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Structure-function relationships of endoglucanase C (CenC) from Cellulomonas fimi Coutinho , John B. 1992

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STRUCTURE-FUNCTION RELATIONSHIPS OF ENDOGLUCANASE C (CENC) FROM CELLULOMONAS FIMI  by  JOHN B. COUTINHO B.Sc., Queen’s University, 1982 M.Sc., Queen’s University, 1986  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  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA September 1992 © John B. Coutinho, 1992  In  presenting  this  thesis  in  partial  fulfilment of  the  requirements  for an  advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study.  I further agree that permission for extensive  copying of this thesis for scholarly purposes may be granted department  or  by  his  or  her  representatives.  It  is  by the head of my  understood  that  copying  or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  SF4’,—  ‘VJ  U  ABSTRACT  The cenC gene of Ceilulomonasfimi, encoding endoglucanase CenC had an open reading frame of 1101 codons closely followed by a 9 bp inverted repeat. The amino acid sequence of mature CenC, which was 1069 amino acids long, is very unusual in that it had a 150 amino acidlong tandem repeat (N1N2) at the N-terminus and an unrelated 100 amino acid-long tandem repeat (C1C2) at the C-terminus. Similarity of the central domain of CenC to the catalytic domains of other endoglucanases placed CenC in subfamily El of the 3-l,4-g1ycanases. CenC could be affinity purified on cellulose or Sephadex. The catalytic properties of recombinant CenC from E. coil, for the substrate carboxymethylcellulose were indistinguishable from those of native CenC from C. fimi. In order to determine which of the repeats N1N2 or C1C2 bind to cellulose or to Sephadex, both repeats were cloned separately. N1N2 mediated binding to both cellulose and Sephadex. Nl or N2 alone did not bind Sephadex but did bind cellulose. The C-terminal repeats, alone or in combination, did not mediate binding to cellulose or to Sephadex. N1N2 bound to regenerated cellulose (phosphoric acid swollen cellulose) but had negligible affinity for bacterial microcrystalline cellulose. To show that the N-terminal repeats could be used as an affinity tag for a polypeptide other than CenC, N1N2 was fused to the catalytic domain of CenA (another endoglucanase from C. fimi). The resulting fusion polypeptide C’ ‘A could be affinity purified on cellulose or Sephadex and retained catalytic activity. C’ ‘A was also used to study the influence of the binding domain on the hydrolysis of cellulosic substrates by comparison to CenA. C’ ‘A had higher activity on the amorphous substrate cellulose azure when compared to CenA. C’ ‘A and CenA had similar activities on regenerated cellulose with the most striking difference being the poor activity of C’ ‘A on crystalline cellulose. The ability (or lack thereof) of the binding domain to adsorb to crystalline cellulose correlated well with the ability of the enzyme to hydrolyze the substrate.  ill  TABLE OF CONTENTS  Page ABSTRACT  ii  TABLE OF CONTENTS  iii  LIST OF TABLES  vi  LIST OF FIGURES  vii  LIST OF ABBREVIATIONS  ix  ACKNOWLEDGEMENTS  xi  1.  1  2.  Introduction 1.1.  Structure of cellulose  I  1.2.  Enzymatic hydrolysis of cellulose  2  1.3.  Measurement of cellulase activities  4  1.4.  Motifs of cellulolytic enzymes  5  1.5.  Cellulases from Cellulomonas fin2i  8  1.6.  Objectives of this thesis  10  Materials and Methods  13  2. 1.  Bacterial strains, plasmids and phage vectors  13  2.2.  Media and growth conditions  13  2.3.  Enzymes and reagents  15  2.4.  DNA preparation and sequence determination  16  2.5.  Cloning the 3’ end of the cenC gene  17  2.6.  Construction of deletion mutants of cenC  17  2.6.1 N-terminal deletions of CenC  17  2.6.2 C-terminal deletions of CenC  18  iv 2.7.  Cloning of the N-tenninal repeats N1N2 and Ni and the C-terminal repeats C1C2 and Ci  18  2.8.  Cloning of N2 using Nuclease Ba131  19  2.9.  Deletion analysis of Ni  19  2.10. Substituting N1N2 for the CBD of endogiucanase CenA  19  2.11. Purification of CenC, CenCA.1 and CenCA2  20  2.12. Expression of C1C2 and Cl  21  2.13. Purification of N1N2, Ni and N2  21  2.14. Purification of C’ ‘A  22  2.15. Measurements of CenC activity  22  2.16. Measurements of C’ ‘A activity  23  2.17. Oligosaccharide products of CenC  23  2.18. Determination of protein concentration  23  2.19. Amino-acid sequence determination  24  2.20. Protein electrophoresis  24  2.21. Immunoblotting  24  2.22. Adsorption of N1N2 or Ni to Avicel and Sephadex  25  2.23. Adsorption of N1N2, Ni or CBDCex to BMCC and PASC 2.24. Adsorption of N1N2, Ni and N2 to PASC  25 26  2.25. Rapid small scale screening procedure for functional binding activity  26  2.26. Adsorption of CenA and C’ ‘A to BMCC and PASC  26  2.27. Hydrolysis of CenA and C’ ‘A by C.Jirni protease  27  2.28. Amino-acid sequence comparisons  27  V  3.  Results 3. 1.  Nucleotide and amino acid sequence of cenC  28  3.2.  Expression of CenC in E. coli  42  3.3.  5’ and 3’ deletions of cenC  51  3.4.  Kinetic parameters for native and recombinant CenC  59  3.5.  Expression of fragments of the cenC gene  65  3.6.  Binding of the N- and C-terminal repeats of CenC to Avicel and Sephadex  3.7.  72  Sequence relatedness between N1N2, Ni and other CBDs  75  3.8.  Expression of N2  80  3.9.  Adsorption of N1N2, Ni and N2 to (PASC)  80  3.10. Consequences of substituting N1N2 for the CBD of endoglucanase CenA  87  4.  Discussion  96  5.  Summary  106  6.  References  107  vi  LIST OF TABLES  Table  Page  1.  Plasmids encoding cenC and its derivatives  14  2.  Kinetic parameters for CenC  60  3.  Partition coefficients of N1N2, Ni and CBDCex for PASC and BMCC  74  4.  Families of cellulose binding domains  76  5.  Adsorption of N1N2, Ni and N2 to PASC  85  6.  Adsorption of CenA and C’ ‘A to BMCC and PASC  7.  91  Activity of CenA and C’ ‘A on cellulosic substrates  92  vu  LIST OF FIGURES Figure  Page  1.  The structure of cellulose  2.  Organization of catalytic and non-catalytic domains in cellulases from C. find  3.  Nuclease Ba131 deletions of eeoC  4.  Mini-preparation of plasmid DNA from clones obtained after Nuclease Ba131 deletions  5.  29  30  31  Identification of clones containing the 3’ end of cenC  7.  Plasmids used in cloning the entire ceiiC gene  8.  Nucleotide sequence of eeoC and its flanking regions and the deduced amino acid sequence of CenC  9.  Repeated amino acid sequences in CenC  10.  Sequence relatedness in the catalytic domains of f3-1,4-glycanases of subfamily El  11.  10  Identification of a DNA fragment containing the 3’ end of cenC  6.  2  32 33  35 38  39  Consensus sequences around the DAGD and H-R peptides in subfamilies El and E2  43  12.  Family E of 13-1,4-glycosidases  45  13.  Expression of CenC in E. coil  47  14.  Expression of CenC in E. coil under different physiological conditions  50  15.  N-terminal deletions of CenC  52  16.  C-terminal deletions of CenC  53  17.  Purification of CenC, CenC CAl and CenC CA2 by affinity chromatography  54  v1u  18.  Purification of CenC by anion exchange chromatography  55  19.  Purification of CenC by gel-filtration  57  20.  SDS-PAGE analysis of purified CenC and its two C-terminal deletions  58  21.  CenC’s oligosaccharide cleavage pattern  61  22.  Temperature and pH optima for CenC  63  23.  Plasmids encoding polypeptides for the N-and C-terminal repeats  24.  66  Western blot of polypeptides N1N2 and Ni synthesized in E. co/i  67  25.  CiC2 and Cl production  26.  Purification of N1N2 and NI by affinity chromatography  27.  Adsorption isotherms for N1N2, Ni and CBDCex to  in  E. co/i  PASC or BMCC 28.  Dendogram of Family II CBDs  29.  Amino acid sequence similarities between Ni, N2 and other CBDs from C. Jinii  68 69  73 78  79  30.  Plasmids encoding polypeptides N1N2, Ni and N2  81  31.  Purification of N2, Ni and Ni N2 by affinity chromatography  82  32.  SDS-PAGE analysis of the adsorption of N1N2, Ni and N2 to regenerated cellulose (PASC)  83  33.  Deletion mutants of Ni  86  34.  Substitution of N1N2 for the CBD of CenA  88  35.  Adsorption of CenA and C’ ‘A to BMCC and PASC  89  36.  Organization of CenA and C’ ‘A  94  37.  Hydrolysis of CenA and C’ ‘A with C. JImi protease  95  38.  Sequence similarities between lectins and C1C2  98  39.  Potential applications of CBDs with different specificities  102  x  LIST OF ABBREVIATIONS  Interposon omega [BI  Bound polypeptide  BMCC  Bacterial microcrystalline cellulose  C1C2  C-terminal repeats of CenC  C’ ‘A  CenC CBD fused to CenA catalytic domain  CBD  Cellulose binding domain  CeID  Endoglucanase D from Clostridium thermocellum  CenA  Endoglucanase A from Cellulomonasfimi  CenC  Endoglucanase C from Cellulomonasfin1i  CenC  Proteolytic derivative of CenC  CMC  Carboxymethylcellulose  CNPC  Chioronitrophenolcellobiose  DNS  Dinitrosalicyclic acid  [F]  Free polypeptide  G3  Cellotetraose  HBAH  Hydrobenzoic acid hydrazide  IPTG  Isopropyl-13-D-thiogalactoside  Kcat  Enzyme turnover number  kDa  Kilodaltons  Km  Michaelis-Menten constant  MUC  Methylumbelliferyl cellobioside  N1N2  N-terminal repeats of CenC  p30  CenA proteolytic fragment  PASC  Phosphoric acid swollen cellulose! regenerated cellulose  pG2  2-chloro-4-nitrophenol cellobiose  p1  Isoelectric point  x PMSF  Phenylmethylsulfonyl fluoride  pNPC  p-nitrophenyl-13-cellobioside  SDS-PAGE  Sodium dodecyl sulfate-polyaciylarnide gel electrophoresis  Vmax  Maximum rate of enzyme reaction  xi  ACKNOWLEDGEMENTS  I would like to thank three very special persons, my supervisors Dr. D.G. Kilburn, Dr. R.C.Miller Jr. and Dr. R.A.J. Warren for giving me the opportunity to work on cellulases. My thanks to my committee members Dr. Speert and Dr. Hancock for their guidance and their useful criticisms and recommendations not only with the project but also in helping me improve my public speaking. My friends who made my stay in Vancouver a very pleasant one, they include Rudy Vlyasevi, Nicky Ahmed, the rookies Dave Nordquist and Howard Damude, the scottish lad pit yer specs on Alasdair MacLeod, the veterans Francois Paradis, Roger Graham, Celia Ramirez, Zahra Assouline and the classmates that I started with Neena Din and Jeffrey Greenwood. Thanks to Norm Greenberg who helped me learn molecular biology techniques and to all persons who contributed in a very big way to this thesis including Bernard Moser, Shen Hua, Emily Kwan, Tara Young, Andreas Meinke, Lando Robillo, Gary Lesnicki, Helen Smith, Donald Trimbur and Neil Gilkes. The persons who made the biggest contributions need a very special thank you and they are Edgar Ong, Peter Tomme and Pat Miller. My thanks also to a very special and caring friend Brigitte Massot who has taught me many things. Finally my very special appreciation for my mother and father and for my brother and sister in law, for their love and encouragement, without whom this surely would not have come to pass.This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada.  1  1. Introduction  1.1. Structure of cellulose Cellulose is a homo-polymer of D-glucose. The linear, unbranched chains of glucose residues are linked to each other by disaccharide is cellobiose. The  f3 -1,4- glucosidic bonds.  f3- linkage (Figure  The basic recurring  la) in cellulose assumes an extended  conformation with parallel cellulose chains being held together by cross-links of hydrogen bonds between the microfibrils (Figure lb). The intramolecular bonds thus help to maintain the rigidity of the cellulose chain. There are two types of cellulose, cellulose I and cellulose II (Sharrock, 1988). Cellulose I, also commonly referred to as ‘native’ celluloses is the form produced naturally by plants, bacteria and algae and is crystalline in nature. Until recently there was general agreement on the structure of native cellulose, however data from 13 C-NMR spectroscopy (Atalla and van der Hart, 1984) support the existence of two distinct crystalline forms of native cellulose, cellulose Io and 1f3 based on differences in resonance patterns. Cellulose lix is predominant in bacterial (Acetobacter) and algal (Valonia) celluloses whereas form I3 is predominant in celluloses from higher plants. Cellulose II on the other hand is a relatively non-crystalline form of cellulose produced by treatment of native cellulose with sodium hydroxide. Within cellulose fibres there are areas of complete order, i.e. crystalline areas, and also less ordered or amorphous regions. The degree of crystallinity within fibres varies with the source of the cellulose and the treatment to which it has been subjected. In the native state, cellulose fibres are associated with other polymers such as hemicellulose, pectin and lignin. The ciystallinity of the native material and its association with lignin are the major factors that inhibit the enzymatic hydrolysis of cellulose (Sharon, 1975; Lehninger, 1982; Coughlan, 1985).  2  —o (a  I  I  (b)  Figure 1. The structure of cellulose a) Cellulose chain; the Dglucose units are in 13-1,4linkage. b) Schematic drawing showing how parallel cellulose chains are held together by a crosslinking of hydrogen bonds (Lehninger, 1982).  1.2. Enzymatic hydrolysis of cellulose Cellulose is a renewable energy resource with approximately 4 x 1010 tons synthesized annually by photosynthesis. By catalyzing the decay of forest and agricultural waste, the cellulases in combination With hemicellulases and ligninases recycle nutrients which would otherwise remain as inert waste for a considerably longer period of time. Equally as important, is the ability to control the activities of cellulolytic organisms in the textile, paper and lumber industries. In the lumber indusy alone, millions of dollars are spent annually on preservatives to protect lumber (Coughlan, 1985). Cellulosic materials  can be pretreated using physicochemical processes to enhance their enzymatic hydrolysis (Millet eta!., 1976; Brownell and Saddler, 1987). Strains yielding high levels of cellulases (Montenecourt, 1983) as well as mutant strains that are resistant to end-product inhibition have been isolated (Stewart and Leatherwood, 1976; Choudhury eta!., 1980).  3 Cellulose can be hydrolyzed and utilized by many bacteria and fungi: Aerobic bacteria such as Cellulonionas species, gliding bacteria (Cytophaga aid Sporocytophaga) and Streptomycetes; anaerobes such as rumen organisms Bacteroides succinogenes,  Butyrvibrio fibrisolvens) and Clostridium thermocellurn; and various fungi of which Trichoderma reesei is most prominent (Gottschalk, 1986). Cellulolytic enzymes have been traditionally divided into three classes, endoglucanases (E.C.3 .2.1.4), exoglucanases or cellobiohydrolases (E.C.3.2. 1.91) and 13-glucosidases (E.C. Cellulases all cleave the same 13-1,4-glycosidic bond, but there must be variation in the microenvironment of these bonds in natural substrates since cellulolytic enzymes are generally induced as multienzyme systems with more than one enyzme representing each class and the different classes of enzymes working in synergy for cellulose hydrolysis (Coughlan and Ljungdahl, 1988; Béguin et al., 1990; Gilkes et at., 1991a). It is not clear what contributes to the differences in the microenvironment of the bond, but accessibility to the substrate, preferences for certain sizes of cellodextrins and the inhibition of a particular enzyme by a certain type of product may play a role. Synergism between endo- arid exo-glucanases has been shown for a number of enzymes (Wood, 1975; Eriksson, 1975; Ryu eta!., 1984; Tomme eta!., 1990). The snynergistic action is most marked on highly crystalline substrates, low with amorphous cellulose and negligible with soluble derivatives (Wood and McCrae, 1979). The first model for the biological degradation of cellulose was put forward by Reese (Reese et al., 1950) in which he proposed a Cl component necessary for the degradation of crystalline cellulose. This contribution stimulated research on the biochemical basis for cellulose hydrolysis and the discovery of different types of cellulases (Nunirni etal., 1981; Klesov eta!., 1983) helped formulate the basis for present day models for the enzymatic hydrolysis of cellulose (Béguin  ci’  al.,1987; Enari and Niku-Paavola, 1987; Stahlberg eta!., 1991). The  generally accepted model that has emerged involves the internal splitting of the cellulose molecule in the amorphous region by endoglucanases. The non-reducing ends generated  4 by this reaction become the substrate for exoglucanases which processively degrade cellulose molecules in stepwise fashion from a non-reducing end liberating cellobiose as a product. Cellobiose and other low molecular weight cellodextrins are then split by  13-  glucosidases to generate glucose thus preventing the buildup of cellobiose which inhibits exoglucanase and endoglucanase activity (Beguin et at., 1987).  1.3. Measurement of cellulase activities Several methods are available for the detection and measurement of the activities of components of the cellulase system (Mullings, 1985; Sharrock, 1988). A simple qualitative assay relies on the staining of carboxymethylcellulose in solid growth media or polyacrylamide gels (Béguin, 1983) with Congo red. Hydrolysis of this substrate followed by Congo red staining provides a zone of clearing (Teather and Wood, 1982). Reducing sugars may be determined by the dinitrosalicylic acid assay (Miller, 1959) or the Nelson-Somogyi assay (Nelson, 1952). The increase in fluidity accompanying hydrolysis of carboxymethylcellulose may be assayed viscometrically (Almin et a!., 1975). A plot of increase in relative fluidity versus time indicates the mode of attack on the substrate and helps to differentiate endoglucanases from exoglucanases. Plots of increase in relative fluidity versus reducing equivalents have been used to compare individual endoglucanases (Gilkes  ci’  al., 1984). Exoglucanase activity has been  determined by measuring the release of p-nitrophenol from p-nitrophenyl-13-cellobioside (pNPC) (Deshpande eta!., 1984) or release of methylumbelliferone from methylumbelliferyl cellobioside (MUC) (van Tilbeurgh  ci’  a!., 1982). 13-glucosidase  activity is determined by measuring the release of p-nitrophenol from p-nitrophenyl-13glucoside or by the release of glucose from cellobiose (Mullings, 1985).  5 1.4. Motifs of cellulolytic enzymes The cloning of cellulase genes, the determination of their nucleotide sequences, and the analysis of the amino acid sequences predicted from the nucleotide sequences have led to a greatly increased understanding of these enzymes. The amino acid sequences of more than sixty  13- 1,4-glycanases have been deduced from the nucleotide sequences of their  genes. Characterization of the enzymes and/or analysis of their amino acid sequences has shown that many of them comprise two or more functional domains (Knowles 1987; Aubert  eta!.,  1988; Béguin, 1990; Gilkes  eta!.,  et a!.,  1991a). In some, if not all, such  enzymes, the domains function independently, and retain their functions when separated by proteolysis (Calza eta!., 1985; Van Tilbeurgh Tomme  eta!.,  1988; Gilkes  eta!.,  eta!.,  1986; Langsford eta!., 1987;  1988; Ghangas and Wilson, 1988; Stahlberg  eta!.  1988; McGavin and Forsberg, 1989). A frequent arrangement is a catalytic domain linked to a cellulose-binding domain (CBD) by a linker sequence rich in proline and/or hydroxyamino acids (Gilbert et al., 1990; Béguin, 1990; Gilkes eta!., 1991a). Catalytic domains: A given microorganism will produce a number of cellulases which generally differ in overall amino acid sequence but which may share short conserved sequences. Cellulases can be grouped into families of related enzymes on the basis of amino acid sequence relatedness in their catalytic domains (Henrissat et a!., 1989). Sequence identity in the catalytic domains of cellulases and xylanases has been reviewed recently and the known sequences can be grouped into nine families A-I which  are quite distinct from one another (Henrissat et a!., 1989; Gilkes et a!., 1991a). Although cellulases cleave the same 13-1 ,4-glycosidic bond, it is postulated that the differences lie in the microenvironment of the cleavage site. Cellulose-binding domains (CBDs): Many cellulases bind to cellulose but the mechanism and significance of this interaction is not clear. Reese’s group was the first to propose a non-enzymatic factor termed Cl that helped with the hydrolysis of crystalline cellulose (Reese ci’ a!., 1950). Klesov and his colleagues working with crude enzyme  6 fractions drew a correlation between the binding strength of an enzyme and the ability to degrade crystalline cellulose (Rabinovitch et a!., 1982; Klesov eta!., 1983; Chernoglazov  et a!., 1983), however, the existence of an essential non-enzymatic factor was demonstrated by Ljungdahl when he isolated the “yellow affinity substance” from culture filtrates of C. thermocellunz which promoted the binding of endoglucanases to cellulose fibres (Ljungdahl eta!., 1983). One year later Griffin isolated a factor from T. reesei filtrates, a factor, that generated microfibrils from filter paper without hydrolysis (Griffin  et a!., 1984). Since then many enzymes have been cloned that have a discrete cellulose binding domain but till the present time, only the CBDs of the exoglucanase/xylanase Cex and the endoglucanase CenA of C.Jmnii and the cellobiohydrolases CBH1 and CBHII of  T. reesei have been characterized in any detail. The CBDs have been reviewed recently (Gilkes eta!., 1991a) and have been grouped into five families I-V based on amino acid sequence similarities (Coutinho eta!., 1992). Most of the CBDs vary in size from 35  -150 amino acids. Although not essential for catalytic activity, the CBDs do influence the activities of the catalytic domains (van Tilbeurgh eta!., 1986; Gilkes eta!., 1988; Tomme  eta!., 1988; Ghangas and Wilson, 1988). The binding of CBDs to cellulose has been analyzed with the native enzymes or with the isolated CBDs (Tomme et a!., 1990; Stahlberg eta!., 1991; Din eta!., 1991; Coutinho eta!., 1992, Gilkes eta!., 1992). It seems likely that the catalytic domains also influence the binding of the CBDs to cellulose Tomnie eta!., 1990; Gilkes eta!,, 1992). The solution of the three dimensional structure of the C-terminal cellulose binding domain of cellobiohydrolase I from T. reesei by nuclear magnetic resonance has been determined (Kraulis eta!., 1989). The protein has a wedgelike shape with an amphiphilic character, one face being predominantly hydrophilic and the other mainly hydrophobic. Linkers: These are short regions of amino acids rich in proline and/or hydroxyaniino acids. Most range in size from 6-40 amino acids and are thought to connect different functional domains (Gilkes et a!., 1991 a). Linker sequences have been observed in many  7 bacterial and fungal cellulases and have been termed a ‘hinge’ (Warren et a!., 1986; Langsford eta!., 1987; Béguin, 1990; Gough eta!., 1990, Gilkes eta!., 1991a) primarily because of their similarity to immunoglobulin molecules (Burton et a!., 1989) where their presence may serve as a hinge to provide flexibility to the two arms. This has allowed speculation that the Pro-Thr like regions may also act as a hinge and thus allow conformational mobility for interactions between the binding domain, the catalytic domain and the substrate (Ferreira et al., 1990; Shen et al., 1991). Linker regions because of their extended hinge like structure are often sensitive to proteolysis. Cleavage sites immediately adjacent to linker regions have been defined for CenA and Cex (Gifices eta!., 1988) and CenB from C.jmnii (Meinke eta!., 199 la,b) and for other cellulolytic enzymes (Tomme et a!., 1988; Gough et a!., 1990). Replacement of the hinge region of CenA with the hinge of immunoglobulin A (IgA) allowed hydrolysis of CenA with an IgA protease (Miller et a!., 1992). Linker regions are also extensively 0-glycosylated (Tomme eta!., 1988; Jentoft, 1990; Williamson eta!., 1992) and this post-transalational modification has been shown to afford protection from proteases (Langsford et at., 1987). Another potential approach to afford protection of enzymes from proteolytic cleavage is by linker replacement. Repeated sequences: Most -1,4-glycanases are in the range 30 kDa to 70 kDa (Aubert eta!., 1988; Béguin, 1990; Gilkes eta!., 1991a). Some f3-1,4- glycanases are larger than this and may contain other domains in addition to a catalytic domain and a CBD. For example, repeated sequences 20 to 150 amino acids long occur in several  I  1,4-glycanases of widely different molecular weights (Hall eta!., 1988, Béguin, 1990; Gilkes et a!., 1991 a). The functions of the repeated sequences are mostly unknown, but in endoglucanase CeIZ from Clostridiuni stercorarium they bind the enzyme to cellulose (Jauris eta!., 1990). The C-terminal repeats of endoglucanase Ce1D from C!ostridium  thermoce!!uni have some sequence similarities to a number of calcium-binding  8 polypeptides, but their removal does not affect calcium binding by the enzyme (Chauvaux eta!.,  1990).  1.5. Cellulases from Cellulonionas flmi Cellulase systems tend to be very complex, and their components can be difficult to purify by biochemical methods. Gene cloning and expression in an appropriate host which is free of cellulase activity facilitates the isolation of a particular enzyme from even trace amounts of the other components. Our group has focused its efforts on gene cloning technology to express genes encoding individual enzymes of the multienzyme cellulase  complex of Cellulomonasfirni in Escherichiu coli. Cellulomonasfirni is a Gram-positive, aerobic, mesophilic, rod-shaped bacterium. Béguin  ci’  at. (1977, 1978) studied the cellulases from Cellulomonas species and reported  two classes of enzymes. One class comprised enzymes which were tightly bound to cellulose and the other enzymes which were found free in the culture supematant. Langsford eta!. (1984) reported the presence of 10 components with cellulase activity in supernatants from cultures of C.fImi grown on Avicel. Four cellulolytic enzymes from  C.firni have been characterized (Gilkes ci’ at., 1991b). Two of them, CenA (418 amino acids) an endoglucanase (Wong et at., 1986) and Cex (443 amino acids) an exoglucanase /xylanase (O’Neill  ci’  at., 1986) are major extracellular enzymes produced when C.Jlmi  is grown on microcrystalline cellulose. Comparison of the amino acid sequences of Cex and CenA show that each enzyme has three distinct regions: a short sequence of about 20 amino acid residues composed only of proline and threonine (Pro-Thr linker), which is conserved almost perfectly in the two enzymes; a region rich in hydroxyamino acids but of low charge density which is 50% conserved (cellulose-binding domain); and a region, comprising about 70% of the polypeptide, the catalytic domain, which is not conserved. The order of the regions is reversed in the two enzymes (Fig. 2) with the Pro-Thr linker separating the catalytic domain from the binding domain in both enzymes (Warren et a!.,  9 1986). Domain shuffling of the catalytic domains of CenA and Cex (Warren et at., 1987) as well as proteolysis with C. firni protease of both these enzymes have confirmed the independent binding ability of the binding domain in the absence of the catalytic domain (Gillces eta!., 1988; 1989). More recently the genes for two other cellulolytic enzymes of C.Jlmi CenB (Owolabi eta!., 1988) and CenC (Moser et al., 1989) have been cloned and expressed in  E. coil. CenB, an endoglucanase, has a molecular mass of 110 kDa and shares similarities with CenA and Cex in that it too can bind microcrystalline cellulose. It has an unusual sequence organization consisting of five domains: a catalytic domain at the Nterminus with an internal cellulose-binding domain, followed by three repeats related to fibronectin type III repeats and lastly a C-terminal cellulose-binding domain (Fig. 2) Meinke eta!., 1991a, b). CenC is an endoglucanase and has an apparent molecular mass of 130 kDa. It binds both to Sephadex and to cellulose. It was purified from C.Jirni culture supematants by virtue of its Sephadex-binding capacity (Moser et at., 1989), and may correspond to enzyme CI, an endoglucanase of Mr 118 purified from the culture supernatant of  CeIlu!omonas sp. IIbc by Sephadex affinity chromatography (Beguin and Eisen, 1978). In C.fimi and in E. coil a truncated form (120 kDa) of the enzyme sharing the same amino-terminus, CenC’, is produced by proteolysis of CenC (Moser et a!., 1989). CenC seems to be a minor component on a percentage weight basis of the cellulase system of C. fimi (Gilkes et al., 1984; Moser eta!., 1989).  10  CfCenA  J  L  Cf Cex  s’\\. ,,,.. ‘‘‘.%  CfCe ri B  ——,,  ..  .‘  •1  1012  Figure 2. Organization of catalytic and non-catalytic domains in cellulases from C. fimi. CfCenA, B and CfCex are the endo-B-1,4-glucanases and exo-13-1,4-glucanase/xylanase of C.firni. The primary structures are drawn approximately to scale and are numbered from the amino terminus of the mature protein. Catalytic domain  ii:i, Linker region  Binding domain (Family III)  ,  ,  Binding domain (Family II) ,  Repeats E+3.  1.6. Objectives of this thesis The goal of this study was to correlate the structure of endoglucanase CenC with its function. In terms of structure CenA (45 kDa) is about half the size of CenC yet both appear to have the same function which is the cleavage of f3-1,4-glycosidic bonds. The primary objective of this thesis was to determine the sequence of CenC which might help explain why CenC is so large. Sequencing of cenC revealed that the gene was incomplete and was terminating in an adjacent sequence of the vector DNA in which it was cloned.  11 The missing sequence of the gene was cloned as described in the section on materials and methods and this was followed by identifying putative domains by sequence comparison. CenC can bind to Sephadex and to cellulose. It was of interest to determine which part of the polypeptide was required for this function. Identification of the amino acid sequence(s) involved in binding is most easily accomplished by examining the isolated domains. Proteolytic separation of domains has been used to dissect the structural and functional organization of endoglucanase CenA (44 kDa) and exoglucanase Cex (47kDa) from C.fImi (Gilkes eta!.  ,  1988, 1989) and of other 3-1,4-glycanases (Tomme eta!.,  1988; Ghangas and Wilson, 1988). For large, multidomain polypeptides such as CenC, however, proteolytic separation of domains is difficult. It is easier to dissect the gene and to express independently the sequences encoding each domain. This approach was used to correlate structure with binding activity either by deletion analysis and/or by expressing the domains alone or in tandem using molecular genetic techniques. To fully understand the mechanisms by which cellulases degrade cellulose, it is necessary to determine the roles of the CBDs. Exoglucanase and endoglucanase from T. reesei require a CBD for efficient hydrolysis of crystalline cellulose (Van Tilbeurgh et a!., 1986; Tomme eta!., 1988) while in C. therinocelluni a CBD increase the affinity of endoglucanase E (EGE) for its substrate (Durrant ci’ a!., 1991). Although the CBDs lack hydrolytic activity, at least one of them, the CBD of CenA, can disrupt cellulose fibres (Din eta!., 1991). One way to compare the different CBDs is to fuse them to the same catalytic domain. This study describes the construction of such a gene fusion which encodes a mutant form of recombinant CenA in which the CBD of CenA is replaced with the CBD of CenC. The influence of the CBD of CenC on the catalytic activity of the mutant enzyme for a number of cellulosic substrates was compared to CenA. Recombinant CenA (non-glycosylated CenA produced in E. coli) is cleaved by C. Jimi  protease adjacent to its linker sequence to generate a p30 fragment that has catalytic  activity but lacks the binding domain (Gilkes eta!., 1988; 1989; Shen eta!., 1991). The  12 mutant form of CenA described above was designed in a manner that the 23 amino acid PT linker of CenA was replaced by the linker lying adjacent to the CenC binding domain. The resulting polypeptide was tested to see if it was more resistant to C. fimi protease than CenA. Using a number of approaches which included studying intact CenC, its deletion mutants and by interchanging domains between CenC and CenA the objective of this thesis is to postulate the special role CenC may play in the cellulase complex of Cellulomonasjmnii.  13 2.  Materials and Methods  2.1. Bacterial strains plasmids and phage vectors Cellulomonasfimi ATCC 484 was used for the work described in this thesis. The Escherichia coli strain JM1O1 has been described previously (Yannish-Perron eta!., 1985). Site-directed mutagenesis was optimized by using the E. co/i host strain RZ1032 (dut, ung-) (Kunkel et al., 1987). The plasmids pTZ-18R and 19R and helper phage M13K07 for the preparation of single-stranded DNA were from Pharmacia (Dorval,  Quebec). Plasmid pUC18-1.6 cenA encoding CenA (Guo eta!., 1988; Wong eta!., 1986) and pUC18-1.6 cenA L\Pro-Thr (Shen et al., 1991) were described previously. Plasmids encoding cenC and its derivatives are presented in Table. 1.  2.2. Media and growth conditions C.fimi ATCC 484 was grown at 30 C in Leatherwood’s basal medium (Stewart and Leatherwood, 1976). The E.  CO/i  strains were grown in Luria-Bertani (LB) medium  (Sambrook et al., 1989) supplemented with 100 jig ampicillin ml at 37 C, for the production of recombinant protein unless stated otherwise or in 2 x YT medium (Messing, 1983) for the production of single-stranded DNA for sequencing. Cultures were induced when the A600 reached 1.0, with 0.1 mM final concentration of isopropyl-13-Dthiogalactoside (IPTG). Solid medium contained 1.5% Difco agar. Cells expressing interposon 2 were selected on LB containing 75 jig ampicillin, 25 jig streptomycin and 25 jig spectinomycin ml-’. Endoglucanase activity was detected on solid medium containing 0.1% carboxyniethylcellulose by staining with Congo red (Teather and Wood, 1982). The interaction of the dye Congo red with intact B-glucans provides the basis of this rapid and sensitive assay for endoglucanases. Potential colonies carrying recombinant plasmids expressing endoglucanase activity were plated on a master plate and a duplicate plate. The plates were incubated at 3T C, overnight. The colonies are then scraped of the plate, the plates were flooded with Congo red 2 mglml and left to rock gently on a platform for 15  14 minutes. The Congo red solution was poured off and the plates were flooded with IM sodium chloride solution to enhance the destaining process. The zones of hydrolysis appeared as a yellow halo against a red background.  Table 1. Plasmids encoding cenC and its derivatives. Plasmid  Description*  Polypeptide  Reference  pTZ-18R-8/5  Plasmid encoding cenC, missing 3’ end: aa 1023-  CenC’  Moser et a!., 1989  CenC CM  Moser Ph.D. thesis, 1988  1069 missing. pTZP-CenC  High-expression plasmid encoding cenC, missing 3’ end: aa 1023-1069 missing.  pTZ-JC1  Plasmid encoding the 3’ end of cenC: aa1023-1069 present.  ‘CenC  Coutinho eta!., 1991  pTZ-JC2  High-expression plasmid encoding ceiiC : CenC LPA  CenC  Coutinho et a!., 1991  CenC CA2  This study  N1N2  Coutinho et a!., 1992  Ni  Coutinho et a!., 1992  C1C2  Coutinho et a!., 1992  +  pTZ-JC2  AKpnI  aa 1-1069.  Internal deletion of C1C2: missing aa 954-1004.  pTZ-JC3  Plasmid encoding both Nterminal repeats N1N2: CenC LP  pTZ-JC6  aa 1-299.  Plasmid encoding Ni: CenC LP  pTZ-J7  +  +  aa 1-150.  Plasmid encoding both Cterminal repeats C1C2: CenCLP+aa 1-11 fused to amino acids 860-1069.  * A  Numbers refer to aa (amino acids) of the mature (processed) polypeptide. LP refers to the (32 amino acid) leader peptide of CenC.  15 Table 1. Plasmids encoding cenC and its derivatives (Contd). Plasmid  Description*  Polypeptide  Reference  pTZ-JC13  Plasmid encoding the fusion of CBD cenC to the catalytic domain of cenA  C’ ‘A  This study  Cl  Coutinho et al., 1992  CenC  LPA +  aa 1-299 of CenC fused to aa 135-4 18 of CenA. pTZ-JC14  Plasmid encoding Cl: CenC LP  +  aa 1-11 fused to  amino acids 860-993. pTZ-JC34  Plasmid encoding N2: N2 CenC LP + aa 1-38 fused to  This study  aa 167-299. * A  Numbers refer to aa (amino acids) of the mature (processed) polypeptide. LP refers to the (32 amino acid) leader peptide of CenC.  2.3. Enzymes and reagents Low-viscosity carboxymethyl cellulose (CMC), dinitrosalicyclic acid (DNS), hydrobenzoic acid hydrazide (HBAH), IPTG and phenylmethylsulfonyl fluoride (PMSF) were from Sigma. The degree of polymerization of the CMC was 400 and the degree of substitution 0.7. Restriction endonucleases, T7 DNA polyrnerase, T4 DNA ligase and deoxyribonucleotides were from Pharmacia (Dorval, Quebec), New England Biolabs Inc. (Beverly, MA, U.S.A.) or Bethesda Research Labs., Inc. (Burlington, Ontario). Radioactive nucleotides were purchase from New England Nuclear Corp. (Dupont Canada Inc. Missisauga, Ontario). Oligodeoxyribonucleotide primers were synthesized on an Applied Biosysterns 380A automated DNA synthesizer (Oligonucleotide synthesis lab, University of British Columbia) and further purified by chromatography on a Sep-Pak C18 reverse phase cartridge (Millipore/Waters Associates, Milford, MA, U.S.A.) (Atkinson and Smith, 1984). Nitrocellulose membranes BA85 were from Schleicher and Schuell  16  Inc. (Keene, NH, U.S.A.). Avicel (microcrystalline cellulose) was from FMC International, Ireland. Sephadex 0-50 was from Pharmacia. Bacterial crystalline cellulose (BMCC) was from the cellulosic pellicle synthesized by Acetobacter xylinum (ATCC 23769) (Hestrin, 1963) and phosphoric acid swollen cellulose (PASC) was prepared by precipitation of a solution of Avicel PH1O1 in 70% (w/v) phosphoric acid (Wood, 1988). Ultra-filtration membranes, PM1O and PM3O were from Amicon (Danvers, MA, U.S.A.). Other chemicals and reagents were from BDH (Vancouver, B.C.) or Sigma Chemical Co. (St. Louis, MO, U.S.A.).  2.4. DNA preparation and sequence determination The following procedures were described previously: isolation of total DNA from C.fimi by a lysozyme-SDS-pronase procedure (Whittle et a!., 1982); isolation of plasmid DNA (Birnboim and Doly, 1979); equilibrium density gradient centrifugation in CsC1ethidiurn bromide (Sambrook eta!., 1989); isolation of single-stranded DNA for sequencing (Sambrook et a!., 1989). The DNA sequence of cenC was determined by Nuclease Ba131 deletions of the EcoRJ-HindJII fragment in plasmid pTZ-l 8R-8/5. 100 ig of CsCI gradient, purified DNA, cut with the appropriate restriction enzyme, was treated with 5U of Nuclease Ba13 1 in the buffer recommended by the supplier for 60 minutes at  37° C and in a final volume of 100 jtl. 5 tl samples were removed at appropriate intervals and the reaction stopped by doing a phenol-chloroform extraction followed by ethanol precipitation (Sarnbrook et a!., 1989). The extent of the deletions was monitored by agarose gel electrophoresis. Conditions were modified as required by either increasing or decreasing the amount of nuclease. Samples with the appropriate level of digestion were pooled and the DNA excised and subcloned into pTZ-18R (Pharmacia). The second strand was sequentially deleted and sequenced in the same way by cloning the fragment in pTZ 19R. Single-stranded DNA was sequenced by the enzymatic method (S anger eta!., 1977) using T7 DNA polymerase and 35 S-cc-dATP (Tabor and Richardson, 1987) and replacing  17 dGTP with 7-deaza-dGTP (Mizusawa et at., 1986). The nucleotide mixes were adjusted for the high G+C content of C.fimi DNA. The synthetic oligonucleotide P-816 was labelled with 32 P using [ctP]-ATP and T4 polynucleotide kinase, then used to screen 32 transformants by hybridization (Sambrook et a!., 1989).  2.5. Cloning the 3’ end of the cenC gene A KpnI site was located close to the end of C.finii DNA in pTZ-18R-8/5. An oligonucleotide (P-8/6) was synthesized which was complementary to the 30 bases at the 3’ end of C.fimi DNA  in  pTZ-18R-815. After labelling with 32 P as described above, the  oligonucleotide was used to probe C. Jimi DNA which had been digested with KpnI and PstI. A 1.8 kb fragment hybridized with the probe. DNA was recovered from this area of the agarose gel, ligated into pTZ-18R which had been digested with KpnI and PstI, then used to transform E. coti JM1O1(Sambrook  ci’  at., 1989). Clones containing potential  inserts were transferred onto nitrocellulose filters, lysed with alkali and the filters probed with the oligonucleotide. Positive clones were sequenced. A clone designated pT’Z-JCl, was obtained which contained the 3’ end of cenC. pTZ-JC1 was digested with KpnI and Hindill and the 1.8 kb fragment recovered and ligated to the large fragment released from pTZP-CenC cut with KpnI and Hindu! to give pTZ-JC2.  2.6. Construction of deletion mutants of cenC  2.6.1. N-terminal deletions of CenC Construction of deletion mutants using Nuclease Ba13 1: N-terminal internal deletion mutants of CenC were created by linearizing plasmid pTZ-JC2 at the unique BamHI restriction site followed by treatment with Nuclease Bal3l. Conditions were as described previously but in this case 20 jig of DNA was digested with 1U of nuclease in a total volume of 20 j.il for 20 minutes. Samples were removed at intervals of 1 minute  18 and analyzed by agarose gel electrophoresis. Samples containing appropriately digested DNA were phenol-chloroform extracted and ethanol precipitated. The DNA was ligated with T4 DNA ligase and transformed into E. coil JM1O1. Ampicillin-resistant colonies were screened for carboxyrnethylcellulase activity. Individual colonies were subjected to mini-preparation of DNA and the DNA cut with restriction endonucleases to detect the extent of deleted DNA. Suitable clones were sequenced to map the precise extent of the deletion.  2.6.2. C-terminal deletions of CenC Plasmid pTZP-CenC (CenC CM), missing the 3’ end of cenC, was obtained in the initial cloning of cenC and has been described. Construction of pTZ-JC2 AKpnI (CenC CA2) a C-terminal internal deletion: pTZ-JC2 carrying the entire coding sequence for cenC has two in-frame KpnI sites separated by 150 base pairs (amino acid 954-1004). Plasmid DNA was digested with KpnI and the fragment recovered after agarose gel electrophoresis of the digest. The  fragment was ligated then used to transform E. coil JM1O1. Ampicillin resistant colonies were screened ([Feather and Wood, 1982) for carboxymethylcellulase (CMCase) activity. The plasmid DNAs from positive clones were sequenced across the junction between the KpnI sites. A plasmid with the correct sequence was designated CenC CA2.  CenC Cz3 was made by inserting the 2 fragment at a HpaI site, (nucleotides 2837-2842) 11 codons downstream from the start of the region encoding Cl.  2.7. Cloning of the N-terminal repeats N1N2 and Ni and the C-terminal repeats C1C2 and Cl pTZ-JC2 and the interposon  were digested with Sn,aI, then ligated together to  give pTZ-JC3. pTZ-JC2 was digested with Sad, then with mung bean nuclease and ligated to SmaI-digested 2 to give pTZ-JC6. A second Apal site was introduced into pTZ  19 JC2 by site-directed loop-out mutagenesis (Zhou et at., 1990). The nucleotides encoding amino acids 859 and 860 of mature CenC were changed from GAGCTC to GGGCCC (Coutinho eta!., 1991). The mutant DNA was digested with Apal and the larger fragment ligated to give pTZ-JC7. pTZ-JC7 was digested with Mitt!; then the ends filled in using the Kienow fragment of DNA polymerasel and the blunt-ended DNA ligated to SmaJ digested 2 to give pTZ-JC14.  2.8. Cloning of N2 using Nuclease Ba131 To obtain N2, an N-terminal internal deletion mutant created in Section 2.6.1. was truncated by introducing the 2 fragment at the Snial site (nucleotides 1050-1055) as described before for generating N1N2.  2.9. Deletion analysis of Ni Ni internal deletions (Ni 3 and A4) were obtained by the same method as described in Section 2.6.1. except that the 2 fragment was introduced at the Sad site (nucleotides 606-6 11), following treatment with mung bean nuclease, as described for generating Ni. C-terminal truncations of Ni (Ni M and A2) were obtained by inserting the Q interposon at sites MtuI (nucleotides 559-564) and BstEJI (nucleotides 466-47 1) respectively.  2.10. Substituting N1N2 for the CBD of endoglucanase CenA pTZ-JC2 encoding cenC has a Snial site between the sequence encoding its CBD and the catalytic domain. pUCi8-i.6 cenAAproT1w encodes a deletion mutant of the cenA gene in which the sequence encoding the linker connecting the CBD and the catalytic  domain is replaced with a HpaI recognition sequence. pTZ-JC2 was digested with Smal and HindIII and the 3.9 kb fragment was recovered after agarose gel electrophoresis of the digest. pUCi8-i.6 cenAApi•oTIr was digested with HpaI and HindIll and the 1.1 kb  20 fragment recovered as above. The fragments were ligated, then used to transform E. coli JM1O1. Ampicillin resistant colonies were screened for carboxymethylcellulase activity. The plasmid DNAs from positive colonies were sequenced across the junction between the cenA and cenC fragments. A plasmid with the correct sequence was designated pTZ JC13 and the fusion polypeptide it encoded CBDCe C CatCenA (C” A). 11 -  2.11. Purification of CenC, CenC CAl and CenC CA2 Cells were grown in LB medium supplemented with 100 .ig ampicillin m1’ at 30° C. After overnight growth, the cells were isolated with a Sharples centrifuge at 33,000g. The cells were ruptured by two passages through a French pressure cell in the presence of 1 mM PMSF and 0.01 mM pepstatin A. Streptomycin sulfate was added to a final concentration of 1.5% (wlv). After overnight precipitation of nucleic acids at 4°C, the extract was clarified by centrifugation twice at 35,000g in a Beckman JA2O rotor. Avicel was added to a final concentration of 1% (w/v). The suspension was stirred at 4° C for 1 hour. The Avicel was recovered by filtration through a Whatman GF-A filter and washed with 1M NaC1-50 mM potassium phosphate, pH 7.0 0.02% sodium azide to remove -  non-specifically adsorbed proteins and then with 50 mM potassium phosphate, pH 7.0  -  0.02% sodium azide till the A280 nm of the filtrate was <0.01. The adsorbed proteins were eluted with water until the A280 nm of the filtrate was again <0.01. The solution of desorbed proteins was concentrated by ultrafiltration through an Amicon PM3O membrane, and the water exchanged with 20 mM potassium phosphate, pH 5.8 0.02% -  sodium azide. This fraction was then subjected to anion-exchange chromatography with the aid of an FPLC using Mono-Q as the matrix (Pharmacia). Bound material was eluted using a linear salt gradient of 0  -  1.0 M NaCI in 20 mM potassium phosphate, pH 5.8  -  0.02% sodium azide over a period of 200 minutes at a flow rate of 1 ml mm . 50 111 of 4 the peak fractions were assayed for activity on a Titertek plate using 50 j.tl CNPC as the colorimetric substrate and then analyzed by SDS-PAGE. The active fraction was  21 concentrated on a PM3O membrane and the buffer exchanged for 50 mM potassium phosphate, pH 7.0 0.02% sodium azide. The concentrated material was made up to a -  final concentration of glycerol of 2% (v/v) and then applied to a P-150, Bio-Rad gelfiltration column, mesh size 100-200 at a flow rate of 0.2 ml min . Active fractions were 1 tested as before, analyzed by SDS-PAGE and concentrated in an Amicon ultrafiltration unit. 2.12. Expression of C1C2 and Cl Cultures of JM1OI/pTZ-JC7 or JM1O1/pTZ-JC14 were grown as above and polypeptides of the expected sizes corresponding to C1C2 and Cl were observed in the culture supernatants. The amino acid sequences of the N-tennini were confirmed by amino acid sequencing.  2.13. Purification of N1N2, Ni and N2 Cultures of JM1O1/pTZ-JC3, JM1O1/pTZ-JC6 or JM1O1/pTZ-JC34 were grown at  370  C to A600 of 1.0, then induced with IPTG. After growth overnight, the cells were  removed by centrifugation. Avicel or Sephadex G-50 was added to the supematant (lg 100 1 m1 ) . The suspension was kept at 40 C for 3 hours with occasional stirring. The Avicel/Sephadex was recovered by filtration through a Whatman GF-A filter and washed as described previously for the purification intact CenC. Adsorbed proteins were recovered by washing the Avicel/Sephadex with water. The water extract was concentrated by ultrafiltration through an Arnicon PM1O membrane and the water exchanged with 50 mM potassium phosphate 0.02% sodium azide. The N-termini of all -  three polypeptides were confirmed by amino acid sequencing. N2 was purified in the same way as N1N2 and Ni.  22 2.14. Purification of C” A C’ A (60.9 kDa) was purified from culture supernatants of E. coli JM1O1/pTZJC13 in the same way as described for N1N2, Ni or N2. The purity of C’ A was monitored by SDS-PAGE and the N-terminal sequence confirmed by amino acid sequencing.  2.15. Measurements of CenC activity The activity of CenC on CMC by the HBAH method was measured as described previously (Moser eta!., 1988; Coutinho et at., 1991). The assay conditions for the HBAH method were as follows: 0.1 ml of appropriately diluted enzyme solution was mixed with 0.4 ml of 0.5% carboxmethylcellulose in 50 mM sodium citrate buffer, pH 6.8. After incubation at 300 C for 30 minutes, the reaction was stopped with 1.0 ml HBAH reagent (Lever, 1973). The tubes were steamed for 12 minutes and the absorbance was read at 420 nm. The kinetic parameters of CenC using chloronitrophenylcellobioside (CNPC) as the substrate were measured and compared to Ce1D (endogiucanase D from C. thermocellurn) another endoglucanase from Family El (Tomme eta!., 1991). Assays were done in triplicate. Briefly a stock concentration of the enzyme 3.85 tM was diluted 1:10 and 20 j.il, added to 80 tl of 50 mM buffer (Na2HPO4 / citric acid buffer) with 100-1000 .jM CNPC as the substrate. The release of 2-chloro-4-nitrophenol was monitored continuously (Hitachi U-2000 spectrophotmeter) at 400 nm. Temperature optimum measurements were done in the same way except 2 mM CNPC was used as the substrate. Temperatures were kept constant by using a circulating water bath (Forma Scientific) directly connected to the spectrophotometer chamber. Buffers for the determination of the pH optimum of CenC were 50 mM: acetate buffer (pH 4.0), citrate-phosphate buffer (pH 5.0), phosphate buffer (pH 6.0, 7.0) and Tris-hydrochioride buffer pH 8.0) (Perrin and Dempsey, 1974).  23 2.16. Measurements of C’ ‘A activity The activity of the purified enzyme on CMC, Avicel, BMCC, cellulose azure and PASC was measured by the HBAH method (Gilkes  et  at., 1988; Shen  et a!.,  1991).  Assays were done in triplicate. Initial rates of hydrolysis were measured by assaying for the amount of reducing sugar released with the HBAH reagent (Lever, 1973). The absorbance of the colored reagent was read at 420 nm against blanks containing equivalent amounts of enzyme added after mixing substrate with HBAH reagent.  2.17. Oligosaccharide products of CenC Oligosaccharides G3-G6 were from Seikagaku (Tokyo, Japan). Cleavage products were separated on a Waters, Dextro-pak column (Millipore) using an HPLC (Waters) and monitored by a Waters 410 differential refractometer. The flow rate was 1 mi/mm at room temperature with water as the solvent. The column was capable of separating the a from the  f3- anomer for oligosaccharides G3 and G4, with the a anomer having a slightly  longer retention time. Reaction mixtures contained substrate concentrations in the range  0.5 2.0 mM and were incubated with 5U of enzyme at 37°C in a final volume of 40 jil -  for times ranging from 2 minutes to 240 minutes. 30 .tl of sample was injected onto the column for analysis. Cleavage, of 2-chloro-4-nitrophenyl glycosides (derived from cellodextrins), at Cl was followed spectrophotometrically at an absorbance of 400 nm, following the release of phenol (Tomme  ci’ a!.,  1991).  2.18. Deteririination of protein concentration Protein concentration was routinely measured by dye binding (Bradford, 1976) using the Bio-Rad protein assay kit. Purified protein concentration was determined by absorbance at 280 nm using the extinction coefficient obtained for the polypeptide (Scopes, 1974).  24  2.19. Amino-acid sequence determination Polypeptides were prepared for sequencing by electrotransfer from SDS-PAGE gels (Laemmli, 1970) on to a polyvinylidine difluoride membrane (PVDF membrane, Millipore) (Matsudaira, 1987, 1990). The N-tenninal amino acid sequences of the excised bands were determined by automated Edman degradation with an Applied Biosystems 470A gas phase sequenator (Protein Sequencing Lab., University of Victoria, B.C.).  2.20. Protein electrophoresis Proteins were analyzed by SDS-PAGE (Laeimrili, 1970) and detected by Coommassie blue staining. Calibration standards were myosin, rabbit muscle (212 kDa); 13-galactosidase, E. coli (130 kDa); phosphorylase B, rabbit muscle (97.4 kDa); albumin, bovine (68 kDa); glutamate dehydrogenase (53 kDa); ovalbumin (43 kDa); glyceraldehyde-3-phosphate dehydrogenase, rabbit muscle (36 kDa) and carbonic anhydrase (29 kDa). Low range calibration standards were from Bethesda Research Labs.: ovalbumin (43 kDa); carbonic anhydrase (29 kDa); 13-lactoglobulin (18 kDa); lysozyme (14 kDa); bovine trypsin inhibitor (6 kDa) and insulin (3 kDa).  2.21. Immunoblotting CenC or its derivatives were detected by immunoblotting (Harlow and Lane, 1988). Bovine serum albumin (3 %) was used for blocking residual sites on the nitrocellulose membrane and washes included 0.05 % Tween 20 to get rid of non-specific adsorption. Western blotting was done using polyclonal sera against CenC (Moser, Ph.D. thesis) and goat anti-rabbit IgG-alkaline phosphatase conjugate as the secondary antibody.  25 2.22. Adsorption of N1N2 or Ni to Avicel and Sephadex The binding to Avicel or Sephadex G-50 of polypeptides in cell extracts or culture supematants was evaluated by allowing the polypeptides to adsorb as described for the purification of Ni and N1N2. The washed adsorbent with adsorbed polypeptides was heated in gel loading buffer (Laemmli, 1970) prior to analysis by SDS-PAGE.  2.23. Adsorption of N1N2, Ni or CBDCex to BMCC and PASC BMCC was extracted from cultures of Acetobacter xylinum (ATCC 23769) grown on peptone, yeast extract, glucose medium (Hestrin, 1963). PASC was prepared by treating Avicel with phosphoric acid (Wood, 1988). CBDCex was used as a comparison and purified polypeptide was provided by Dr. E. Ong (E. Ong, Ph.D. thesis). N1N2, Ni and CBDCex were stable for 36 hours under the test conditions. The optimal times for equilibration were determined by incubating saturating levels of purified polypeptide with cellulose (1 mg of PASC or 1 mg of BMCC) in a total volume of 1 ml 50 mM potassium phosphate buffer pH 7.0 0.02% sodium -  azide in a 1.5 ml Eppendorf tube at 30° C with gentle agitation in a rotary mixer. Equilibration was reached between 30 minutes and 1 hour after adding the polypeptide; the amount of unbound polypeptide in the supernatant changed negligibly between 1 hour and 36 hours after addition. For convenience, mixtures were allowed to equilibrate for 24 hours at 30° C in all subsequent experiments. Tubes containing substrate and no polypeptide were used as controls. After equilibration, the tubes were centrifuged at 15,000g for 10 minutes. The supernatant liquid was transferred to another tube and centrifuged again for 10 minutes. The absorbance of the supernatant fluid was measured at 280 nm. Protein concentrations were determined from the extinction coefficients at this wavelength. Adsorption was expressed as jamoles protein bound g 1 cellulose. All assays were done in triplicate.  26  2.24. Adsorption of N1N2, Ni and N2 to PASC N1N2, Ni and N2 were incubated with or without 1 mg of PASC at concentrations of 25, 100 and 250 ig m1’ in a total volume of 1 ml of 50 mM potassium phosphate buffer, pH 7.0 0.02% sodium azide. To allow for equilibration the samples -  were incubated at room temperature in a rotary shaker for a period of 24 hours in 1.5 ml Eppendorf tubes. The tubes were centrifuged at 15,000g for 10  mm. Samples were  analyzed by SDS-PAGE (Laemmli, 1970). Gels were scanned by a Bio-Image (Millipore) computer assisted densitometer to get a relative estimate of the percent of free polypeptide present in solution.  2.25. Rapid small scale screening procedure for functional binding activity Cultures carrying individual deletion mutants were grown in 500 ml of culture medium at 37° C to A600 of 1.0, then induced with IPTG. The cells were removed by centrifugation and the culture supernatant was mixed with 10 g of Avicel. The Avicel was washed and the proteins desorbed with 100 ml of water. The eluted material was then concentrated and exchanged with 50 mM potassium phosphate 0.02% sodium azide by -  ultrafiltration through an Amicon PM 10 membrane and analyzed on a SDS-PAGE gel.  2.26. Adsorption of CenA and C’’ A to BMCC and PASC Increasing amounts (25, 100 and 250 j.ig) of purified CenA and C” A were mixed with 1 mg of BMCC in 1 ml of 50 mM potassium phosphate, pH 7.0 0.02% sodium -  azide in 1.5 ml Eppendorf tubes. Controls did not contain BMCC. The tubes were incubated at room temperature on a rotary mixer. Both polypeptides were stable for at least 24 hours under these conditions. After 8 hours, the tubes were centrifuged at 15, 000g for 10 mm. The supernatants were transferred to fresh tubes and centrifuged again at 15, 000g for 10 mm. Then a 500 jil sample of each supernatant was transferred to an Eppendorf tube containing 500 .ig PASC. The second set of tubes, together with those  27 containing the remainder of the supematants from the first set of control tubes, were incubated as before for 8 hours. The supernatants were recovered after two centrifugations as before. Equal quantities of all supernatants, including the residual supematants from the first set of tubes, were analyzed by SDS-PAGE. Gels were scanned by a Bio-Image Millipore) computer assisted densitometer to get a relative estimate of the percent of free polypeptide present in solution.  2.27. Hydrolysis of CenA and C’’ A by C.fimi protease  C.firni protease was prepared from culture supernatant as described previously (Gilkes eta!., 1988). The activity of the preparation was measured by incubating samples with 10 mg of hide powder azure in 1.5 ml of 50 mM phosphate buffer pH 7.0 at 370 C. The A585 nm of the supernatant was measured at intervals. One unit of activity gave an increase in A585 nm of 1.0 Fr . 80 .tg samples of CenA and C’’ A were incubated with 1  C.Jirni protease for 3 hours at 30° C in 50 iI 50 mM potassium phosphate, pH 7.0  -  0.02% azide. After incubation, samples from each tube were analyzed by SDS-PAGE.  2.28. Amino acid sequence comparisons Analysis were done on a PC-AT compatible microcomputer, using programs in PC-Gene (Intelligenetics Inc., Mountain View, California). PCLUSTAL was used for multiple sequence alignments and generation of dendogranis.  28 3. Results  3.1. Nucleotide and amino acid sequence of cenC CenC was cloned initially into 2gt1 1, using a partial Saullia digest of C.fimi DNA. Screening of the library was done using a restricted pooi of oligonucleotides corresponding to the codons for amino acids 7-11 of the N-terminal of CenC. The fragment encoding CenC was then subcloned into pTZ18R to give pTZ18R-8/5 (Moser et at., 1989). Sequencing of the C. fIrni DNA after Nuclease Ba13 1 deletions of pTZ1 8R8/5 (Fig. 3,4) showed cenC to be incomplete. The 18 amino acids, sequence RLPFTSCAVCLQDSMRRR, at the 3’ end of the open reading frame established by the N-terminal amino acid sequence of CenC (Moser et at., 1989), corresponded to codons from ?.gt1 1. The missing fragment of the gene was cloned as described in Materials and Methods. Briefly, a 30 mer synthetic oligonucleotide P-8/6 corresponding to the nucleotide sequence at the 3’end of the truncated gene was used to probe C.fimi genomic DNA cut with restriction enzymes KpnI and PstI. A 1.8 kb KpnI/PstI fragment was obtained after digestion of C.fin2i genomic DNA (encoding the 3’ end of the gene) (Fig.5) was recovered and cloned into pTZ- 1 8R. Colony hybridizations of potential clones gave four colonies which carried the correct insert (Fig.6) and the plasmid encoding the 3’ end of the gene was designated pTZ-JC1(Fig. 7). Sequencing of the C.firni DNA in pTZ-JC1 showed that 46 codons were missing from the cenC sequence in pTZ18R-8/5. Plasmid pTZ-JC2 (Fig. 7) encodes complete CenC and was generated by subcloning the KpnI Hindlil fragment from pTZ-JC1 into pTZP-CenC (which carries a portable transalation initiation site for improved expression). Codon usage in cenC was very similar to that in cex, cenA and cenB Meinke et a!., 1991a; data not shown), reflecting the 71 moles% G+C in C.fimi DNA.  29 Figure 3. Nuclease Ba13 I deletion of plasmid p1Z— I XR—X/5 to generate deletion clones for the sequencin of cenC. The DNA molecular  v. eight markers:X/I  1 and X/E. H refer to  ‘ambda DNA cut with Hi,ullhI and I-/inS/li ILcuRI respectively. Time course digestion of pTZ— I 8R—/5. in  buffer recommended  b the supplier, from 0—60 minutes at 37 C using 5  U of Nuclease Bal3 I I 00u of DNA. in a total volume of 100 p.1. .  pTZ 18R_815  kb  23.1  .3.50  0 HEIH  Bal31  30 Figure 4. Mini-preparation of plasmid DNA from clones generated after Nuclease Ba13 1 deletions and cut with BarnHI. The DNA molecular weight markers: to lambda DNA cut with Hind!!!! EcoR! and PstI respectively.  kb  23 1  A)’ HIE P  DELETED CLONES  “N”  2 19  a  0 56  a a  HIE and  P refer  31  Figure 5. Southern blot of C.Jirni DNA cut with K (KpnI), P (PstI), K/P (KpnI and Pstf) and probed with the synthetic oligonucleotide P-8/6 labelled with [ccP]-ATP to locate 32 the fragment encoding the 3’ end of cenC. Uncut plasmid pTZ-18R-8/5 was used as a control. A unique band corresponding to a size of 1.8 kb was excised and cloned into pTZ-18R also cut with KpnI and PstI, and then transformed into E. coli JM1O1.  I8R8/5  C.timi kb  K  P  KIP  —  r  -—  72  3.8  1.8  j  32 Figure 6. Colony hybridization of potential clones carrying the 1.8 kb KpnIIPstI fragment encoding the 3’ end of cenC with oligonucleotide P-816. Nitrocellulose filters were probed in duplicate to minimize the possibility of picking non-specific background spots as potential clones. C refers to JM1O1/pTZ18R-815 as control.  I  •( : ‘V  .  •1  33 Figure 7. Plasmids used for the cloning of complete cenC and for its high-expression. C. fimi DNA is indicated by the double line, with the cenC coding sequence in black. The arrow shows the direction of transcription of cenC. Plasmids: pTZ18R-8/5, encodes cenC but missing the 3’ end of the gene. pTZP-CenC is the same clone engineered for highexpression with a portable transalation initiation site. pTZ-JC1 encodes the 3’ end of cenC and pTZ-JC2 encodes complete cenC. Blunt! BamHI  .HiIII I-lindlll  Blunt BamHl  Hindill  34 Twenty-one codons were not used in cenC, and of these, twenty were not used in any of the genes. Endoglucanase C from Cellulornonasfirni is one of the largest 3-1,4-g1ycanases characterized to date. The mature polypeptide is 1069 amino acids long, preceded by a leader peptide of 32 amino acids (Fig. 8). The p1 of CenC calculated from its predicted amino acid sequence is 4.1. This may contribute to the discrepancy between the molecular mass of 112, 969 calculated from the predicted amino acid sequence and the molecular mass of 130 kDa observed by SDS-PAGE. Acidic proteins can give Mrs by SDS-PAGE which are significantly greater than their actual molecular masses (Kaufman eta!., 1984).The promoter and ribosome binding site of CenC were identified previously (Fig. 8; Moser et at., 1989). A 9 bp inverted repeat 7 nucleotides downstream of the putative translational stop codon could be a transcription termination signal (Fig. 8), which would give a mRNA of —3.37 kb. This was in good agreement with the size of 3.5 kb determined for CenC rnRNA from C.JInii (Moser ci’ at., 1989). The most striking features of the predicted amino acid sequence of CenC were the long tandem repeats at both ends. Two contiguous repeats of —150 amino acids at the Nterminus (Ni, N2) were 50% similar, and two unrelated, contiguous repeats of —100 amino acids at the C-terminus (Cl,C2) were 60% similar (Fig. 9) (Coutinho eta!., 1991). It is rare amongst 13-1 ,4-glycanases for the catalytic domain to be flanked at both ends by extended sequences of amino acids (Beguin, 1990; Gilkes et a!. 1991a). ,  Amino acids 300-880 of CenC shared greater than 30% similarity with the catalytic domains of f3-1,4-glycanases of sub-family El (Fig.8, 10; Henrissat eta!. 1989; ,  Béguin, 1990; Gilkes eta!., 1991a). There was much less similarity with the catalytic domains of members of sub-family E2 (data not shown). Interestingly, the tetrapeptide DAGD (amino acids 47 1-474 in CenC) occurs 305-329 amino acids from the tripeptide H R (amino acids 799-80 1 in CenC) in all of the enzymes in family E. The consensus sequences around these motifs in subfamilies El and E2 share some similarity  35 Figure 8. Nucleotide sequence of cenC and its flanking regions, and the deduced amino acid sequence of CenC. The nucleotide sequence is numbered with +1 as the transcriptional start site. The amino acid sequence is numbered with +1 as the N-terminal amino acid of mature CenC. -10 and -35 are the promoter sequences, and S.D. is the ribosome binding site identified previously (Moser et at., 1989). The leader peptide, 32 amino acids, precedes A, the leader peptide processing site. The inverted repeats downstream of the translational stop codon are underlined with arrows. The repeats in the amino acid sequence are boxed. AV and BV are the junctions between the N-terminal and C-terminal repeats, respectively. The KpnI site near the 3’ end of the coding sequence is boxed. P-8/6 indicates the sequence of the oligonucleotide probe used to clone the 3’ terminus of the cenC coding sequence. The underlined amino acid sequence Ti 15 matches completely, the sequence of the tryptic peptide determined from the digestion of CenC previously, and also used for generating an oligonucleotide probe in the initial cloning of cenC (Moser et at., 1989). This sequence appears in the EMBL/GenBankIDDBJ Nucleotide Sequence Data Libraries under the accession number  X57858.  G  +1  A  T S  T  G A  L C  V A  V P  A  0 S A  Q  Y  0  V  0  V  VI  A  S  A  S  Y  P  L  IP  IV  N  F  F  C  L  V  Q  R  T  D  A  L  V  Y  R  T  V  0  T  E  P  T  R  A  W  S  S  V  R  0  C  S  T  W  L  C  A H  I  C  S  A  C  Q  T  L  C  R  L  Q  N  A  C  S  Y  R  G  0  A  T  D  0  L  PD  P  V  0  L  A  T  P  A  R  V  V  0  0  L  V  A  0  L  P  PG  A  P  0  D  P  P  A  0  C  0  V  0  L  V  K  0  V  A  S  K  K  V  TO  0  0  0  V  P  A  P  D  S  V  A  P  0  0  A  S  N  P  W  R  R  0  RV  A  L  V  K  P  P  R  0  KG  QON  BIlE  A  RVLVO  L  V  S  PD  P  PV  VD  DO  V  YE  P  H  RF4C  F  P  PP  S  T  S  A  W  P  V  P  ATIP  A  C  T  S  SAT  S  Y  A  T  FAD  0  L  PA  PV  S  P  SF  F  S  S  A  L  T  L  C  T  L  Y  L  P  P  A  H  P  P  P  3  A  F  A  A  0  K  0  S  P  L  S  0  0  P  Q  V A  F  GAY  H  D  0  A  F  S  V  A  C  0  S  P  0  H  I  P  B  0  V  D  R  H  II  C  0  0  C  0  V  0  V  Y  I.  N  K  A  L  K  L  0  N  V  P  Y  P  P  K  RPAF  VNQVG  Q  0  0  V  V  N  Q  S  P  V  Q  Q  A  PG  A  0  N  C  L  A  P  A  EQG  V  0  0  R  Q  S  A  KG  W  I  S  L  P  K  L  0  P  Y  A  C  0  I  0  0  V  B  A  V  A  H  AT  P  S  QI  I  S  I  A  V  P  L  V  R  C  C  N  L  w  Q  I  Y  N  LVT  I  APLTG  KR  Y  S  PWDAGLVYNGVPVG  S  P  TSR  0  IC  L  C  P  IL  G  A  1)  0  T  IL  G  REGTTYTLRYTATASTDV?VRALVGQNGAPYGTVI  Y  I  A  ZGTI  ILNGVA  V  PIG  SD.  G  W  SQARGALTAVVxrLALALAasGTALAIAs  -10  5  RS  P  p  0  F’  0  R  MVS  -35  1739 528  1619 488  1499 448  1379 408  1259 368  1139 329  1019 288  999 248  779 209  659 168  539 128  419 88  299 48  179 8  59  -62  K  T  0  V  V  W  L  Q  T  V  R  I  P  K  V  R  P-ale  V  AG  Q  A  P  S  P  Q  0  L  C  W C  RIIA  T  0  V  A  S  W  C  A  C  A  T  C  P  P  C  P  T  T  N  S  C  P  L  V  V  E  S  V  U  V  L  A  V  P  L  A  N  W  E  L  R  V  C  .R  T  P  Q  TV  L  P  P  H  I  A  0  W  N  Q  R  A  N  V  C  P  C  C  P  N  S  S  L  S  L  C  T  A  C  P  L  C  P  P  S  P  K  N  V  P  A  L  0  L  S  S  N  Q  N  P  S  Q  T  F  W  A  A  0  P  V  A  S  P  0  S  R  P  A  TN  L  A  A  A  TAR  A  N  S  V  S V  A  V  Q  P  H  L  P  5  P  C  A  S  K  V  A  A  P  V  V  P  V  C  0  V  L  W  W  W  L  V  R  P  T  C  K  A  A  P  C  K  VV  S  V  P  >  V  A  R C  P  A  P  YR  V  C S  A  V  R  U  S  A  P  K  V  0  A  P  V  V  A  H  A  Q  F  Y  P  A  C  A  C  P  K  C  W  C  0  L  K  L  D  R  It  A  C  A  Q  K  V  A  K  A  H  H  V  V  P  C  V  H  A  0  V  A  A  F  P  A  A  0  E  C  T  T  L  Y  L  A  S  L  N  Y  C  A  Y  K  L  L  G  A  P  E  H  A  P  Q  A  A  T  A  V  T  T  I  S  H  A  P  S  W  L  R  H  H  L  W  ITNAVGTAATEPAELAVQRPRSI*  V  L  S  P  LV  E  L  T  R  ‘1  1.  S  IV  R  C  L  0  K  A  A  K  P  AR  1w  S  R  A  Z  L  E  K  Q  0  R  0  A  W  P  S  0  L  -<  V  L  A  A  R  F  A  A  A  R  V P  TN  S C  P  P  Q  H  Q  0  C  C  A  R  V  P  0  K  C  S  R  P  V  I  K  V  P  T  V  A  A  I  V  N  A  C  A  A  A  Q  3536  1069  3419  3299 1048  3179 1008 Cj  F  968  3059  2939 928  2819 888  2699 848  2579 808  2459 768  2339 728  2219 688  2099 648  1979 608  1859 568  V  R]  S  C  A  PIT V P  A  C A  S P  A  L  Q  F  W  H  K  A  A  P  C Q  R  A  V S  W  Y  P  K  P  L  0  L  0  F  C  C  K  P  A  R  A  W  0  Q  P  D  Q  AG  A  A  Y  C  L  F  C  D  A  A  V  Q  L  A  L  L  Q  Ti 15  P  S  P  L  N  0  C  F  P  0  A  Q  V  K  P  N  Y  T  38 Figure 9. Repeated amino acid sequences in CenC.  A N-terminal repeats. .  C-terminal repeats.  :, identical amino acids; , conservative changes; -,  gaps left to improve the alignment.  Amino acids said to be ‘similar’ are: A,S,T; D,E; N,Q; R,K; I,L,M,V; F,Y,W  60  1 .  *  .  *  *  •  *  *..  *.  •  149 EvErLpHTSF?ESLGPWSLYGTSEPVF2D-GRMCVDLPGGQ 61 TLRrrATAST •  *••*.*  *  *  .*.  ..•  *  4DGLVYNGVPVGEXESY 209  VRALVGGAPYG1VLDT-SPALTSEPRQVTErFTASATYPATPAADDP 121 *  .*  *. .*  *.*.  .  210 VLSFrASATPEt1PVRVLVGGGAYRTFEQS?PLTGEPATREYAFrSNLTFP--PD3DAP 269 122 BQIAFQLGGFS7DAWTFCI.DDVALDS •.*..  ..  .*  •.*  .*.  148  *  270 -GQVFHI1KAG---AYE’CISQVSLTTSAT 296  886 TAP  VRAGX-WVAGATGrTLTVRAT  rEQ-P  945  *.  *•*  *  976 AAPVVr-QHPADVRA’GTPAVAAADY?TPC\M VGGSWRPIP3ATSTTLSV?%1T 1036 946 ?IDTRYRAV?rNAAGSVESAVVRLTVER *  .:““  :*  *  975  :*:  1037 VLAAGTEYRAWrNAVGTAATEPAEUVQRPRS 1069  39 Figure 10. Sequence similarity in the catalytic domains of 3-1,4-g1ycanases of subfamily El. Symbols for identity, conservative changes and gaps, as for Fig. 9. CfCenC, endoglucanase C of C. flnii; PfEndA, endoglucanase A of Pseudomonasfluorescens subsp. cellulosa (Hall and Gilbert, 1988); CtCe1D, endoglucanase D of Clostridium thermocellum (Joliff et at., 1986a); BfCedl, cellodextrinase of Butyrivibriofibrisolvens (Berger et a!., 1990). The sequences are numbered from the N-terminal amino acids of the mature enzymes, except where marked  A  In these cases the numbering is from the  start of the signal sequence because the sites of processing have not been determined.  40  CfCenC:  300  GYEPUrG-PRVRVNQVGYLPFGPKRATLVTDAAEPVAWELRflJDGVVVAD3TSEPR *  :  ::*::*:  :::  *  :  :  *  *:  *  :*  :*  *  :  PfidA:  29  CtCe1D:  44’ ITBYQRIRLNSIGFIPNHSKKA- --TIA?\NCSTFYVVKEDTIVYTGTATSM  BfCed:  38  **  *:*.:  :*  *  *  *  *:  :***  *  CfCenC:  AFtX3KVEHFGT----  GVEPSAAQAVHVLDFSDTQGAG’X7LVAETSRPFDIDLYQQLRYD.LNYFY •  PfEndA:  *  *.  .*  *.  •  ..*  •.  .**.  .*.*..*  **  .  GSDASSGLZIHIDLSSVTATGSGFrLIVGGDSSYPFSISSAFYDALKYF1 * * * * :* :* * : : : ::: :  CCCe1D: ...  **  *  .  *  ..  .  .  .*  .*  BfCed:  --DEISGED1’YVADFSLTEEGKYKI-VADGQESVLFSISNDAYDKI21KDICKcFY  CfCenC:  LARSGI’EIEADVVGEE---YA  PfEndA:  .  ...*  HSGIAIE1’flI’GGGGSYASMSPWSRPAGHLNQGANKGDMNVPCWSGTcNYS-:***  CtCe1D:  REAGHVGVAPNQGLYIWPCIGPRDYYD3-  ..  .  *  *  :  :  GPCHDAYLt71I  ILRCGTSVSA--TYNG-IHY SM **.  *  . •  •  •  •  KPCHTI’EA1V--Y  BfCed:  YLRCGDALSX--EFAG-EYY- H  CfCenC:  WrCDYRLDVSGYDAGDHGKYVVNGGIAVGQLLQrYERALHAGTAD-LAIXTLD  PfEndA:  LNVTKGDAGDHGKYVVNGGISVPLt2’IYERAQHITGNLAAVA3SMN  CtCe1D:  NGQHTKKDSTKDAGDYNKYVVNAGITVGSMFLAWE---H--FKEQ-LEPVALE  BfCed:  GEDVEPVDGGWHDGDYGRYSTAGAVAVANILYGVR---F- -FKGL-LD-VHYD  CfCenC:  VP---DVPDVLDEARWELThLSMIVPEG-EYAGN*XVFDEGWTGLPLLP  *.  *.  *  .  .*  ..  *  .  IP---ESGNGVADILDEARWQMANQVPQGQAKAGMAHHICIIWVGW1’GLPLAP .  ****•*. .  **  CtCe1D:  **  *  *  ..  *••.**  •  *.  **  **.  *.*..*.  PfEndA:  •  ..*  *.  • .*  *.  .  *  *  •  IP---ESIPDFLDELKYEIEThTMQYPIDG-- -SGRWHKVSTRNFGGF-IMP .*•  •*•**  ...*.  •  .  ..  .  .  *•.  BfCed:  IPK/AGDKLPEILAEVKVELDFL1QRG---S4--H?ITFNHAPF-LMP  C fCeriC:  ADDPQARSLPSTAATLNLSAVAAARLLEPYDPQLTLLEAARPTWAAQEH  PfEnA:  HDPQQRALVPPSTAATULAATAAQAARIWKDID1.GFAALCLTAAERAAAQAN ** • *• • . ** * :: EDERFF’JWSSAATADFV7TTAMAARIFRPYDPQYAEKCINAAKVSYEFUN *: * ..*: * **. * :* :*: : DDREELFLFSVSSLATADIAVFALAYTTYDAEYADKU1QKSLLAYKWLLtJ  *  CtCe1D: BfCed:  *  *  .  .  .  ..  *.  41  CfCenC:  PAL-YAPGEAGA3GGAYNDSQVADEAELYLTI’GEDAFATA---VflSPLH  PfEridA:  PND-IYSGGGGG-YGDRFDEFWAAAELYITrGDSRYLFr-- -INNYTLE * * ::* :::::** :: : :  •  *  • *..•*  .  *  *  CtCe1D: •  •  *  •  .••  •  **•*  .  BfCed:  PDELLFRPGQ-YDFDISRFACALYEATSrCzYSDAQEL}RLEE  CfCenC:  TADVFI’AD  GFGGSVAALGRLDLTVPNELPGLDAVQSSVVEGAQEYL  PfEndA:  RTDFGWPD  TELL  CtCe1D:  FSKKIEAD  BfCed:  FDKNAQ1GYQGECLAEVAGLGSLSLLUR--EEN-ALCSLARNSFVED  CfCenC:  AAQAGQGFGSLYSPPG-GEYW3SSSQV?NNLVVVATAYDLTGDERFRAATLEGLD  *  **  FVNIJWfYLLSERPGKNPALVQSIKDSLLSTAD *  •  •.  **  PfEndA:  ASGYPAPLSSLE *  *  *.*:  *  **  •  *  MSLAWPATHTNSLRIAAPHWIASTHLTS  *  . . .  •  . •  *.  **  •  YSNSVINKLVLNGLYDFSGNQNFALGVSKGIN *** *: * • :* * :  CtCe1D: BiCed:  *: *: * *: * :*: :*: :: RLVVSGFGLGDFILUC1LAIRIPEYKLALEGLD  CfCenC: *.*.  *  PfdA:  YLFGSNVLSTSFITGLGVAQPHPFtZAGALNSNYPWAPPGALSGGPN---AGL  CtCe1D:  HVFGBNYRSYVrGrJ3flJPPt?HDRR-SGtX--IWEPWPWfLVGG  •  •  *  BfCed:  *..  .**  ...  .  .  .  *..  *  •  *  YILXNSMDISYNGEKAPHRP-TAVDD--IEEV1PGLVSGGPNSGLFUE  881  C fCenC: *  *  .*  ..*  .**.**  *.*.•.*  •*•.  **  •  *  PfdA:  EDSLSASPLSGCTSRPATCWDSIWSEITflAPLWVLGFYNDFAAT 604”  CtCe1D:  GWPGPVDIQDSYQTNEIAINZAALIYALAGFVNYNSA 577”  *  *  *.  BfCed:  ..*  •  .**..*•  *  *  **  *  RAQTLRGK- ---GLPPMKCYIDHIDLYSLNEITIYSPLVFALSGILE  547”  42 (Fig. 11,12) (Coutinho etal., 1991). Similar considerations apply to three recent additions to family E, EndAFS from Fibrobacter succinogenes AR1(Cavicchioli eta!., 1991) and enzymes El and E4 from Thernionosporafusca (Wilson, D.B., 1992). Domains in Cex, CenA and CenB are separated by linkers, short sequences rich in proline and/or hydroxyamino acids (O’Neill ci’ al., 1986; Wong et a!., 1986; Meinke  eta!.,  1991a). In CenC, the sequences SLTTSATPPP (amino acids 292-299) and PVPTAP (amino acids 983-988) linked the N-terminal and C-terminal repeats, respectively, to the remainder of the polypeptide (Fig. 8). Amino acids 809-818, with the sequence PSLPSPPPGS, may delineate a domain of -60 amino acids adjacent to the C-terminal repeats (Fig. 8).  3.2. Expression of CenC in E. coli The fragment of C.fimi DNA in pTZ18R-8/5 was manipulated to increase the level of expression of cenC before it was learned that the gene lacked its 3’ end (Moser, Ph.D. Thesis, 1988; Coutinho eta!., 1991). In pTZ18R-8/5, the Plac on the vector was separated from the 5-end of cenC by some 600 bp of C.Jirni DNA containing several inverted repeats, which could interfere with transcription and/or translation in E. co/i. Furthermore, C.firni promoters for other cellulase genes function poorly in E. co/i (O’Neill  et  a!., 1986; Wong eta!., 1986; Owolabi et al., 1988). Therefore, the  presumed coding sequence of cenC, except for the GTG translational start codon, was fused to an ATG codon at the 3’-end of a portable translation initiation site (PTIS), and the fusion put under the control of Plac on pTZ-1 8R to give pTZP-cenC (Moser,, 1988; Coutinho  eta!.,  1991). This reduced the size of the C.finii DNA insert to about 3.6 kbp.  Subsequently, the 3’ end of the gene was subcloned from pTZ-JC1 into pTZP-cenC to give pTZ-JC2 (Fig. 7, Coutinho et at., 1991). Fig. 13 (top panel) shows the expression of pTZ- 1 8R as the control (with no insert), pTZP-CenC and pTZ-JC2 respectively. While the level of expression of CenC as  43 Figure 11. Consensus sequences around the DAGD and H-R peptides in subfamilies El  and E2. Symbols as for Fig. 9. 0 and B in the amino acid sequences denote (aromatic amino acids and histidine) and basic amino acids, respectively. Enzymes names are as in Fig. 10.  44  SUBFAMILY El:  WrCDYRLDVSGGWYDAGDffGK’xVVNGGIAV 486  CfCenC:  457  PfEndA:  186” GTCNYSLNVTKGWYDAGDf-{GKYWNGG ISV 215”  CCCe1D:  184” NGQHKK1DSTKGWHTYGDYNKYVVNGITV 213”  BfCed:  132” GEDVEPVDVTGGWHDGDYGRYSTAGAVAV 161” CWODAGEO-BY  El CONSENSUS  V  SUBFAMILY E2: 42  CfCenB:  71  ADVGDLTGGWDGDHVKFGFPMAFSA **  *  94”  65” 1XSSYNVDLVGG1fDAGLLKFGLPMATr  PaCe 1:  *  **  *  ...*_..  .  D270 -6:  68” KNGIYNLSGGYFD?GDVKFGLPMAYSM  97”  CtCe1Z:  60” LX3ADVGLDLTGGWYDGDHVKFNLFMAYSQ  96”  L-GG000AGD--KF--PMAO--  E2 CONSENSUS  GOODaGO-  E1/E2 CONSENSUS  SUBFAMILY El:  CfCenC:  778  GAIQSYGEVASHQQHSIFAHQLDPSLPSPPPGSLGG 822 *  502” C  CtCe1D:  495” GRNTfNRS •  BfCed:  TGLGINPPMNPHDRR-SGAtX--IWEPWPGYLVGG 536” •..  .  453” GCNSMDISY  El CONSENSUS  ALl SNYPPPGALSGG 546”  STFITGtJ3TAQP  PfEndA:  *  .  *  .  ONGEKAFPHLRP-TAVDD-- IPWPGLVSGG 494” PG---GG  H-R  G-N----SO-TG-G  SUBFAMILY E2:  G4PRSSSYVVGFCANPPTAPNHRTAHGSWLDSITPAQSRHVLY 400  CfCenB:  356  PaCel:  390” GQNPA  t2 70-6  369” C  CtCe1Z  379” ALSSGRSYVVGFGVNPPUPHHRTAHSSWADSMSVPDYHPHVLI 422”  •  •  . .  ..  . .*. • • •  .  .. .  •**  *  ...  PNQQSFVGMGPNYPIPH  E1/E2 CONSENSUS  SO-VG-G---P---HHP.  SO--G-G  *  .  *  *  AAHS1TNDI1NWNLYLLK 413” *..  E2 CONSSUS  *  *  SYMVGFGERYPQHVHHRGSSLPSVQVHPNSIPQAGFQ 434”  H-R  •  *  *  .  45  Figure 12. Family E of 13-1 ,4-glycosidases. The numbers refer to the positions of amino acids.  i’’’’  subfamily E2; repeated sequences;  catalytic domains of subfamily El.  “‘‘i  catalytic domains of  sequences common to both types of catalytic domain; linker sequences;  ‘  cellulose-binding domains.  CfCenC, PfEndA, CtCe1D and BfCedl as in Fig. 8. CfCenB, endoglucanase B of C. fimi (Meinke et a!., 1991a). CsCe1Z, endoglucanase Z of Clostridium stercorarium (Jauris et al., 1990). Dd270-6, spore germination specific protein of Dictyostelium discoideum (Giorda et at., 1990). PaCel, endoglucanase of Persea americana (Tucker eta!., 1987).  PaCel  Dd270-6  CsCeIZ  CfConB  BfCod  CICoID  PEndA  CfCenC 150  I  300  [  810  380  I  35  rA%%.  40  ireeezsj.  40  820  •  I  520  465  LW////AIi  45  rF4W///A1I  105  150  150  490 980  680  935  I H  1069  956  1012  47 Figure 13. Expression of CenC in E. coli. Top panel. Arrows indicate the location of CenC in JM1O1/pTZP-CenC and JM1O1/pT’Z-JC2 on a Comassie stained SDS-PAGE gel. JM1O1/pTZ-18R (no insert) was used as control. Cultures were grown in the absence of inducer IPTG at  300  C. Cells were harvested and fractionated as described previously  (Moser, 1988). and the fractions were analyzed by SDS-PAGE. pf: periplasmic fraction obtained by osmotic shock (Nossal and Heppel, 1966); cf: cytoplasmic fraction (obtained by French press of osmotically shocked cells) and  Ce:  whole cell extract (obtained by  French press of whole cells). Bottom panel. Western blot of identical gel probed with anti CenC serum showing a large number of degradation products, specific to CenC, as these were not present in the fractions of the control culture JM1O1 carrying the plasmid pTZ 18R (Pharmacia) with no insert.  48  pTZP  18R pf  ci  ce  p1  ci  JC2 ce  pt  ci  ce  kDa 205 116  66  36 29  WESTERN  BLOTIAntI CenC  —  -m  —  — — —  —  — —  —  .-  —  49 observed on SDS-PAGE gels of crude cell extracts, was high, most of the CenC accumulated in the cytoplasm in an inactive form (Moser, 1988). Furthermore the polypeptide was very unstable as depicted by the Western blot of an identical gel (lower panel) probed with anti-CenC serum. In order to improve the expression a number of physiological parameters were tested which included: a) altering the growth temperature Fig. 14 (lanes 1, 2 and 3 represent CenC purified from cultures grown at 25, 30 and 37° C respectively. b) the absence of the inducer TPTG or its addition at A600 of 1.0 Fig. 14 (lanes 4 and 5 respectively) and c) the age of the inoculum (lane 6 represents the initial starter culture grown for less than 3 hours and A600 less than 0.4 and lane 7 represents the initial starter culture grown overnight). Lanes 8 and 9  represent  CenC and CenC purified fromCfimi.  Despite the difficulty in obtaining reproducible results for the expression of CenC, there were three physiological parameters that were ascertained over the course of these experiments that favoured the expression of CenC (i) Growth of the culture at 30° C (ii) The absence of the inducer IPTG and (iii) The use of a fresh starter inoculum. These conditions were used for all subsequent purifications. Two additional problems in the purification of CenC were that some of the degradation products of CenC also had catalytic activity  (much of the activity remained in the cell extracts even after Avicel  binding) and furthermore despite the amount of intact protein seen in crude cell extracts and the ability of the antibody to recognize the polypeptide, most of this material did not bind Avicel. The yields of purified CenC from a 10 1 culture varied from 20 j.ig to 100 .tg and this agreed well with previous results obtained in the lab (Moser Ph.D. thesis, 1988).  50 Figure 14. Production and purification of CenC from E. coli grown under different physiological conditions. Comassie stained, SDS-PAGE gel of the fraction from whole cell extracts, remaining bound to Avicel and desorbed with water. The location of full length CenC is indicated by an arrow. The lower bands are degradation products of CenC which retain the capacity to bind Avicel. Lanes 1, 2 and 3: Effect of growth temperature,  25, 30 and 37°C respectively; Lanes 4 and 5: Effect of the absence or presence of the inducer IPTG, Lane 4, IPTG and 5, -  +  IPTG; Lanes 6 and 7: Effect of the age of the  starter culture, Lane 6, starter culture grown for 3 hours, Lane 7, starter culture grown overnight. Lanes 8 and 9: C3.2 and C3.1 are native forms of CenC purified from C.Jlmi.  kDa  1  2  3  4  5  6  789  205  .-  c_-  116 -.  66  I.  --  51  3.3. 5’ and 3’ deletions of cenC Sequence comparisons with other cellulases suggested that the central domain (amino acids 300-880) was responsible for catalytic activity. Attempts were made to try and see how much of the the N-terminal and C-terminal repeats could be deleted without loss of catalytic activity as assayed by CMCase activity on a Congo red plate. Fig. 15 depicts the results obtained for the N-terminal deletions. Fig. 16 depicts the results obtained for deletions made in the C-terminal repeats of cenC. The properties of the C-terminal deletion mutants (Fig. 16) revealed deletions of amino acids 954-1004 and deletion of amino acids 1022-1069 did not result in a loss of CMCase activity. In order to test if the deletion mutants could still bind to cellulose and Sephadex, the two constructs, CenC CAl and CenC CA2 were chosen for further study. A three step purification protocol employing cellulose affinity, anion-exchange and gelfiltration chromatography was used to purify whole CenC and the two C-terminal deletions from their breakdown products. Fig. 17 shows the polypeptides obtained after affinity chromatography (CB: cellulose binding). The lower bands are breakdown products of CenC which still have the capacity to bind cellulose. Figures 18 and 19 show the active fractions obtained, after FPLC assisted ion-exchange chromatography on a Mono-Q anion-exchange column followed by gel-filtration on a bio-gel matrix with a exclusion of 150 kDa, for CenC. Similar results using this purification protocol were obtained for the two C-terminal deletions as well (Data not shown). Lanes 1-8 show (Fig.18) active fractions eluted from the Mono-Q column using a linear increasing salt gradient. A further purification step  52 Figure 15. N-teririinal deletions of CenC. CMC activity: Carboxyrnethylcellulase activity  was detected qualitatively, by Congo red staining, and then looking for the absence or presence of halos around the colony. Dotted lines refer to internal deletions obtained by Nuclease Bal. 31 deletions. Numbers refer to the amino acid sequence of mature (processed) CenC.  Cl  N2  Ni  CMC Activity  C2  CenC  + 886  1069  CenC N1  + 1  38138  300  886  1069  300  886  1069  CenC NA2 1 28  CenC Nz3  167  [:ZZZZ__________  1 11  498  886  1069  53 Figure 16. C-terminal deletions of CenC. Captions as in Fig. 15. Deletion I was obtained as a result of the initial cloning of CenC and deletion 3 was constructed by insertion of the omega interposon. The internal deletion, deletion 2, was constructed by excision of a  KpnI-KpnJ fragment as described in materials and methods.  Ni  N2  Ci  CMC Activity  C2  CenC  + 1  CenC CAl  300  —% 1  300  886  1069  + 886  1022  +  CenC CA2 1  300  886 / \ 1069 954 1004  CenC CA3 300  897  54 Figure 17. Cellulose-affinity chromatography of CenC, CenC CAl and CenC CA2, analyzed by SDS-PAGE. CB: cellulose bound. The location of CenC and its deletions are shown by an arrow. M: molecular mass marker. Lanes I and 2: CenC and CenC’ purified from C.fimi. Lanes 3,4 and 5 are fractions of CenC, CenC CAl and CenC CA2 obtained after binding cell extract to Avicel followed by washing of bound material with high salt and desorption with water.  kDa  M  I  3  2  205 116  U  29  CB  4  5  55 Figure 18. SDS-PAGE of CenC purified by FPLC assisted anion exchange chromatography on Mono-Q. The location of CenC is shown by an arrow. Lanes 1-8 are fractions of active CenC eluted by an increasing linear salt gradient. Mono-Q: Anion exchange matrix (Pharnacia).  kDa  1  2  3  4  205 116  7  6 ::-  66  41  5  -R—-  MONO..Q  —  8 —  56 involving gel—filtration allowed the  separation  from other contaminatin degradation  ot the top band (Fig. 19. lanes 1. 2 and 3)  products  (Fig. 19. lanes 4  —  ). Fig. 20 shows the  tinal products obtained for all three polypeptides. Polvpeptides encoded by both C— terminal deletion mutants retained the ability to hind both Avicel and Sephadex indicating that the  presence of  much of the C—terminal repeats (either amino acids 954—1004 or amino  acids 1022—1069) are not essential br bindin to these substrates.  57 Figure 19. SDS-PAGE of CenC after gel-filtration on P-150. Lanes 1-8 are fractions of active CenC eluted from the gel-filtration column. P-150: Polyacrylarnide bio-gel, 150,000 mol. mass. size exclusion matrix (Biorad). The location of CenC is shown by an arrow.  kDal 205 116  2345  678  —  —  66  41 29  P. 150  —.  —  58 Figure 20. SDS-PAGE analysis of intact CenC (Lane 1) and its two C-terminal deletions CenC CM (Lane 2) and CenC Cz\2 (Lane 3). M: Molecular mass marker. The location of CenC and the two deletion derivatives are indicated by an arrow.  kDa 205 116  66  Ml  2  3  59 3.4. Kinetic parameters for native and recombinant CenC The kinetic parameters for hydrolysis of CMC by native and recombinant CenC are presented in Table 2A.and compared to those of CenA and CenB. CenC is the most active component of the C.fImi cellulase system isolated to date (Moser et a!., 1989; Coutinho eta!., 1991). The hydrolysis of 2-chloro-4-nitrophenyl cellobioside by native CenC is compared to Ce1D (endoglucanase from Clostridiurn therrnoce!lum ) and are presented in Table 2B. The oligosaccharide cleavage patterns for CenC on the paranitrophenol derivatives: 2-chloro-4-nitrophenol lactoside; 2-chloro-4-nitrophenol cellobioside (pG2), trioside (pG3) and tetraoside (pG4) and on the cellodextrins G3-G6 (detected by HPLC) are shown in Fig. 21. The lower panel shows a sample chromatograrn for G6 cleavage in which the sample was injected onto the HPLC column, immediately after enzyme and substrate were mixed together. pG2 and the 2-chloro-4-nitrophenol lactoside were cleaved at Gi but pG3 and p04 were not. The addition of cellobiose to the reaction mixture inhibited the cleavage at Cl of pG2. CenC gave several products from cellohexaose and cellopentaose. The generation and interconversion of the anomeric forms of G3 indicated that CenC cleaved the 13-1 ,4-glucosidic bond with inversion of the anomeric configuration. The theoretical cx-OH: 13-OH ratio at G3 after hydrolysis of cellohexaose or cellopentaose should be greater than 1 shortly after cleavage (before the two anomeric forms reach equilibrium) if it is an inverting enzyme and less than 1 if it is a retaining enzyme. The measured ratio was 2.2 indicating the change in configuration from  f3 to cx. Racemization  is complete within about 40 minutes and the measurements were made at intervals between  5 and 20 minutes (by integrating the area under the peak). CenC cleaved cellotetraose preferentially to yield cellobiose. G3 was hydrolyzed to G2 and 01 but the preferred point of hydrolysis (Cl or C3) could not be deten’nined by HPLC. The temperature and pH optima of CenC on CNPC were 45CC and pH 5.0 respectively (Fig.22).  60  TABLE 2A. Kinetic parameters for hydrolysis of CMC by native and recombinant endoglucanases.  Enzyme  Source  CenC” CenC’ CenC CenC’ ±CenA CenB 0  C.fimi C.fimi E. coli E. coli E. coli E. coil  *Km  0.15 0.10 0.13 0.30 0.17 0.51  ± ± ± ± ± ±  0.02 0.02 0.03 0.03 0.10 0.05  12.33 11.39 12.69 14.86 2.76 2.87  ± ± ± ± ± ±  0.21 0.31 0.44 0.30 0.05 0.12  Initial velocities for the production of reducing sugars at 30°C were determined with HBAH over a substrate range of 0.8 to 13.0 Km. Values were derived by weighted linear regression analyses of Lineweaver-Burke plots. *Km values: mgCMC/ml +Vm values: imoles reducing sugar produced per nmole protein. “The author acknowledges the help of B. Moser for these measurements. ± From Langsford eta!, 1984. o J.B. Owolabi, personal communication. TABLE 2B. Kinetic parameters for hydrolysis of CNPC by CenC and Ce1D Enzyme  Km (riM) Kcat (s1) KcatlKm (s’M’)  CeLD  98  7.9  80,000  CenC  479  60.6  126,600  Activity measurements were performed at 2YC, pH 5.0 with CNPC (100-1000 pM) as the substrate. Kinetic parameters were derived from (S/V) vs (S) plots (Hanes, 1932).  The values for Ce1D from Clostridiuni therniocellum were reported previously Tomme et al., 1991).  61 Figure 21. Oligosaccharide cleavage patterns of CenC for various cellodextrins. Cleavage of the p-nitrophenol conjugated cellodextrins were followed by the absence or presence of color. Cleavage sites for the non-conjugated cellodextrins was monitored by separation of the cleavage products by HPLC and the separated products monitored by a diffractometer. A sample chromatograrn showing the separation of the cleavage products, including the separation of the alpha from the beta anorner for cellodextrins G3 and G4, on the HPLC is presented in the lower panel.  62  EEEFLEJEEI L-L-L-L1 •  p-nitrophenol glucose residue  A  galactose residue f3-1 ,4-glucosidic bond cleavage site  6.00  0  5.00  G3  x G4 4.00  3.00  -  0.50  1.50  1.00 :<  11j  .Ofl L  2.5u  63 Figure 22. Temperature and pH optima for the hydrolysis of chioronitrophenylcellobiose CNPC at Cl by native CenC. The reaction,  monitoring  the release of phenol, was  followed spectrophotometrically at an absorbance of 400 nrn. The enzyme had a temperature optimum of 45CC and a pH optimum of 5.5  64  30  -  E 20 E > 10  0— 3.0  5.0  4.0  6.0  7.0  8.0  9.0  pH  160  —  120  C  80  -  > 40  020  30  40 Temp (C)  50  60  65 3.5. Expression of fragments of the cenC gene. The N-terminal repeats of CenC were designated Ni (amino acids 1-148) and N2 (amino acids 149-296); the C-terminal repeats were designated Cl (amino acids 886-975) and C2 (amino acids 976-1069). Amino acid sequence comparison revealed that the Nterminal repeats (N1N2) were distantly related to the cellulose-binding domains of Cex, CenA and CenB (Fig. 29). Sequence comparisons of the C-terminal repeats (C1C2) did not reveal any obvious siniilarites to any other cellulase sequences reported to date. Deletion mutants of the cenC gene were constructed which encoded the leader peptide plus either Ni (amino acids 1-150), N1N2 (amino acids 1-299), Cl (amino acids 860-993) or C1C2 (amino acids 860-1069) (Fig. 23). Polypeptides corresponding to Ni and N1N2 were detected on Western blots of fractions from cultures of E. coil JM1O1 carrying plasmids pTZ-JC6 and pTZ-JC3, respectively, using anti-CenC serum (Fig. 24). The low molecular mass band seen in cell extracts of E. coil JMIO1/pTZ-JC3 was also seen in the E. coil JM1O1 pTZ-18R control cell extract and was considered to represent the non-specific interaction of the antiserum with an E. coil protein. Polypeptides Cl and CIC2 reacted poorly with anti CenC’ serum (data not shown); however they were detected as unique bands by SDS-PAGE in appropriate E. coli extracts following induction with IPTG (Fig. 25). The extinction coefficients for N1N2 and Ni as determined by UV absorbance (Scopes, 1974) were 1.06 and 1.10 respectively, in good agreement with the values predicted from the tryptophan and tyrosine content (Cantor and Schimmel, 1980) of the proteins (1.21 and 1.20 respectively). The apparent molecular masses of N1N2 and Ni (20.0 and 40.0 kDa, respectively) were significantly greater than those predicted from their corresponding DNA sequences (15.4 kDa and 30.4 kDa) (Fig. 26). The predicted pT’s of Ni and N1N2 (3.54 and 3.48, respectively) showed that both  are  highly acidic polypeptides and this  may account for the discrepancies between their predicted and apparent molecular masses. Similar considerations apply to intact CenC (Coutinho  ci’  at., 1991).  66 Figure 23. Polypeptides encoded by the various plasmids. The plasmid encoding CenC or the CenC polypeptide is shown in parenthesis. The locations are indicated of the restriction endonuclease sites used to construct the various plasmids. repeats;  ,  ,  N-terminal  C-terminal repeats. The numbers refer to the amino acid residues,  starting from the N-terminus of mature CenC (Coutinho et al., 1991). The transcriptional and transalatiorial stops were from interposon omega and the promoter used was the lac promoter.  Smal  Sad  Apal  //  CenC (pTZ-JC2) 1  148  1  150  300  N1N2 (pTZ-JC3)  Ni (pTZ-JC6)  Cl C2 (pTZ-JC7) 860  975  860  993  Cl (pTZ-JC14)  1069  MM  Apal’  \/ 886  975  1069  67 Figure 24. Intracellular and extracellular distribution of polypeptides N1N2 and Ni synthesized in E. coli JM 101 by Western blotting. Polypeptides from cell extracts (ce) and culture supernatants (cs) were analyzed by SDS-PAGE followed by Western blotting and detection with polyclonal anti-CenC’ serum. Plasmids pTZ-JC3 and pTZ-JC6 encode N1N2 and Ni, respectively; pTZ-18R is the control plasrnid, without cenC DNA. Ni was detected wealdy by the antibody. The low molecular weight band seen in cell extracts of  E. coil JMiO1/pTZ-JC3 was also seen in theE. coil JM1O1/pTZ-18R and was considered to represent the non-specific interaction of the antiserum with an E. coil protein.  JC3  JC6  cecscecs  18R  ce  68 Figure 25. Intracellular and extracellular distribution of polypeptides C1C2 and Cl synthesized in E. coli JM1O1. Polypeptides from cell extracts (ce) and culture supernatants (cs) were analyzed by SDS-PAGE, 6h after induction with 0.1 mM IPTG. The left lane of each pair corresponds to induction when the A600 of the culture supematant reached 0.4; the right corresponds to induction when the A600 reached 1.0 Polypeptides C1C2 and Cl, indicated by arrows, are encoded by pTZ-JC7 and pTZ-JC14 respectively. Molecular mass markers are shown in the far left lane.  JC7 kDa  Cs  JC14 ce  Cs  ce —  -  116 66  36 29  4’  I•  “  69 Figure 26. Purified N1N2 and Ni obtained by affinity chromatography. A. Polypeptides from E. coli JM1O1 cell extracts (ce) and culture supernatants (cs), and the purified polypeptides (pp) analyzed by SDS-PAGE. Plasmids pTZ-JC3 and pTZ-JC6 encode N1N2 and Ni respectively; pTZ-18R is the control plasmid, without cenC DNA. N1N2 and Ni were purified by affinity chromatography on Sephadex G-50 and Avicel PHiO1, respectively, as described in Materials and Methods. B. SDS-PAGE analysis of 10, 20  and 40 .tg of Ni purified from E. coli JM1O1 (pTZ-JC6), and 10 and 40 jig of N1N2 purified from E. coli JM1O1 (pTZ-JC3), to evaluate levels of contamination with other polypeptides. The far left lane of each panel shows the molecular mass markers.  70  18R ce  kDa  JC3 Cs  JC6  pp  cs  pp  97 66 41 29  A  kDa 97 66  JC3  JC6  — .  *.  41 29  —  j  -—  . B  71 The apparent molecular masses of Ci and C1C2 (16.0 and 28.0 kDa, respectively) agreed well with those predicted from their corresponding DNA sequences (16.2 and 26.4 kDa, respectively); their predicted p1’s are 5.07 and 8.76, respectively. N-terminal sequence analysis revealed that Ni, N1N2, Ci and C1C2 all had the same N-terminus (ASPIG), as expected (Fig. 23). Ni, N1N2, Ci and C1C2 were devoid of detectable carboxymethylcellulase activity (Data not shown).  72 3.6. Binding of the N- and C-terminal repeats to cellulose and to Sephadex. Polypeptides N1N2 and Ni (Fig. 26 A) both bound to Avicel (a heterogenous microcrystalline cellulose preparation containing both crystalline and non-crystalline regions), but only N1N2 bound to Sephadex G-50. Neither C1C2 nor Ci bound to Avicel or to Sephadex G-50 (data not shown). N1N2 and Ni were purified to virtual homogeneity by affinity chromatography on Avicel or Sephadex G-50 (Fig. 26B). The binding specificities of N1N2 and Ni for two different cellulose allomorphs were investigated by determination of their adsorption to PASC and to BMCC. The adsorption of the CBD of Cex (CBDCex) was also included for comparison. CBDCex (11.1 kDa) had an extinction coefficient of 2.30, in agreement with its predicted value (E. Ong, Ph.D. thesis). Adsorption of the three polypeptides to the two forms of cellulose is described by isotherms bound [B] vs free [F]) shown in Fig. 27. The partition coefficient (the initial slope of the adsorption isotherm) was determined as a measure of the relative affinities of Ni, N1N2 and CBDCex for a particular cellulose allomorph (Table 3). All three polypeptides adsorbed to regenerated cellulose (Fig. 27). The partition coefficients for N1N2 and NI for regenerated cellulose (Table 3) were significantly different (approximately three fold lower) than the corresponding values for CBDCex with a level of confidence of> 95% (the difference in the partition coefficients between the two means was greater than 2.5 standard deviations). Saturation of regenerated cellulose was not obtained with Ni, N1N2 or CBDCex at the highest polypeptide concentration tested. In contrast to the adsorption of N1N2 and Ni to regenerated cellulose, there was no apparent adsorption of either polypeptide to bacterial crystalline cellulose; however, CBDCex adsorbed well to this substrate (Fig. 27, Table 3). There was no detectable binding of the C-terminal repeats (Ci C2 and Ci) to either substrate confiririing what was observed earlier, in that C-tenriinal deletions of CenC, retained both catalytic activity and binding to cellulose and to Sephadex.  73 Figure 27. Adsorption isotherms for N1N2, Ni and CBDCex. Adsorption of the various polypeptides to PASC (Panels A,B,C (i) ) or BMCC (Panels A,B,C (ii)) was determined as described in Materials and Methods. [F] is the free polypeptide concentration (tiM) and [B] is the bound polypeptide concentration (jimol per g cellulose) at equilibrium. PASC refers to phosphoric acid swollen cellulose and BMCC refers to bacterial microcrystalline cellulose.  A(ii)  A(i) 60  60  q) 0  E 0  aS  0 -PASC CBDc  50 40 30  40  .  .  .  30  •.  20  I  fO  0 200  150  100  50  0  0  300  250  0  0  0  10  0  0  0  20  ••  10  CB0X -BMCC  50  20  40  60  80  100  B(ii)  B(i) 20  20  NIN2-BMCC  N1N2-PASC 15  15 0  E  10  10  CS  a-  ...  5  .  .  .  .  . 5  ••• 0  0 20  0  80  60  40  100  0  20  60  40  80  100  C(ii)  C(i) 20  20  N1-PASC  N1-BMcc  15  15 0  E  :i  10  .  .  10  0  CS  a-  5  ••  •  5  I  0 0  0 50  100  [P] jiM  150  200  0  50  100  [P] iM  150  200  74  Table 3. Partition coefficients of N1N2, Ni and CBDCex for PASC and BMCC. PASC refers to phosphoric acid swollen cellulose and BMCC refers to bacterial microcrystalline cellulose.  Polypeptide  Partition Coefficient ) 1 (L.g PASC  BMCC  CBDCex  0.598  0.967  N1N2  0.203  0.001  Ni  0.236  0.001  l1 nitial slope of adsorption isotherm plot ([P1 ad VS. [P1).  75 3.7. Sequence relatedness between Ni, N2 and other CBDs  f3-1,4-glycanases can be grouped into families according to amino acid sequence relatedness within their catalytic domains (Henrissat et at., 1989; Beguin, 1990; Gilkes et a!., 1991a). As more amino acid sequences are deduced from the nucleotide sequences of genes, it is becoming clear that there are also families of CBDs (Gilkes eta!., 199 1a. At present, however, some of the families contain only one or two members. The CBDs within a family are quite uniform in size and amino-acid sequence, but there are considerable differences between the families (Table 4). Since the catalytic domain families are designated by letters, a different designation should be used for the CBD families; Roman numerals are used in this study (Coutinho et a!., 1992). Ni and N2 of CenC, and the T. reesei EglIl sequence, could be in a sub-family of family II (Table 4, Fig. 28). Although there is little overall sequence identity between Ni, N2 and the other CBDs, all of them contain two cysteines —iOO amino acids apart, with aromatic, aliphatic and hydroxyamino acids at the same or similar locations between them (Fig. 29). However, the other CBDs are —l06 amino acids long, whereas Ni and N2 are —150 amino acids long. The dendogram also suggests that the D. discoideurn 270-il sequences a, b are in a third sub-family (Fig. 28).  Microbisporn bis porn Pseudornonas fluoresceus subsp. cellulosn P. fluorescens subsp. cellulosa P. fluorescens subsp. cellulosa P. fluorcsccns subsp. ccllulosa Therruornonosporn fusca T. fuscn T. reesei  Cellulo,nonns finvigena Dictyostelium discoideum  Butyrivibrio fibrisolvens Cellulomonas firni C. firni C. firni C. fimi  II  Organism  Hurnicoin grisen var. thermoidea Phanerochnete chrysosporium Trichoderma reesei T. reesei T. reesei T. reesei Trichoderma viride  -  Families of cellulose-binding domains  I  Family  Table 4  95 106 103 106 148 148 106 98 106 100 100 102 101 99 86 103 90  EgIB XynA XynB/C 2 E 5 E EglIl  36 35 36 36 36 36 36  End! CenA CenB Cex CenC NI • CenC N2 C1fX 270-Ha 270-lIb CeIA EgIA  Cbhl CbhI CbhI CbhII EglI EglIl CbhI  Enzyme Amino acids*  N N N C N I  C N C C N I C C I C C  C C C N C N C  +  +  +  +  +  +  +  +  +  +  +  17 18 19 20 20 6,21  15 16  13 14  8 9 10 11 12  1 2 3 4 5 6 7  Terminus Binding# Reference  ,  ,  ,  ,  ,  EgZ  CelE  ,  End End End CeIB CenB Ce1ZC Ce1ZC  ,  63  240  132 132 132 136 131 144 133  ,  ,  Enzyme Amino acids*  ,  ,  C  1  C C C I I I C  ,  +  +  +  +  ,  31  29,30  22 23 24 25,26 27 28  Terminus Binding# Reference  ,  ,  ,  ,  ,  ,  *Nljmber of amino acids in binding domain; ±C and C are in a 50 kDa Iragment el CeIZ which binds cellulose hut is catalytically inactive; # binding of the domain to cellulose has actuaHy been demonstrated; Othe binding domain is somewhere within a sequence of 240 amino acids. N: binding domain is at the N-terminus; C: binding domain is at the C-terminus; I: binding domain is internal. References: (1) Azvedo ci at. 1990 (2) Sims ci at. 1988 (3)Shoemaker ci at. 1983 (4) Chen ci at. 1987 (5) Penttila ct at. 1986 (6) Teen et at. 1987 (7) Cheng ci at. 1990 (8) l3erger ci (it. 1989 (9) ‘vVong ci at. 1986 (10) Meinke et al. 1991a (11) O’Neill ci al. 1986 (12) Coutinho c a!. 1991 (13) Al Tawheed, 1988 (14) Giorda et a!., 1990 (15) Yablonsky et at. 1988 (16) 1-lall and Gilbert, 1988 (17) Gilbert ci a!. 1990 (18) Hall et al. 1989 (19) Kellett et al. 1990 (20) Lao et a!. 1991 (21) Meinke ci a!. 1992 (22) Robson and Chambliss, 1986 (23) Nakamura ci a!., 1987 (24) Mackay ci a!. , 1986 (25) Saul et nI., 1989 (26) Saul ci at., 1990 (27) Meinke et at. 1991b (28) Jauris et al, 1990 (29) Hall ci at. 1988 (30) Durrant eta!. 1991, (31) Py ci a!., 1991.  Erwinia chrysanthemi  V  therrnocel!urn°  Clostridium  Bacillus subtilis DLG B. subtilis N-24 B. subtilis PAP115 Caldocellurn saccharblyticum C. fimi Clostridiurn stercorariurn+  Organism  Families of cellulose-binding domains (cont’d)  IV  III  Family  Table 4  78 Figure 28. Dendogram of Family II CBDs. Complete sequence data are contained in the references listed in Table 4 (Bfi, B.Jlbrisolvens: Cfi, C.fimi; Cfl, C.fiavigena; Ddi, D.  discoideum; M bi, M. bispora; Pfl, P. fluorescens su bsp. cellulosa; Tfu, T. fusca; Tre, T. reesei.).  Cfi CenC Ni Cfi CenC N2  Tre Egi II Cfi CenA  Mbi CeIA Cfl CenB Cfl CIfX  Cfi Cex 2 TfuE 5 Tfu E Ff1 EgIA Pfl EgIB  Pfl XynA PfI XynB  Ff1 XynC Sf1 End I Ddi 270-1 la Ddi 270-1 lb  79 Figure 29. Amino acid sequence similarities between Ni, N2 and other CBDs from C. Jimi. Boxes enclose three or more identical amino acids;• indicates similar amino acids.  The numbers at the ends of the sequences refer to the positions of those amino acids in the sequences of the mature enzymes. Amino acids said to be ‘similar’ are: A,S,T; D,E; N,Q; R,K; I,L,M,V; F,Y,W.  CeriCNi  33  CenCN2  181  C-V--DLPGGQGNPWDAGL-VYN_GVPVGEGESYV  4  CRVDYAVTNQWPGGFGANVTITNLGD_PVS_SWK  C-V--APAGSAQ-YGVG-VLN_GVAIEEGTTY  •  • •  CenA  •  F-i  • •  ••  Cex  341  C Q V L W G V  •  CenB  913  -  N Q W N T G F T A N V  • TI!IK  •  •  N T S S A P V D  •  -  -  C W T  •  CTVVYS-TNSWNVGFTGSVKITNTTTPLT___WT  •  •• CenC Ni  ••  •  •  L R Y T A T A S T DV R A L V G Q N G A P Y G T V L D  •  •  •  •  -  P 5 P A L  ••  •  CenC N2  L S F T A S A T P D M P V R V L V C E C G C A Y R T A F EG S A P L  CenA  L D W T Y T A G Q R I Q  Q L W N G T A S T N G G Q V  Cex  LTFSFPSGQQVT  QAWSSTVTQSQS  •  •  ••  •• CenB  •  •  •  •  •  •  •  T S E P R Q V T E P F  •  -  •  V  ••  QGWSATWSQTGT  • -  -  •  CenCN2  ••  •  LGFAFPSGQQVT  •• CenC Ni  •  -  •  •  V  ••  T A S A T Y P A T P A A D D P E C Q I A  •  •  TGEPATREYAF____TSNLT[P__PDGDAPGQ  CenA SVTSLPWNCSIPTGCTASFFFNGSWACSNPT_  A  PAS  Cex  TVRNAPWNGS IPACGTAQFGFNGSHTGTNAA- PT  CenB  TATGLSWNATLQPCQSTDIGFNGSHPGTNTN_ PAS  ••  • ••  •• CenCN1  FQLGGFSADAWFC  140  CenCN2  FHL-GK-AGAYEFC  285  CenA  FSLNG  TTC  103  TPC  440  EVC  1011  • Cex  FSLNG  • CenB  FTVNG  ••  •  •  • ••  80 3.8. Expression of N2 A deletion mutant encoding N2 was constructed by linearizing the fragment encoding N1N2 at the unique BamHI site followed by digestion with NucleaseBal3l (Fig.30). The polypeptide could be purified by affinity adsorption to Avicel (Fig. 31) but not to Sephadex G-50 (Data not shown). N-terminal sequence analysis revealed that N2 had the expected N-terminus (ASPIG) (Fig. 30). The polypeptide (17.3 kDa) had an extinction coefficient of 1.09 as determined by UV absorbance (Scopes, 1974) and in good agreement with the value predicted from the tryptophan and tyrosine contents (Cantor and Schimmel, 1980) of the polypeptide (1.18).  3.9. Adsorption of N1N2, Ni and N2 to PASC Fig. 32, Panel A shows the relative adsorption (pg/rng) of N1N2, Ni and N2 on PASC. The capacity of NIN2 and N2 to adsorb to PASC was greater than Ni. Panel B shows the free polypeptide in solution when N1N2 and N2 are compared with each other with N1N2 having a higher capacity to adsorb to PASC. Panel C shows competition between N2 and Ni with N2 having a slightly higher capacity of adsorption over Ni. The relative adsorption capacities of the three polypeptides in the presence of one another (based on Jig/mg of PASC) are N1N2  >  N2  >  Ni. Table 5 summarizes the results, using  a densitometer, to scan the SDS-PAGE gels for the relative amount of free polypeptide left in solution post-binding. It must be understood however that on a molar basis, because N1N2 is about twice the size of Ni or N2, it would have half the number of molecules. The assumption in the above experiment that the number of binding equivalents is the same for all three polypeptides, with N1N2 having 2 and Nl or N2 having 1, does not  take into account the fact that steric hindrance between the binding sites in N1N2 might affect the adsorption process. The experiment should be redone using equivalent molar amounts of the three polypeptides which would make up for the difference in the number of molecules. None of the Ni deletion derivatives bound to Avicel (Fig. 33).  81 Figure 30. Polypeptides encoded by the various plasmids. The plasmid encoding each polypeptide appears in parentheses. The locations of the restriction endonuclease sites used to construct the plasmids are indicated. The stippled regions indicate the N-terminal repeats. The dotted lines indicate the extent of deletion using Nuclease Ba131. The numbers refer to the amino acid residues of CenC.  N1N2 (pTZ-JC3)  Ni (pTZ-JC6) I  ISO  N2 (pTZ-JC34) 138  167  299  Figure 31. SDS-PAGE analysis ol purilied Ni NI and Xl N2 Polypeplides from E. .  uuii Thl 101 culture supernatants (cs) were puriIc1 b affinity chromatography on Avicel.The far left Jane shuw the molecular mass marlers.  kDa 43•  29  18 14  N2  Ni  N1N2  83 Figure 32. SDS-PAGE analysis of the adsorption of N1N2, Ni and N2 to 1 mg of (PASC). From left to right, increasing concentrations of polypeptide in paired lanes (Materials and Methods): Open circles are control samples incubated without cellulose and  run on a gel to determine the amount of starting material and closed circles are samples which were run after adsorption to cellulose to ascertain the amount of polypeptide remaining in supernatants after binding. Panel A: N1N2 vs Ni vs N2. Panel B: N1N2 vs N2. Panel C: N2 vs Ni. PASC: Phosphoric acid swollen cellulose. The far left lane shows the molecular mass markers. An estimate of the relative amount of free polypeptide remaining in solution, by densitornetric scanning of the SDS-PAGE gel, before and after binding, appears in Table 5.  A  o• o•o•  kDa 43  —  29  ___—  ——  —  —  — —  —  N1N2 N2 Ni  B O•O•O• k Da  —  —  29%  — —  N1N2  —  N2  -  —  —  C kOa  o•o•o•  29%  — •  —  —  __--  PASC  N2 NI  85  Table 5: Adsorption of N1N2, Ni and N2 to PASC. Free polypeptide (% of starting material) in supernatant after adsorption.  Polypeptide concentration [25]  [100]  [250]  % free polypeptide, after adsorptionb  Substrate  Polypeptide  Panel A N.D.C  20  60  N1N2  N.D.  45  90  N2  40  85  90  Ni  PASCd  Panel B PASC  N.D.  10  40  N1N2  N.D.  40  85  N2  Panel C PASC  N.D.  30  75  N2  25  65  90  Ni  a [Concentration] jig/mi b Amount in supernatant before adsorption was taken as 100% c None detected d Phosphoric acid swollen cellulose (regenerated cellulose)  86 Figure 33. Deletions of Ni. Restriction endonuclease sites used to generate the deletions are included. Binding: As detected by adsorption to Avicel PH 101. Dotted lines refer to the extent of each deletion. NI Al and NI A2 were created by truncation at Miul and BstEll respectively using the 2 fragment. NI A3 and Ni A4 were created by digestion with BaniHi followed by treatment with Nuclease Ba131. Numbers refer to the amino acid residues of mature (processed) CenC.  Binding  Ni (pTZ-.JC6)  + — — —  N1M 1  133  150  N1A2 102  1  N1A3  j3  N1A4 1  34  150  150  I  64  150  87 3.10. Consequences of substituting N1N2 for the CBD of endoglucanase CenA Fig. 34 shows the cloning strategy for fusing the sequence encoding the catalytic domain of cenA with the sequence encoding the cellulose binding domain of cenC resulting in plasmid pTZ-JC13 which encodes CBDCenC CatCenA (C’ ‘A). The mutant -  enzyme retained both the binding properties of the CenC binding domain as well the catalytic activity borne by the CenA catalytic domain. N-terminal sequencing of the polypeptide confirmed the expected N-terminal sequence (ASPIG). The extinction coefficient (1 cm, 1 mg i1, 280 nm) for the polypeptide was determined by far UV absorbance. The value of 2.12 obtained was in good agreement with its predicted value of 1.81 (Scopes, 1974). Results with N1N2 showed that the CenC CBD had negligible affinity for BMCC. To test if the same held true for C’ ‘A, a mixture containing CenA (non-glycosylated CenA produced in E. coil) and C’ ‘A were tested for binding to BMCC and then to PASC. Fig. 35A shows the removal of CenA from a mixture of CenA and C’ ‘A when BMCC is used as the adsorbent followed in panel B by the removal of both CenA and C’ ‘A when PASC is used as the affinity adsorbent. These results confirmed that the binding domain of CenC retained its specificity (negligible affinity for BMCC) in the fusion polypeptide C’ ‘A. The relative amount of free polypeptide remaining in solution (determined by scanning the SDS-PAGE gel) before and after binding is given in Table 6. The activities of CenA on a range of substrates were compared to those of C’ ‘A (Table 7). CenA produced in E. co/i is sensitive to hydrolysis in its linker region and is converted into a 30 kDa fragment (p30) which has catalytic activity but lacks the binding domain (Gilkes et a!., 1988). The activity of p30 against the soluble substrate CMC was higher than that of either C’ ‘A or CenA. Higher activities for p30 were also observed against PASC and cellulose azure. The activity of p30 against Avicel was lower than that of C’ ‘A or CenA. The activity of C’ ‘A on Avicel was significantly higher than that of  88 Figure 34. Construction scheme for domain swapping of CBDCenC (NIN2) with CBDCenA. Plasmid pUC18-1.6 cenA encoding polypeptide CenA was digested with  HpaI and Hind!!!. Plasmid pTZ-JC2 encoding polypeptide CenC was digested with Sma! and Hind!!!. The fragments were isolated, then ligated and the plasmid encoding polypeptide C ‘A, designated pTZ-JC13. PT 23 refers to the 23 amino acid PT linker in CenA and PPP refers to the triproline linker in CenC.  Hpal Smal Hind Ill Hind Ifl  Isolate small fragmect  soata large fragment  T4 DNA Ligase  .PPP rer  •TGA  89 Figure 35. Adsorption of CenA and C’ ‘A to BMCC and PASC. Panel A. Increasing amounts (paired lanes) of purified CenA and C’ ‘A were incubated without BMCC  (-) (as  controls, to determine the amount of starting material) or mixed with 1 mg of BMCC (+) to ascertain the amount of free polypeptide present in solution, post-binding. CenA was preferentially adsorbed by BMCC from a solution bearing both CenA and C’ ‘A. Panel B. Samples after the adsorption lanes  experiment to  BMCC were rebound to PASC. Paired  (-) and (+) refer to incubations without or with PASC. Both CenA and C ‘A  adsorbed to PASC. The far left lane in each panel shows the molecular mass markers. BMCC: Bacterial microcrystalline cellulose. PASC: Phosphoric acid swollen cellulose (Regenerated cellulose).  90  BMCC  A kDa_  -  +  +  -  +  -  116  C’ ‘A 66 43  Ce n A  —  —  —  36  PASC  B kDa  -  +  -  +  -  +  116  c”A 66  Cen A  36  91  Table 6: Adsorption of CenA and C’ ‘A to BMCC and PASC. Free polypeptide (% of starting material) in supernatant after adsorption.  Polypeptide concentration [25]  [1001  [250]  % free polypeptide, after adsorptionb  Substrate  Polypeptide  BMCCcI 100  100  100  C’ ‘A  N.D.C  15  30  CenA  10  50  80  C’A  N.D.  N.D.  5  CenA  PASC  a [Concentration] jig/mi b Amount in supernatant before adsorption was taken as 100% C  None detected  d Bacterial microcrystalline cellulose e Phosphoric acid swollen cellulose (regenerated cellulose)  4.5 ± 0.20 1.2 ± 0.05 1.0 ± 0.04  39.1 ± 0.4 31.2 ± 0.2 19.3 ± 0.6  3.6 ± 0.2 3.7 ± 0.2 4.6 ± 0.2  145.5 ± 1 327.8 ± 3 586.7 ± 4  22.5 ± 0.1 26.1 ± 0.4 33.2 ± 0.4  ngCenA C’ ‘A p30  aCai.boxyietlylcelft1lose bpljosphoric acid swollen cellulose. CBactei.jal microcrystalline cellulose. dkatal/mol, activity of I mol product/s/mo] enzyme. eUnits/nmiol, production of A595 nm = 1/h/mmol enzyme.  BMCCC millikatalJrnol  Avicel millikatal/mol  PASCb katal/mol  Cellulose azure units/mole  Activity on CMCa katal/mold  Enzyme  Table 7: Enzyme activity of CenA and C’ ‘A against a range of cellulosic substrates  93 p30 alone indicating that the CenCCBD did influence the activity of the fusion polypeptide.The results for CenA and p30 were similar to those obtained previously (Gilkes eta!., 1988; Shen eta!., 1991). The activities of C’ ‘A against the amorphous substrates PASC and cellulose azure were compared to those of CenA. C’ ‘A had identical activity on PASC however it had greater than twice the activity on cellulose azure. Another striking difference was the poorer ability of C’ ‘A to hydrolyze crystalline substrates. The activity of C’ ‘A was 20%  and 70% lower on Avicel and BMCC respectively as compared to CenA. CenA had the highest activities on crystalline cellulose, C’ ‘A had intermediate activity on crystalline cellulose and p30 had highest activity on soluble and amorphous substrates. In the cloning strategy in Fig. 34 the 23 amino-acid PT linker separating the CenA catalytic domain from its cellulose binding domain (which is sensitive to cleavage by C.  fimi protease to generate p30) was removed and replaced by a linker (PPP) directly following the N-terminal repeats (Fig.36). Fig. 37 shows the effect of increasing amounts of C.Jmnii protease on CenA and C’ ‘A. Removal of the protease site from C’ ‘A resulted in enhanced stability of the polypeptide to C.JIrni protease as compared to CenA.  94  Figure 36. Bar diagram indicating features of CenA and C’ ‘A. V indicates the location of the 23 amino acid PT linker in CenA and the PPP linker in C’ ‘A. TheC. fimi protease sensitive site in CenA is located adjacent to the PT linker. Cleavage of  CenA by C.fimi protease results in a P30 proteolytic fragment.  domain; E%1 CenC binding domain;  linker region; I  renA binding  1 CenA catalytic  domain.  PT linker V C. Ini pco(ease se  P30  CenA 1  112 134  418  PPP linker  C ‘A 1  Ni  150  N2  300  584  95 Figure 37. Hydrolysis of CenA and C’ ‘A with C.fInii protease. Both polypeptides were treated with 0 (Control: Starting material, untreated with protease), 0.1, 0.5 and 1.0 units of C.fimi protease and the products of hydrolysis analyzed by SDS-PAGE. P30 is a proteolytic fragment derived from the hydrolysis of CenA. The far left lane shows the molecular mass markers.  C”A  Cen A  k Da 116  66  43  —  —  p30  29  1.0  0.5  0.1  C. fimi  0  1.0  0.5  protease (U)  0.1  0  96  4. Discussion  Identities within the amino acid sequences of the catalytic domains of 13-1,4glycanases allow grouping of the enzymes into families (Knowles et a!., 1987; Henrissat  eta!. 1989; Béguin, 1990; Gilkes et al., 1991a). The enzymes of family E can be grouped into sub-families El and E2 (Béguin, 1990). Amino acids 300-880 of CenC contain the catalytic domain; this sequence places CenC in sub-family El (Fig. 10). Although the similarities between the sub-families are much less than those between enzymes within a sub-family, the sequence DAGD occurs in all members of the family, and may be part of the active site because carboxyl groups are involved in glycosidic bond cleavage (Sinnott, 1987; Chauvaux et at., 1992). In enzymes from sub-family El it is about 150 amino acids from the N-terminus of the catalytic domain, for those of E2 it is about 40 amino acids from this terminus (Joliff ci’ at., 1986a; Tucker eta!. 1987; Hall ,  and Gilbert, 1988; Berger ci’ a!., 1990; Giorda ci’ at. 1990; Meinke ,  Coutinho  eta!.,  et a!.,  1991a;  1991). The common sequence H-R, —320 amino acids away, may also  be important for catalytic activity. Residues near its C-terminus are required for the catalytic activity of Ce1D of Ctostridiuni thermocellun? (Béguin, 1990; Tomme et a!., 1991). In CbhII of Trichodernia reesei, for which the three-dimensional structure is known, the substrate binding site of the catalytic domain involves amino acids 135-428 (Rouvinen  et  al., 1990). Aspartic acid residues 175 and 221 are involved in glycosidic  bond cleavage. Asp 175 occurs in the sequence V-Y--P-RDC, which is conserved in all members of its family; Asp221, however, is not conserved in all members of the family (Rouvinen  ci’  al., 1990). Interestingly, the sequence Y-D-GH--W is conserved in all  members of the family. In CbhII this histidine is His266. It is conceivable, therefore, that the DAGD and H-R sequences, although separated by >300 amino acids in the enzymes of family E, are close to each other in the three-dimensional structures and form parts of their active sites. The crystallization of CelD, a member of family El, whose three  97 dimensional structure is known confirms the location of DAGD and the H-R sequences in the same vicinity (Juy et al., 1992). Many proteins accumulate in the cytoplasm as granules when the genes encoding them are overexpressed in E. coli (Kane and Hartley, 1988; Wilcox and Studnicka, 1988), including Cex (O’Neill et al.  ,  1986) and CenA (Z.M. Guo, personal communication).  The proteins in these inclusion bodies can be inactive, but granule formation alone may not cause inactivation. CenC unlike Ce1D from Clostridium thermocellum (Joliff et a!., 1986b), does not form granules, but most of it is inactive and appears as a soluble form in the cytoplasm (Coutinho  ci’  al., 1991). As with Cex, CenA and CenB, E. coli exports  some CenC to the periplasm. CenC is very susceptible to proteolysis in E. coli. Deletions into both the Nterminal or C-terminal repeats did not result in  improved  stability of the enzyme when it  was produced in E. coli. C-terminal deletions revealed that most of C1C2 is not essential for catalytic activity. The functions of the C-terminal repeats in CenC are not known at present. Neither C1C2 nor Cl could be affinity purified on Avicel or Sephadex. A search of the entire SWISS PROT data-bank of protein sequences with Cl or C2 (approximately 100 aminoacids in size each) revealed no obvious similarities but there was one interesting aspect of the alignments that allows speculation as to what the repeats may be doing. There is a perfectly conserved region of 7 amino acids in both Cl and C2 (YRAVFTNA). This region was observed to be very similar to a region in rat and human hepatic lectins which too are well conserved in this region (Patthy, 1987). The alignments are shown in Fig. 38. This region in the rat and human hepatic lectins comprises part of the carbohydrate recognition domain (CRD) of these proteins and is similar to a number of other lectins such as mannose-binding protein (Drickamer, 1989). It is interesting to note that despite the small size of 10 amino-acids of this conserved region, a peptide isolated by proteolytic  98 digestion from Cytisus sessilifolius anti-H(O) lectin (DTYFGKTYNPW) of 11 aminoacids retains the ability to bind carbohydrate (Konarni et al., 1992) The component enzymes in some cellulase systems form multienzyme complexes (Lamed and Bayer, 1988). Although each of the C.JInii cellulases examined to date has a discrete CBD (O’Neill et at. 1986; Wong et at., 1986; Meinke et at., 1991a; Coutinho et ,  a!., 1992) there may be interaction between the enzymes on the substrate. The C-terminal repeats might be involved in such interactions. One possible function of CenC’s Cterminal repeats may be the recognition and attachment to other glycosylated enzymes which might improve synergy in the hydrolysis of cellulose. Another possibility is that CenC via its C-terminal repeats may allow binding to as yet unidentified substrates.  Figure 38. Similarity of the conserved region within the C-terminal repeats to a conserved region withing the carbohydrate recognition domains (CRDs) of lectins. B, charged residue; 0, aromatic residue; : identical residue; ,  *  ,  conserved residue  HHL1,2 and RHL1,2: human hepatic lectins and rat hepatic lectins respectively. The sequences are numbered from the N-terminal amino acid.  HHL1  CR0  220  GTDYETGFKN  229  *..  HHL2  CR0  243  GTDYRHNYKN  252  *..  RHL1  CR0  226  GTDYETGFKN .*.  235  *  RHL2  CR0  192  GTEYRSNFKN  201  CenC  Cl  950  GTRYRAVFTN  959  CenC  C2  1041  GTEYRAVFTN  1050  CONSENSUS  GTBYB- -0-N  99  CenC is the most active endoglucanase isolated from C.fImi to date, but it seems to be a minor component of the cellulase system of this organism (Moser et at. 1989). ,  Endoglucanase D, whose catalytic domain is also in Family El like CenC, is also the most active endoglucanase from C. thermocetlum (Joliff eta!., 1986b; Tomme et at., 1991; Table 2). Both enzymes have similar cleavage specificities (Fig.17). Cex, CenA and CenB of C.firni have a discrete CBD of about 110 amino acids, at the N-terminus in CenA, at the C-tenriini in Cex and CenB (Gilkes et at., 1988; Gilkes et a!. 1991a). The sequences of the CBDs of the three enzymes are about 50% similar ,  (Meinke et al. 1991a), with four conserved tryptophans, two conserved cysteines and a ,  number of asparagine and glycine residues at several other sites (Gilkes eta!., 1991a). The N-terminal repeats of CenC are 150 amino acids in length. Sequence comparison of the N-terminal repeats of CenC with other C.JInii CBDs revealed that the cysteine residues in both Ni and N2 were conserved and were also separated by about 100 amino acids as was seen for the other CBDs. Interestingly, most of the tryptophans in the binding domains of CenA, CenB and Cex were substituted for tyrosines in the CenC CBD. There were a number of other residues that were also conserved suggesting that both Ni and N2 were cellulose-binding domains which were similar to but distantly related to the cellulose-binding domains of Cex and C.fimi endoglucanases A and B. CenC can bind both to cellulose and to Sephadex. In order to confirm that the N-terminal repeats could bind independently to cellulose or Sephadex, polypeptides corresponding to Ni, N2 and N1N2 were affinity purified on Avicel and/or Sephadex, proving that the Nterminal repeats of CenC constitute a cellulose- and a Sephadex-binding domain. It is noteworthy that both repeats are necessary for binding to Sephadex whereas a single repeat mediated binding to cellulose. Cellulose is a polymer of -D-glucopyranosy1 units joined by 1 ,4-glycosidic bonds. The backbone of the polymer has a fully extended, flat conformation (Gardner and Blackwell, 1974). Sephadex is prepared by cross-linking a  100 bacterial dextran with epichiorohydrin. The native dextran is a polymer of cL-D glucopyranosyl units. The units in the backbone of the polymer are linked by a,1—6 bonds, with sidechains attached to the backbone by CL,1—>3 bonds (Gasciolli, eta!., 1991). The 1,6 linkages give the backbone a loosely jointed conformation because the glucosyl units are separated by three rather than two bonds. This conformation may allow binding by the paired but not the single repeats. Whatever the explanation, the ability of the paired N-terminal repeats of CenC to bind both CL-linked and 13-linked glucose polymers is striking. CenC had no detectable activity on Sephadex or raw starch. Binding to Sephadex has also been observed for CenA (N.R. Gilkes, personal communication), concavalin A (Agarwal and Goldstein, 1967) and pea lectin (Stubbs eta!., 1986). Adsorption of N1N2, Ni or N2 to regenerated cellulose, but not to bacterial crystalline cellulose indicates a specificity of these domains for cellulose II and/or amorphous cellulose. This is the first reported example of a CBD showing specificity for a particular structural form of cellulose. Avicel is a heterogeneous form of cellulose prepared by partial acid hydrolysis of wood fibres and contains both crystalline and amorphous components (Ooshima eta!., 1983; Marshall and Sixsmith, 1974). Presumably, adsorption of N1N2, Ni or N2 to Avicel involves binding to the noncrystalline regions of this substrate. It is possible that adsorption of CenC in vivo  is  dependent on prior disruption of crystalline cellulose by other cellulolytic components; for example the CBD of CenA has been shown to disrupt the surface structure of cotton fibres, apparently by a non-catalytic process (Din eta!., 1991). Although it is not known whether such disruption enhances the adsorption of CenC, mechanisms of this kind may contribute to the observed synergism between some cellulolytic enzymes. The fusion of CBDCenC (N1N2) to the catalytic domain of CenA was stable when expressed and could be purified from the culture supernatants of E. co!i using Avicel as the affinity adsorbent. The endoglucanase activity borne by the catalytic domain of CenA and the lack of affinity of CBDCenC for BMCC were retained by the fusion polypeptide  101 indicating the tri-proline linker of CenC allowed the separate and independent folding of the catalytic and cellulose binding domains. The use of CBD’s bearing different specificities as affinity tags has potential practical application in biotechnology for the selective binding of different CBD fusion proteins. This was demonstrated by the selective removal of CenA from a mixture bearing both CenA and C’ ‘A using BMCC as the affinity adsorbent. The introduction of a specific protease site such as IEGR for Factor X between the CBD and the tagged polypeptide facilitates cleavage of the purified fusion polypeptide (Ong eta!, 1989; Sassenfeld, 1990). In such situations it is preferable not only to recover the CBD after cleavage of the fusion polypeptide by the protease (thus leaving the polypeptide of interest in solution) but it is also advantageous to recover the protease used for the digestion. To recover the protease itself in as pure a form as possible it would be preferable to tag the protease with a CBD having a different specificity to that of the CBD used for fusion to the polypeptide of interest (Fig. 39). The use of CBD’s bearing different specificities may also have applications in industry for example the brewing industry, where it would be advantageous not only to recover enzymes but also to allow addition or removal of enzymes at different times both during the fermentation process and in the downstream processing of the brew prior to bottling. The preferential tagging of polypeptides may also have applications in the automation of diagnostic tests and techniques in molecular biology techniques, for example, the sequential treatment of DNA by restriction and modification enzymes in cloning experiments. This is the first time that by using separate cellulose binding domains and using a preferred adsorbent for one of the two CBD’s that each can be differentially removed from a single solution bearing both enzymes.  102  Contaminating protein  Protease site  V a  CBD A  Polwep(ide P  4  a  Micro crystalline cellulose  Wash  / rA  L  Protease  CBD NI N2  / / Amorphous cellulose  / iF Purified poIeptide P  Figure. 39 Purification scheme for fusion polypeptides bearing CBDs with different specificities.  103 The activity of C’ A was tested on a variety of cellulosic substrates and compared to CenA: BMCC (predominantly form I, highly crystalline cellulose) (Henrissat and Chanzy, 1986; Ross etal., 1991); Avicel (heterogeneous cellulose containing both crystalline and amorphous components); PASC (regenerated cellulose, thought to be predominantly form II) (Sarko, 1986) although other workers have concluded that it is amorphous (Lee eta!., 1982; Ooshima  ci’  at., 1983); Cellulose azure (dye-linked  amorphous cellulose) (Fernley, 1962) and CMC (methyl substituted soluble cellulose). C’ ‘A had higher activity on the amorphous substrate cellulose azure but a similar activity on PASC as compared to CenA. The isolated catalytic domain of CenA, p30, had the highest activity on both soluble and amorphous substrates. C’ ‘A was more active on Avicel than p30 alone but the  most  striking difference was the poor activity of C’ ‘A on  crystalline cellulose as compared to CenA. The results indicate that CBDs have a marked influence on catalytic activity only when the substrate is of an insoluble relatively crystalline nature such as Avicel and BMCC. It is remarkable that Klesov and his colleagues, with great insight, were able to predict the correlation between the ability of enzymes to adsorb to crystalline cellulose and the hydrolyis of crystalline cellulose (Rabinovitch eta!., 1982; Klesov eta!., 1983; Chernoglazov eta!., 1983). Almost a decade later and with a better understanding of the molecular basis of attachment between enzyme and substrate, it is much easier to appreciate their contribution in understanding the hydrolysis of cellulose. The ability of the catalytic domain to function on different substrates based on the specificity of the binding domain permits the engineering of enzymes suited for the hydrolysis of a particular substrate. The study of such engineered enzymes along with natural isolates should lead to an even greater understanding of cellulose hydrolysis. Cellulases may consist of two or more domains linked to each other by linker sequences rich in Ser, Thr and Pro residues (Gilkes etal., 1991a). Low angle X-ray studies (Schmuck et at., 1986; Abuja eta!., 1988) suggest that these regions are flexible.  104 A recent search of natural linker sequences found at domain interfaces of proteins in the Brookhaven protein structural databank revealed that so far  oniy  relatively short linkers of  five or six amino acids have been identified as general candidates to link protein molecules or domains through gene fusion (Argos, 1990), therefore detailed information concerning the structures of flexible linker regions long enough to be useful for the design and construction of novel fusion proteins is not yet available (Takkinen et aL, 1991). Sequences composed of 3 or more consecutive proline residues have been observed in the linker regions of cellulases (Gilkes  ci’  a!., 1991a). The use of proline in  linker oligopeptides was considered controversial as linkers maintain an extended conformation (Jentoft, 1990; Williamson et at., 1992) and proline can be considered hydrophobic, tends to bend and kink the main chain and is rigid in dihedral conformation. Nonetheless it was a preferred residue amongst the natural linkers and may thus be considered desirable (Argos, 1990). A conserved triproline sequence has been postulated to generate a sharp bend in the polypeptide chain of myelin basic protein (Brostoff and Eylar, 1971). Subsequent nuclear magnetic resonance experiments failed to confirm the presence of cis-proline bonds indicating instead that the proline residues had an all-trans conformation in aqueous solution (Fraser and Deber, 1985) inconsistent with a sharp bend with chain reversal (Brostoff and Eylar, 1971). NMR work done on a separate triproline peptide also indicated the absence of any sharp bend at the PPP sequence and indicated that the segments of the peptide on opposite sides of PPP were distinctly separated from each other (Nygaard eta!., 1984). These results suggest that the triproline sequence may act as a rigid spacer that separates parts of the protein. This may help explain why the conformation of the polypeptide C’ ‘A in this region is more resistant to C.flmi protease. The high activity of CenC on soluble substrate, its inability to bind to highly crystalline cellulose and the modified activity of the catalytic domain of CenA influenced by the CBD of CenC suggest that the role of CenC in the C.Jmnii cellulase system maybe for the hydrolysis of noncrystalline and or amorphous cellulose. Further improvements in  105 the stable expression of CenC would allow for some very interesting experiments on the synergy of CenC with enzymes that prefer crystalline cellulose. Furthermore unlike CenA and Cex, which were recovered from the residual cellulose in cultures of C.fimi grown with Avicel (Langsford  ci’  at. 1984), CenC and CenC’ remained in the supernatant of  such cultures and were isolated by virtue of their capacities for binding to Sephadex (Moser eta!., 1989). It was not clear at that time why CenC and CenC’ were not recovered on the residual Avicel. The present study suggests that Cex and CenA, which are major components of the C.finii cellulase system, may saturate the available binding sites and this may have prevented the recovery of CenC under such conditions. This thesis has attempted to correlate amino-acid sequence with function for the endoglucanase CenC. The large size of CenC is contributed to by the presence of repeating sequences at the N and C-terminal of the catalytic domain. It is postulated that the hydrolysis of crystalline cellulose requires the presence of endoglucanase A and B as well as exoglucanase/xylanase Cex. As digestion proceeds, more amorphous regions are exposed and soluble cellodextrins are produced; it is suggested that CenC may play a greater role at this stage of the digestion process. The function of the C-terminal repeats of CenC are not known at present but they may have a role in protein-protein interactions.  106 5.  Summary The cenC gene was sequenced. The missing Y end was identified by Southern blotting of C.fimi genomic DNA. Sequence comparison to other cellulolytic enzymes revealed three regions, Nterminal repeats, C-terminal repeats and the central region comprising the catalytic domain. The N-terminals repeats were cloned and expressed separately and shown to have binding independent of the catalytic domain. The binding domain of CenC is unusual, the first described to have affinity for amorphous cellulose but negligible affinity for bacterial crystalline cellulose. It can be produced in large amounts (>20 mg/I) in E. co/i. The industrial advantage of using the CenC CBD as an affinity tag is that it can be eluted from cellulose with water unlike most other CBD fusions which require much harsher and more expensive conditions for elution using guanadinium hydrochloride. Binding domains with different specificities allowed the preferential adsorption of polypeptides from a mixture by use of the appropriate affinity adsorbent. Although the function of the C-terminal repeats is not known at the present, it was shown that most of the C-terminal repeats could be deleted without any observable effect on catalytic or binding activity. Replacement of CBDCenA with CBDCenC in CenA allowed the role of the CBDs in cellulose hydrolysis to be examined. The CBDs have a significant influence on catalytic activity especially on insoluble substrates. 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