG AAA AAG ATG TTT TTC CCT  ccc  CCA CCG TCT TCC TCA TCG AGT AGC ACT ACC ACA ACG TGG TAT TAC GTT GTC  1 17 1 12 0 1 0 42 1 61 0 6 1 39 0 26 5 90 2 6 0 10 0 24 0 0 1 0 32 0 17 0 38 12 0 31 1 18 0 40 0 25 1 43 0 22 0 68 1 71 37 0 55 0 51  cex 2 28 2 34 1 12 0 6 0 1 0 23 0 28 0 6 0 22 0 14 1 38 0 3 0 7 0 9 0 0 0 0 16 0 9 0 19 6 0 22 1 10 0 22 0 8 0 13 0 9 0 22 0 27 12 0 10 0 27  110  Codon usage continued number of codons amino acid codon  Stop  GTA GTG TAA TGA TAG  cenA 0 6 0 1 0  cenB  cenC  cenD  cbhA  cbhB  0 24 0 1 0  0 45 0 1 0  0 23 0 1 0  l 28 0 1 0  l 19 0 1 0  cex 0 14 0 l 0  


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