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Biochemical characterization and site-directed mutational analysis of the double chitin-binding domain from chitinase 92 of Aeromonas hydrophila JP101
FEMS Microbiology Letters 232 (2004) 61^66
www.fems-microbiology.org
Biochemical characterization and site-directed mutational analysis of the double chitin-binding domain from chitinase 92 of Aeromonas hydrophila JP101 Ming Chung Chang a , Pe Lin Lai b , Mei Li Wu b
c;
a Department of Biochemistry, Medical College, National Cheng Kung University, Tainan 701, Taiwan Graduate Institute of Biotechnology, National Pingtung University of Science and Technology, Pingtung 912, Taiwan c Department of Food Science, National Pingtung University of Science and Technology, Pingtung 912, Taiwan
Received 24 September 2003; received in revised form 15 December 2003; accepted 7 January 2004 First published online 3 February 2004
Abstract Chitinase 92 from Aeromonas hydrophila JP101 contains C-terminal repeated chitin-binding domains (ChBDs) which were named ChBDCI and ChBDCII and classified into family 5 carbohydrate-binding modules on the basis of sequence. In this work, we constructed single and double ChBD by use of the pET system, which expressed as isolated ChBDCII or ChBDCICII . Polysaccharide-binding studies revealed that ChBDCICII not only bound to chitin, but also to other insoluble polysaccharides such as cellulose (Avicel) and xylan. In comparison with ChBDCII , the binding affinities of ChBDCICII are about 10- and 12-fold greater toward colloidal and powdered chitin, indicating that a cooperative interaction exists between ChBDCI and ChBDCII . In order to investigate the roles of the highly conserved aromatic amino acids in the interaction of ChBDCICII and chitin, we have performed site-directed mutagenesis. The data showed that W773A, W792A, Y796A and W797A mutant proteins exhibited a much weaker affinity for chitin than wild-type protein, suggesting that these residues play important roles in chitin binding. 9 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords : Chitin-binding domain ; Carbohydrate-binding module; Chitinase; Site-directed mutagenesis; Aromatic amino acid residue
1. Introduction Chitin, a L-1, 4-linked biopolymer of N-acetylglucosamine (GlcNAc), is a major component of insect exoskeletons, shells of crustaceans, and fungal cell walls. Chitinases (EC 3.2.1.14) catalyze the hydrolysis of L-1,4glycosidic bonds of chitin, which have been found in a wide variety of organisms, including viruses, bacteria, fungi, insects, plants, and animals. Bacteria produce chitinases to degrade chitin as a source of energy. Many bacterial chitinases, like other glycoside hydrolases that hydrolyze insoluble polysaccharides, such as cellulases and xylanases, contain a catalytic domain and one or more non-catalytic carbohydrate-binding modules (CBMs). To date, many CBMs have been investigated
* Corresponding author. Tel. : +886 (8) 7740524; Fax : +886 (8) 7740213. E-mail address : [email protected] (M.L. Wu).
and can be classi¢ed into approximately 33 di¡erent families on the basis of the amino acid sequence and structural similarities (http://afmb.cnrs-mrs.fr/CAZY/CBM_intro.html and [1]). Several studies have shown that CBMs mediate adsorption to the target substrates, and can enhance the catalytic activity. In our previous studies, [2] showed that Chi92 from Aeromonas hydrophila JP101 has also been found to be a modular enzyme, having a catalytic domain of family 18 type, an all L-strand N-terminal domain, a domain of unknown function (A domain), and repeated chitin-binding domains (ChBDs). The C-terminal repeated CBDs, named ChBDCI and ChBDCII , were classi¢ed as members of the family 5 CBMs on the basis of sequence homology. Recently, the ChBDs of chitinases have been classi¢ed into three types based on sequence homology by the NCBI conserved domain database (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml). The ChBDCI and ChBDCII are both members of CBM family 5 and can be reclassi¢ed as ChBD type 3 that includes chitinases found in bacteria.
0378-1097 / 04 / $22.00 9 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0378-1097(04)00014-X
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Chitin binding and enzyme activity studies with C-terminal truncated Chi92 derivatives lacking ChBDs demonstrated that the ChBDs are responsible for its adhesion to chitins. Adsorption experiments with GST fusion proteins demonstrated that ChBD could promote e⁄cient chitin and cellulose binding. Three-dimensional structures of family 5 CBMs have been solved [3,4]. These include the nuclear magnetic resonance (NMR) structure of cellulose-binding domain (CBDEGZ ) from Erwinia chrysanthemi CelZ and the crystal structure of ChBDChiB from Serratia marcescens ChiB. The structural analysis has shown that ChBDChiB exhibits similar folding to CBDEGZ and has two surface-exposed aromatic residues, Trp479 and Tyr481 (corresponding to the two aromatic residues in the AKWWTQG motif), which are localized on the putative chitin-binding site. As expected from their signi¢cant sequence similarity, the tertiary structures of ChBDCI and ChBDCII closely resemble those of ChBDChiB and CBDEGZ . The adsorption of Chi92 to chitin, likely mediated by ChBDCI and ChBDCII , has been identi¢ed earlier [2], but the binding properties have not been elucidated in detail. Our objectives in this study were (1) to determine the binding speci¢city and properties of ChBDCICII and ChBDCII in the absence of the other domains of Chi92, (2) to further con¢rm the cooperative e¡ect between ChBDCI and ChBDCII by determining the adsorption isotherms for the double ChBD (ChBDCICII ) and single ChBD (ChBDCII ) on insoluble chitin, and (3) to examine whether the aromatic amino acids play important roles in the interaction of ChBDCICII and chitin by individually mutating the four highly conserved aromatic residues (Trp773, Trp792, Tyr796 and Trp797) in ChBDCI domain to alanine or phenylalanine, and performing the adsorption isotherm experiments on the mutant proteins.
2. Materials and methods 2.1. Bacterial strains, plasmids, and cultivation conditions Escherichia coli strain JM109 [5] and XL1-Blue (Stratagene) were used as hosts for cloning, and E. coli BL21 (DE3) :pLysS (Novagen) was used as the host for protein production. All E. coli strains were grown in Luria^Bertani (LB) medium [6] at 37‡C. When necessary, the medium was supplemented with ampicillin (50 Wg ml31 ). Plasmid pET21b (Novagen) was used as a cloning and expression vector. 2.2. Construction of ChBDCICII and ChBDCII Plasmids pET-CICII and pET-CII, which encoded ChBDCICII and ChBDCII , respectively, were constructed by PCR ampli¢cation of the 3P region of Chi92 in plasmid pHX [2]. The ChBDCICII - and ChBDCII -encoding regions
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(positions 2246^2610 and positions 2444^2610) of chi92 were ampli¢ed using the forward primers (primer 1, 5PTG ACC GTG GAT CCG GCC AGT AC-3P; primer 3, 5P-AG GTG GAT CCG GGT TGG GAT GC-3P; the BamHI is underlined) and reverse primers (primer 2, 5PCAT CAC GGC CTC GAG GTT GCA GCT CGC-3P; the XhoI site is underlined). These two PCR products were digested with BamHI and XhoI and fused in frame to the coding region of T7 tag and His6 tag of pET21b. 2.3. Construction of ChBDCICII mutants Site-directed mutants of ChBDCICII were constructed by PCR overlap extension methods [7]. The following mutagenic primers were used to introduce point mutations (the altered bases are in boldface type): W773F (forward), 5PGAT CCG GCC TTT AGT GCA GG-3P; W773F (reverse), 5P-CC TGC ACT AAA GGC CGG ATG-3P; W773A (forward), 5P-GAT CCG GCC GCG AGT GCA GG-3P; W773A (reverse), 5P-CC TGC ACT CGC GGC CGG ATG-3P; W792A (forward), 5P-CAA CTG GTG GCG CAG GCC AA-3P; W792A (reverse), 5P-TT GGC CTG CGC CAC CAG TTG-3P; Y796A (forward), 5PCAG GCC AAG GCG TGG ACC CAG-3P; W796A (reverse), 5P-CTG GGT CCA CGC CTT GGC CTG-3P; W797A (forward), 5P-CAG GCC AAG TAT GCG ACC CAG-3P; W797A (reverse), 5P-CTG GGT CGC ATA CTT GGC CTG-3P. In the ¢rst PCR, the two slightly overlapping PCR products were prepared with two pairs of primers (primers 1 and reversed mutagenic primer ampli¢ed the region from the N-terminus to the mutation site, and forward mutagenic primer and primer 2 ampli¢ed the region from the mutation site to the C-terminus). In the secondround PCR, the two PCR products were used as the DNA templates with primers 1 and 2, and gave a full-length DNA sequence containing the desired mutation and the restriction sites. The PCR products encoding the mutant forms of ChBDCICII were digested with BamHI and XhoI and ligated into expression vector pET21b. All sequences were determined with an Applied Biosystems Model 377 sequencer to verify that only the desired mutations were present in the sequence. 2.4. Expression and puri¢cation of ChBDCII , ChBDCICII , and ChBDCICII mutant proteins The proteins were puri¢ed from cell-free extract by nickel ion a⁄nity chromatography. ChBDCII , ChBDCICII , and ChBDCICII mutant proteins were expressed in E. coli BL21 (DE3) containing the appropriate plasmids. The E. coli strains were grown at 37‡C in LB medium containing ampicillin to an optical density at 595 nm of 0.8. The culture was induced with 1 mM IPTG (isopropyl-L-D-thiogalactopyranoside) for 4 h. The cells were harvested and resuspended in 20 mM Tris (pH 8.0), and disrupted by sonication. After centrifugation, the crude extracts were applied
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to a 5-ml HiTrap metal chelating column (Amersham Biosciences) charged with Ni2þ . The protein was eluted with a linear gradient of 0^300 mM imidazole containing 0.5 M NaCl and 20 mM Tris^HCl (pH 7.9). The eluted protein fractions were dialyzed against 10 mM ammonium bicarbonate bu¡er (pH 7.9) to remove salts, and the puri¢ed proteins were lyophilized and stored at 320‡C. The puri¢ed proteins were separated by sodium dodecyl sulfate^ 15% polyacrylamide gel electrophoresis (SDS^PAGE) [8]. Protein concentration was determined according to the Bradford method [9].
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an N-terminal T7 tag and a C-terminal His6 tag, derived from pET21b. After expression in E. coli BL21 (DE3), these protein products were puri¢ed from cell extract by nickel ion a⁄nity column chromatography. The yield of the recombinant proteins was 30^50 mg l31 E. coli culture. The puri¢ed protein products showed a single band on SDS^PAGE, and their molecular sizes were 14.6 kDa for ChBDCICII and ChBDCICII mutants, and 7.5 kDa for ChBDCII , respectively (Fig. 1). These were in agreement with the expected values for ChBDCICII and ChBDCII . 3.2. Binding speci¢city and properties of isolated ChBDCICII and ChBDCII on insoluble polysaccharides
2.5. Polysaccharide-binding studies Powdered chitin from crab shells (Sigma), colloidal chitin [10], chitosan (Sigma), cellulose (Avicel), starch (Wako), and xylan from beechwood (Sigma) were used as polysaccharide substrates. For binding assay, 50 Wg isolated ChBDCICII was mixed with 1 mg of insoluble polysaccharides, in a ¢nal volume of 1 ml of Tris^HCl bu¡er (pH 8.0). The mixture was kept under constant agitation at 4‡C for 1 h, and then centrifuged. The concentration of unbound enzyme was determined from absorbance by the Bradford method, and used to calculate the amount of enzyme bound to the polysaccharide. Adsorption isotherm measurements were used to generate binding data for ChBDCII , ChBDCICII , and ChBDCICII mutant proteins on colloidal chitin and powdered chitin. Protein solution (0.2^16 WM) was incubated with 1 mg of chitin substrate in a ¢nal volume of 1 ml of Tris^HCl bu¡er (pH 8.0) at 4‡C. After 1 h, chitin was removed by centrifugation (12 000Ug, 4‡C), and the concentration of unbound protein was determined by the Bradford method. The amount of bound protein was quanti¢ed by the initial protein subtracted from unbound protein concentrations. All experiments were done in triplicate. Partition coe⁄cients were obtained from the initial slopes of the curve of adsorption isotherm [11].
To determine the binding capacity and speci¢city of ChBDCICII and ChBDCII , we measured the adsorption of ChBDCICII and ChBDCII with various insoluble polysaccharides, including powdered chitin, colloidal chitin, chitosan, cellulose (Avicel), xylan, and starch. As shown in Fig. 2, the levels of adsorption of ChBDCICII to colloidal and powdered chitin were 86% and 64%, respectively. The results indicated that ChBDCICII not only bound to various forms of insoluble chitin, but it also bound to other insoluble polysaccharides such as cellulose (Avicel), and xylan. The control protein, bovine serum albumin, did not exhibit signi¢cant binding to insoluble polysaccharides. Furthermore, a time course experiment showed that the binding of ChBDCICII was completed within 5 min of incubation, and did not change upon extending incubation up to 2 h (data not shown). This suggests that ChBDCICII bound rapidly and binding equilibrium was reached almost instantly. The data shown in Fig. 2 also indicate that isolated ChBDCICII preferentially binds to colloidal chitin, followed by powdered chitin and exhibited weak but signi¢cant binding to cellulose. Similarly, isolated ChBDChiC (from Streptomyces griseus ChiC) and GST fusion protein of ChBD (from Alteromonas sp. O-7
3. Results and discussion 3.1. Expression and puri¢cation of ChBDCICII , ChBDCII and ChBDCICII mutant proteins To investigate the binding properties of ChBDCICII and ChBDCII , we constructed single and double ChBD by the pET system. In the construction of pET-CICII and pETCII, the DNA regions of the chi92 gene corresponding to ChBDCICII (Asn769 to Asn865 of Chi92) and ChBDCII (Asn817 to Asn865 of Chi92) were inserted into pET21b and expressed as isolated domains. In addition, three tryptophan residues and one tyrosine residue were individually mutated to alanine or phenylalanine to create the following mutations in ChBDCICII : W773F, W773A, W792A, Y796A, and W797A. These protein products all contained
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Fig. 1. SDS^PAGE analysis of ChBDCII , ChBDCICII , and ChBDCICII mutants. Proteins were puri¢ed by nickel ion a⁄nity column chromatography, as described in Section 2. The proteins were analyzed by 15% SDS^PAGE, and stained with Coomassie brilliant blue R-250. Lane 1, molecular mass markers (Invitrogen); lane 2, ChBDCICII ; lane 3, ChBDCICII W773F; lane 4, ChBDCICII W773A; lane 5, ChBDCICII W792A; lane 6, ChBDCICII Y796A; lane7, ChBDCICII W797A; lane 8, ChBDCII .
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Fig. 2. Binding assays of ChBDCICII on di¡erent polysaccharide substrates. Binding assay of ChBDCICII to various insoluble polysaccharides was measured in 1 ml 20 mM Tris^HCl bu¡er (pH 8.0) as described in Section 2. The concentration of ChBDCICII was 50 Wg ml31 ; the concentrations of insoluble polysaccharide substrates were in 1 mg ml31 . Vertical bars indicate standard deviations.
ChiC), both belonging to family 5 CBMs, exhibited binding activity toward chitin and cellulose [12,13]. In contrast to the adsorption of ChBDs belonging to family 5 CBMs to cellulose, Masayuki et al. [14] have reported that isolated ChBDChiA1 , which has been classi¢ed as a family 12 CBM, binds speci¢cally to insoluble chitin, with no significant binding to cellulose. This suggests that members of the family 12 CMBs have similar binding properties. Further research is necessary to produce additional evidences. We determined the in£uences of pH and salt on the binding of ChBDCICII to chitin. Binding is strongest at pH 8.0 and decreased slightly with changing pH between 5.0 and 8.0 and between 8.0 and 11.0 (Fig. 3). The e¡ect of salt concentration was examined by binding ChBDCICII to colloidal chitin at pH 7.0 in the presence of 0.5 M NaCl. As shown in Fig. 4, addition of 0.5 M NaCl increased the level of adsorption of ChBDCICII to colloidal chitin from 70% to 90%. This result indicated that the binding capacity of ChBDCICII was improved in the presence of salt ions.
Fig. 3. In£uence of pH on the binding of ChBDCICII to colloidal chitin. ChBDCICII (50 Wg ml31 ) was incubated with 1 mg (dry weight) of colloidal chitin in 1-ml bu¡ers at various pHs. Vertical bars indicate standard deviations.
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Fig. 4. The e¡ects of salt on the binding of ChBDCICII to colloidal chitin at pH 7.0. ChBDCICII (50 Wg ml31 ) was incubated with 1 mg (dry weight) of colloidal chitin in a ¢nal volume of 1 ml phosphate bu¡ers (pH 7.0) (b) or 1 ml phosphate bu¡ers (pH 7.0) with 0.5 M NaCl (F).
3.3. Chitin-binding a⁄nities of ChBDCII , ChBDCICII and ChBDCICII mutant proteins To determine the binding a⁄nity of ChBDCII , ChBDCICII , and ChBDCICII mutant proteins on chitin, the adsorption isotherms were measured at pH 8.0 (Fig. 5). The adsorption isotherms for the double ChBD and single ChBD clearly showed that the binding a⁄nities of ChBDCII to colloidal and powdered chitin were markedly lower than that of the ChBDCICII . In order to further
Fig. 5. Adsorption isotherms for ChBDCII , ChBDCICII , and ChBDCICII mutants on colloidal chitin (A) and powdered chitin (B). (B) and (F) are the concentrations of bound and free protein, respectively.
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quantify the binding a⁄nities, the partition coe⁄cients (Kp ) were measured for ChBDCII , ChBDCICII , and ChBDCICII mutants from the initial slopes of adsorption isotherms shown in Fig. 5. As shown in Table 1, the partition coe⁄cients of ChBDCICII are about 10- and 12-fold greater toward colloidal and powdered chitin than those of ChBDCII . A similar result has been observed for a recombinant double CBD carrying two CBDs from Trichoderma reesei CBHI and CBHII [15]. The binding a⁄nity showed a 5^10-fold increase for the double CBD as compared with the single CBD toward Avicel. Linder et al. proposed a two-step model to describe the binding behavior of the double CBD [15]. Based on the model, the much higher a⁄nity of the double ChBD for chitin means that a cooperative interaction exists between ChBDCI and ChBDCII . Aromatic amino acids have been shown to play a prominent role in carbohydrate-binding domains, by forming hydrophobic stacking interaction with the sugar rings of their target substrates. In order to investigate the importance of the highly conserved aromatic amino acids in the interaction of ChBDCICII with chitin, we substituted Tyr796 and Trp797 in ChBDCI domain with alanine, which correspond to the surface aromatic residues located in the AKWWTQG motif. As seen in Table 1, the ChBDCICII mutants, W773A and W792A, only retained 16^23% and 20^23% of their binding a⁄nities on colloidal and powdered chitin. Therefore, Tyr796 and Trp797 are likely to be involved directly in chitin binding because of their positions on the surface. Site-directed mutagenesis studies of CBDEGZ showed that the equivalent residues (Trp43 and Trp44 in the AKWWTQG motif) play critical roles in cellulose binding [16]. In accordance with the NMR structure of CBDEGZ [3], Trp773 and Trp792 (corresponding to Trp13 and Tyr39 of CBDEGZ ) were predicted to be located in the hydrophobic core surrounding Pro803 (corresponding to Pro49 of CBDEGZ ). Therefore, we replaced Trp773 and Trp792 with alanine, and also replaced Trp773 with phenylalanine. As shown in Table 1, W773A and W792A mutation led to a dramatic loss of binding a⁄nity for colloidal and powdered chitin, while W792F mutant lost only about
Table 1 Partitioning coe⁄cients (Kp ) for the adsorption of ChBDCII , ChBDCICII , and ChBDCICII mutants to colloidal chitin and powdered chitin Protein
Kp (l g31 ) on colloidal chitin
ChBDCICII ChBDCII ChBDCICII ChBDCICII ChBDCICII ChBDCICII ChBDCICII
7.45 0.71 4.26 1.14 0.41 1.22 1.73
a
W773F W773A W792A Y796A W797A
(100)a (10) (57) (15) (6) (16) (23)
Kp (l g31 ) on powdered chitin 3.05 0.25 1.70 0.36 0.16 0.62 0.70
(100) (8) (56) (12) (5) (20) (23)
Values in parentheses represent percent relative adsorption a⁄nity.
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50% of its binding a⁄nity on chitin. Therefore, the phenylalanine in position 773 could retain some of the capability of forming hydrophobic interaction which results in a smaller decrease in chitin binding. Based on the structure of CBDEGZ [3], these data described above imply that residues Trp773 and Trp792 do not play direct roles in chitin binding, but are important in the correct folding of the protein. Further structural studies are required to determine if the reduced a⁄nities are a conformation change or a direct consequence of the mutation of the respective aromatic residues.
References [1] Tomme, P., Warren, R.A., Miller Jr., R.C., Kilburn, D.G. and Gilkes, N.R. (1995) Cellulose-binding domains: classi¢cation and properties. In: Enzymatic Degradation of Insoluble Polysaccharides (Saddler, J.N. and Penner, M., Eds.), pp. 142^163. American Chemical Society, Washington, DC. [2] Wu, M.L., Chuang, Y.C., Chen, J.P., Chen, C.S. and Chang, M.C. (2001) Identi¢cation and characterization of the three chitin-binding domains within the multidomain chitinase Chi92 from Aeromonas hydrophila JP101. Appl. Environ. Microbiol. 67, 5100^5106. [3] Brun, E., Moriaud, F., Gans, P., Blackledge, M.J., Barras, F. and Marion, D. (1997) Solution structure of the cellulose-binding domain of the endoglucanase Z secreted by Erwinia chrysanthemi. Biochemistry 36, 16074^16086. [4] van Aalten, D.M.F., Synstad, B., Brurberg, M.B., Hough, E., Riise, B.W., Eijsink, V.G.H. and Wierenga, R.K. (2000) Structure of a twoU resolution. domain chitotriosidase from Serratia marcescens at 1.9-A Proc. Natl. Acad. Sci. USA 97, 5842^5847. [5] Yanisch, P.C., Vieira, J. and Messing, J. (1985) Improved M13 phage cloning vectors and host strains : nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103^119. [6] Sambrook, J. and Russel, D.W. (2001) Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, Plainview, NY. [7] Higuchi, R., Krummel, B. and Saiki, R.K. (1988) A general method of in vitro preparation and speci¢c mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 16, 7351^ 7367. [8] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277, 680^685. [9] Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248^254. [10] Shimahara, K. and Takiguchi, Y. (1988) Preparation of crustacean chitin. Methods Enzymol. 161, 417^423. [11] Linder, M., Lindeberg, G., Reinikainen, T., Teeri, T.T. and Pettersson, G. (1995) The di¡erence in a⁄nity between two fungal cellulosebinding domain is dominated by a single amino acid substitution. FEBS Lett. 372, 96^98. [12] Itoh, Y., Kawase, T., Nikaidou, N., Fukada, H., Mitsutomi, M., Watanabe, T. and Itoh, Y. (2002) Functional analysis of the chitinbinding domain of a family 19 chitinase from Streptomyces griseus HUT6037: substrate-binding a⁄nity and cis-dominant increase of antifungal function. Biosci. Biotechnol. Biochem. 66, 1084^1092. [13] Tsujibo, H., Orikoshi, H., Shiotani, K., Hayashi, M., Umeda, J., Miyamoto, K., Imada, C., Okami, Y. and Inamori, Y. (1998) Characterization of chitinase C from a marine bacterium, Alteromonas sp. strain O-7, and its corresponding gene and domain structure. Appl. Environ. Microbiol. 64, 472^478.
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[14] Hashimoto, M., Ikegami, T., Seino, S., Ohuchi, N., Fukada, H., Sugiyama, J., Shirakawa, M. and Watanabe, T. (2000) Expression and characterization of the Chitin-binding domain of chitinase A1 from Bacillus circulans WL-12. J. Bacteriol. 182, 3045^3054. [15] Linder, M., Salovuori, I., Ruohonen, L. and Teeri, T.T. (1996) Characterization of a double cellulose-binding domain. Synergistic high
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a⁄nity binding to crystalline cellulose. J. Biol. Chem. 271, 21268^ 21272. [16] Helen, D.S. and Frederic, B. (1999) Functional analysis of the carbohydrate-binding domains of Erwinia chrysanthemi Cel5 (Endoglucanase Z) and an Escherichia coli putative chitinase. J. Bacteriol. 181, 4611^4616.
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Biochemical characterization and site-directed mutational analysis of the double chitin-binding domain from chitinase 92 of Aeromonas hydrophila JP101 Ming Chung Chang a , Pe Lin Lai b , Mei Li Wu b
c;
a Department of Biochemistry, Medical College, National Cheng Kung University, Tainan 701, Taiwan Graduate Institute of Biotechnology, National Pingtung University of Science and Technology, Pingtung 912, Taiwan c Department of Food Science, National Pingtung University of Science and Technology, Pingtung 912, Taiwan
Received 24 September 2003; received in revised form 15 December 2003; accepted 7 January 2004 First published online 3 February 2004
Abstract Chitinase 92 from Aeromonas hydrophila JP101 contains C-terminal repeated chitin-binding domains (ChBDs) which were named ChBDCI and ChBDCII and classified into family 5 carbohydrate-binding modules on the basis of sequence. In this work, we constructed single and double ChBD by use of the pET system, which expressed as isolated ChBDCII or ChBDCICII . Polysaccharide-binding studies revealed that ChBDCICII not only bound to chitin, but also to other insoluble polysaccharides such as cellulose (Avicel) and xylan. In comparison with ChBDCII , the binding affinities of ChBDCICII are about 10- and 12-fold greater toward colloidal and powdered chitin, indicating that a cooperative interaction exists between ChBDCI and ChBDCII . In order to investigate the roles of the highly conserved aromatic amino acids in the interaction of ChBDCICII and chitin, we have performed site-directed mutagenesis. The data showed that W773A, W792A, Y796A and W797A mutant proteins exhibited a much weaker affinity for chitin than wild-type protein, suggesting that these residues play important roles in chitin binding. 9 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords : Chitin-binding domain ; Carbohydrate-binding module; Chitinase; Site-directed mutagenesis; Aromatic amino acid residue
1. Introduction Chitin, a L-1, 4-linked biopolymer of N-acetylglucosamine (GlcNAc), is a major component of insect exoskeletons, shells of crustaceans, and fungal cell walls. Chitinases (EC 3.2.1.14) catalyze the hydrolysis of L-1,4glycosidic bonds of chitin, which have been found in a wide variety of organisms, including viruses, bacteria, fungi, insects, plants, and animals. Bacteria produce chitinases to degrade chitin as a source of energy. Many bacterial chitinases, like other glycoside hydrolases that hydrolyze insoluble polysaccharides, such as cellulases and xylanases, contain a catalytic domain and one or more non-catalytic carbohydrate-binding modules (CBMs). To date, many CBMs have been investigated
* Corresponding author. Tel. : +886 (8) 7740524; Fax : +886 (8) 7740213. E-mail address : [email protected] (M.L. Wu).
and can be classi¢ed into approximately 33 di¡erent families on the basis of the amino acid sequence and structural similarities (http://afmb.cnrs-mrs.fr/CAZY/CBM_intro.html and [1]). Several studies have shown that CBMs mediate adsorption to the target substrates, and can enhance the catalytic activity. In our previous studies, [2] showed that Chi92 from Aeromonas hydrophila JP101 has also been found to be a modular enzyme, having a catalytic domain of family 18 type, an all L-strand N-terminal domain, a domain of unknown function (A domain), and repeated chitin-binding domains (ChBDs). The C-terminal repeated CBDs, named ChBDCI and ChBDCII , were classi¢ed as members of the family 5 CBMs on the basis of sequence homology. Recently, the ChBDs of chitinases have been classi¢ed into three types based on sequence homology by the NCBI conserved domain database (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml). The ChBDCI and ChBDCII are both members of CBM family 5 and can be reclassi¢ed as ChBD type 3 that includes chitinases found in bacteria.
0378-1097 / 04 / $22.00 9 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0378-1097(04)00014-X
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Chitin binding and enzyme activity studies with C-terminal truncated Chi92 derivatives lacking ChBDs demonstrated that the ChBDs are responsible for its adhesion to chitins. Adsorption experiments with GST fusion proteins demonstrated that ChBD could promote e⁄cient chitin and cellulose binding. Three-dimensional structures of family 5 CBMs have been solved [3,4]. These include the nuclear magnetic resonance (NMR) structure of cellulose-binding domain (CBDEGZ ) from Erwinia chrysanthemi CelZ and the crystal structure of ChBDChiB from Serratia marcescens ChiB. The structural analysis has shown that ChBDChiB exhibits similar folding to CBDEGZ and has two surface-exposed aromatic residues, Trp479 and Tyr481 (corresponding to the two aromatic residues in the AKWWTQG motif), which are localized on the putative chitin-binding site. As expected from their signi¢cant sequence similarity, the tertiary structures of ChBDCI and ChBDCII closely resemble those of ChBDChiB and CBDEGZ . The adsorption of Chi92 to chitin, likely mediated by ChBDCI and ChBDCII , has been identi¢ed earlier [2], but the binding properties have not been elucidated in detail. Our objectives in this study were (1) to determine the binding speci¢city and properties of ChBDCICII and ChBDCII in the absence of the other domains of Chi92, (2) to further con¢rm the cooperative e¡ect between ChBDCI and ChBDCII by determining the adsorption isotherms for the double ChBD (ChBDCICII ) and single ChBD (ChBDCII ) on insoluble chitin, and (3) to examine whether the aromatic amino acids play important roles in the interaction of ChBDCICII and chitin by individually mutating the four highly conserved aromatic residues (Trp773, Trp792, Tyr796 and Trp797) in ChBDCI domain to alanine or phenylalanine, and performing the adsorption isotherm experiments on the mutant proteins.
2. Materials and methods 2.1. Bacterial strains, plasmids, and cultivation conditions Escherichia coli strain JM109 [5] and XL1-Blue (Stratagene) were used as hosts for cloning, and E. coli BL21 (DE3) :pLysS (Novagen) was used as the host for protein production. All E. coli strains were grown in Luria^Bertani (LB) medium [6] at 37‡C. When necessary, the medium was supplemented with ampicillin (50 Wg ml31 ). Plasmid pET21b (Novagen) was used as a cloning and expression vector. 2.2. Construction of ChBDCICII and ChBDCII Plasmids pET-CICII and pET-CII, which encoded ChBDCICII and ChBDCII , respectively, were constructed by PCR ampli¢cation of the 3P region of Chi92 in plasmid pHX [2]. The ChBDCICII - and ChBDCII -encoding regions
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(positions 2246^2610 and positions 2444^2610) of chi92 were ampli¢ed using the forward primers (primer 1, 5PTG ACC GTG GAT CCG GCC AGT AC-3P; primer 3, 5P-AG GTG GAT CCG GGT TGG GAT GC-3P; the BamHI is underlined) and reverse primers (primer 2, 5PCAT CAC GGC CTC GAG GTT GCA GCT CGC-3P; the XhoI site is underlined). These two PCR products were digested with BamHI and XhoI and fused in frame to the coding region of T7 tag and His6 tag of pET21b. 2.3. Construction of ChBDCICII mutants Site-directed mutants of ChBDCICII were constructed by PCR overlap extension methods [7]. The following mutagenic primers were used to introduce point mutations (the altered bases are in boldface type): W773F (forward), 5PGAT CCG GCC TTT AGT GCA GG-3P; W773F (reverse), 5P-CC TGC ACT AAA GGC CGG ATG-3P; W773A (forward), 5P-GAT CCG GCC GCG AGT GCA GG-3P; W773A (reverse), 5P-CC TGC ACT CGC GGC CGG ATG-3P; W792A (forward), 5P-CAA CTG GTG GCG CAG GCC AA-3P; W792A (reverse), 5P-TT GGC CTG CGC CAC CAG TTG-3P; Y796A (forward), 5PCAG GCC AAG GCG TGG ACC CAG-3P; W796A (reverse), 5P-CTG GGT CCA CGC CTT GGC CTG-3P; W797A (forward), 5P-CAG GCC AAG TAT GCG ACC CAG-3P; W797A (reverse), 5P-CTG GGT CGC ATA CTT GGC CTG-3P. In the ¢rst PCR, the two slightly overlapping PCR products were prepared with two pairs of primers (primers 1 and reversed mutagenic primer ampli¢ed the region from the N-terminus to the mutation site, and forward mutagenic primer and primer 2 ampli¢ed the region from the mutation site to the C-terminus). In the secondround PCR, the two PCR products were used as the DNA templates with primers 1 and 2, and gave a full-length DNA sequence containing the desired mutation and the restriction sites. The PCR products encoding the mutant forms of ChBDCICII were digested with BamHI and XhoI and ligated into expression vector pET21b. All sequences were determined with an Applied Biosystems Model 377 sequencer to verify that only the desired mutations were present in the sequence. 2.4. Expression and puri¢cation of ChBDCII , ChBDCICII , and ChBDCICII mutant proteins The proteins were puri¢ed from cell-free extract by nickel ion a⁄nity chromatography. ChBDCII , ChBDCICII , and ChBDCICII mutant proteins were expressed in E. coli BL21 (DE3) containing the appropriate plasmids. The E. coli strains were grown at 37‡C in LB medium containing ampicillin to an optical density at 595 nm of 0.8. The culture was induced with 1 mM IPTG (isopropyl-L-D-thiogalactopyranoside) for 4 h. The cells were harvested and resuspended in 20 mM Tris (pH 8.0), and disrupted by sonication. After centrifugation, the crude extracts were applied
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to a 5-ml HiTrap metal chelating column (Amersham Biosciences) charged with Ni2þ . The protein was eluted with a linear gradient of 0^300 mM imidazole containing 0.5 M NaCl and 20 mM Tris^HCl (pH 7.9). The eluted protein fractions were dialyzed against 10 mM ammonium bicarbonate bu¡er (pH 7.9) to remove salts, and the puri¢ed proteins were lyophilized and stored at 320‡C. The puri¢ed proteins were separated by sodium dodecyl sulfate^ 15% polyacrylamide gel electrophoresis (SDS^PAGE) [8]. Protein concentration was determined according to the Bradford method [9].
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an N-terminal T7 tag and a C-terminal His6 tag, derived from pET21b. After expression in E. coli BL21 (DE3), these protein products were puri¢ed from cell extract by nickel ion a⁄nity column chromatography. The yield of the recombinant proteins was 30^50 mg l31 E. coli culture. The puri¢ed protein products showed a single band on SDS^PAGE, and their molecular sizes were 14.6 kDa for ChBDCICII and ChBDCICII mutants, and 7.5 kDa for ChBDCII , respectively (Fig. 1). These were in agreement with the expected values for ChBDCICII and ChBDCII . 3.2. Binding speci¢city and properties of isolated ChBDCICII and ChBDCII on insoluble polysaccharides
2.5. Polysaccharide-binding studies Powdered chitin from crab shells (Sigma), colloidal chitin [10], chitosan (Sigma), cellulose (Avicel), starch (Wako), and xylan from beechwood (Sigma) were used as polysaccharide substrates. For binding assay, 50 Wg isolated ChBDCICII was mixed with 1 mg of insoluble polysaccharides, in a ¢nal volume of 1 ml of Tris^HCl bu¡er (pH 8.0). The mixture was kept under constant agitation at 4‡C for 1 h, and then centrifuged. The concentration of unbound enzyme was determined from absorbance by the Bradford method, and used to calculate the amount of enzyme bound to the polysaccharide. Adsorption isotherm measurements were used to generate binding data for ChBDCII , ChBDCICII , and ChBDCICII mutant proteins on colloidal chitin and powdered chitin. Protein solution (0.2^16 WM) was incubated with 1 mg of chitin substrate in a ¢nal volume of 1 ml of Tris^HCl bu¡er (pH 8.0) at 4‡C. After 1 h, chitin was removed by centrifugation (12 000Ug, 4‡C), and the concentration of unbound protein was determined by the Bradford method. The amount of bound protein was quanti¢ed by the initial protein subtracted from unbound protein concentrations. All experiments were done in triplicate. Partition coe⁄cients were obtained from the initial slopes of the curve of adsorption isotherm [11].
To determine the binding capacity and speci¢city of ChBDCICII and ChBDCII , we measured the adsorption of ChBDCICII and ChBDCII with various insoluble polysaccharides, including powdered chitin, colloidal chitin, chitosan, cellulose (Avicel), xylan, and starch. As shown in Fig. 2, the levels of adsorption of ChBDCICII to colloidal and powdered chitin were 86% and 64%, respectively. The results indicated that ChBDCICII not only bound to various forms of insoluble chitin, but it also bound to other insoluble polysaccharides such as cellulose (Avicel), and xylan. The control protein, bovine serum albumin, did not exhibit signi¢cant binding to insoluble polysaccharides. Furthermore, a time course experiment showed that the binding of ChBDCICII was completed within 5 min of incubation, and did not change upon extending incubation up to 2 h (data not shown). This suggests that ChBDCICII bound rapidly and binding equilibrium was reached almost instantly. The data shown in Fig. 2 also indicate that isolated ChBDCICII preferentially binds to colloidal chitin, followed by powdered chitin and exhibited weak but signi¢cant binding to cellulose. Similarly, isolated ChBDChiC (from Streptomyces griseus ChiC) and GST fusion protein of ChBD (from Alteromonas sp. O-7
3. Results and discussion 3.1. Expression and puri¢cation of ChBDCICII , ChBDCII and ChBDCICII mutant proteins To investigate the binding properties of ChBDCICII and ChBDCII , we constructed single and double ChBD by the pET system. In the construction of pET-CICII and pETCII, the DNA regions of the chi92 gene corresponding to ChBDCICII (Asn769 to Asn865 of Chi92) and ChBDCII (Asn817 to Asn865 of Chi92) were inserted into pET21b and expressed as isolated domains. In addition, three tryptophan residues and one tyrosine residue were individually mutated to alanine or phenylalanine to create the following mutations in ChBDCICII : W773F, W773A, W792A, Y796A, and W797A. These protein products all contained
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Fig. 1. SDS^PAGE analysis of ChBDCII , ChBDCICII , and ChBDCICII mutants. Proteins were puri¢ed by nickel ion a⁄nity column chromatography, as described in Section 2. The proteins were analyzed by 15% SDS^PAGE, and stained with Coomassie brilliant blue R-250. Lane 1, molecular mass markers (Invitrogen); lane 2, ChBDCICII ; lane 3, ChBDCICII W773F; lane 4, ChBDCICII W773A; lane 5, ChBDCICII W792A; lane 6, ChBDCICII Y796A; lane7, ChBDCICII W797A; lane 8, ChBDCII .
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Fig. 2. Binding assays of ChBDCICII on di¡erent polysaccharide substrates. Binding assay of ChBDCICII to various insoluble polysaccharides was measured in 1 ml 20 mM Tris^HCl bu¡er (pH 8.0) as described in Section 2. The concentration of ChBDCICII was 50 Wg ml31 ; the concentrations of insoluble polysaccharide substrates were in 1 mg ml31 . Vertical bars indicate standard deviations.
ChiC), both belonging to family 5 CBMs, exhibited binding activity toward chitin and cellulose [12,13]. In contrast to the adsorption of ChBDs belonging to family 5 CBMs to cellulose, Masayuki et al. [14] have reported that isolated ChBDChiA1 , which has been classi¢ed as a family 12 CBM, binds speci¢cally to insoluble chitin, with no significant binding to cellulose. This suggests that members of the family 12 CMBs have similar binding properties. Further research is necessary to produce additional evidences. We determined the in£uences of pH and salt on the binding of ChBDCICII to chitin. Binding is strongest at pH 8.0 and decreased slightly with changing pH between 5.0 and 8.0 and between 8.0 and 11.0 (Fig. 3). The e¡ect of salt concentration was examined by binding ChBDCICII to colloidal chitin at pH 7.0 in the presence of 0.5 M NaCl. As shown in Fig. 4, addition of 0.5 M NaCl increased the level of adsorption of ChBDCICII to colloidal chitin from 70% to 90%. This result indicated that the binding capacity of ChBDCICII was improved in the presence of salt ions.
Fig. 3. In£uence of pH on the binding of ChBDCICII to colloidal chitin. ChBDCICII (50 Wg ml31 ) was incubated with 1 mg (dry weight) of colloidal chitin in 1-ml bu¡ers at various pHs. Vertical bars indicate standard deviations.
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Fig. 4. The e¡ects of salt on the binding of ChBDCICII to colloidal chitin at pH 7.0. ChBDCICII (50 Wg ml31 ) was incubated with 1 mg (dry weight) of colloidal chitin in a ¢nal volume of 1 ml phosphate bu¡ers (pH 7.0) (b) or 1 ml phosphate bu¡ers (pH 7.0) with 0.5 M NaCl (F).
3.3. Chitin-binding a⁄nities of ChBDCII , ChBDCICII and ChBDCICII mutant proteins To determine the binding a⁄nity of ChBDCII , ChBDCICII , and ChBDCICII mutant proteins on chitin, the adsorption isotherms were measured at pH 8.0 (Fig. 5). The adsorption isotherms for the double ChBD and single ChBD clearly showed that the binding a⁄nities of ChBDCII to colloidal and powdered chitin were markedly lower than that of the ChBDCICII . In order to further
Fig. 5. Adsorption isotherms for ChBDCII , ChBDCICII , and ChBDCICII mutants on colloidal chitin (A) and powdered chitin (B). (B) and (F) are the concentrations of bound and free protein, respectively.
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quantify the binding a⁄nities, the partition coe⁄cients (Kp ) were measured for ChBDCII , ChBDCICII , and ChBDCICII mutants from the initial slopes of adsorption isotherms shown in Fig. 5. As shown in Table 1, the partition coe⁄cients of ChBDCICII are about 10- and 12-fold greater toward colloidal and powdered chitin than those of ChBDCII . A similar result has been observed for a recombinant double CBD carrying two CBDs from Trichoderma reesei CBHI and CBHII [15]. The binding a⁄nity showed a 5^10-fold increase for the double CBD as compared with the single CBD toward Avicel. Linder et al. proposed a two-step model to describe the binding behavior of the double CBD [15]. Based on the model, the much higher a⁄nity of the double ChBD for chitin means that a cooperative interaction exists between ChBDCI and ChBDCII . Aromatic amino acids have been shown to play a prominent role in carbohydrate-binding domains, by forming hydrophobic stacking interaction with the sugar rings of their target substrates. In order to investigate the importance of the highly conserved aromatic amino acids in the interaction of ChBDCICII with chitin, we substituted Tyr796 and Trp797 in ChBDCI domain with alanine, which correspond to the surface aromatic residues located in the AKWWTQG motif. As seen in Table 1, the ChBDCICII mutants, W773A and W792A, only retained 16^23% and 20^23% of their binding a⁄nities on colloidal and powdered chitin. Therefore, Tyr796 and Trp797 are likely to be involved directly in chitin binding because of their positions on the surface. Site-directed mutagenesis studies of CBDEGZ showed that the equivalent residues (Trp43 and Trp44 in the AKWWTQG motif) play critical roles in cellulose binding [16]. In accordance with the NMR structure of CBDEGZ [3], Trp773 and Trp792 (corresponding to Trp13 and Tyr39 of CBDEGZ ) were predicted to be located in the hydrophobic core surrounding Pro803 (corresponding to Pro49 of CBDEGZ ). Therefore, we replaced Trp773 and Trp792 with alanine, and also replaced Trp773 with phenylalanine. As shown in Table 1, W773A and W792A mutation led to a dramatic loss of binding a⁄nity for colloidal and powdered chitin, while W792F mutant lost only about
Table 1 Partitioning coe⁄cients (Kp ) for the adsorption of ChBDCII , ChBDCICII , and ChBDCICII mutants to colloidal chitin and powdered chitin Protein
Kp (l g31 ) on colloidal chitin
ChBDCICII ChBDCII ChBDCICII ChBDCICII ChBDCICII ChBDCICII ChBDCICII
7.45 0.71 4.26 1.14 0.41 1.22 1.73
a
W773F W773A W792A Y796A W797A
(100)a (10) (57) (15) (6) (16) (23)
Kp (l g31 ) on powdered chitin 3.05 0.25 1.70 0.36 0.16 0.62 0.70
(100) (8) (56) (12) (5) (20) (23)
Values in parentheses represent percent relative adsorption a⁄nity.
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50% of its binding a⁄nity on chitin. Therefore, the phenylalanine in position 773 could retain some of the capability of forming hydrophobic interaction which results in a smaller decrease in chitin binding. Based on the structure of CBDEGZ [3], these data described above imply that residues Trp773 and Trp792 do not play direct roles in chitin binding, but are important in the correct folding of the protein. Further structural studies are required to determine if the reduced a⁄nities are a conformation change or a direct consequence of the mutation of the respective aromatic residues.
References [1] Tomme, P., Warren, R.A., Miller Jr., R.C., Kilburn, D.G. and Gilkes, N.R. (1995) Cellulose-binding domains: classi¢cation and properties. In: Enzymatic Degradation of Insoluble Polysaccharides (Saddler, J.N. and Penner, M., Eds.), pp. 142^163. American Chemical Society, Washington, DC. [2] Wu, M.L., Chuang, Y.C., Chen, J.P., Chen, C.S. and Chang, M.C. (2001) Identi¢cation and characterization of the three chitin-binding domains within the multidomain chitinase Chi92 from Aeromonas hydrophila JP101. Appl. Environ. Microbiol. 67, 5100^5106. [3] Brun, E., Moriaud, F., Gans, P., Blackledge, M.J., Barras, F. and Marion, D. (1997) Solution structure of the cellulose-binding domain of the endoglucanase Z secreted by Erwinia chrysanthemi. Biochemistry 36, 16074^16086. [4] van Aalten, D.M.F., Synstad, B., Brurberg, M.B., Hough, E., Riise, B.W., Eijsink, V.G.H. and Wierenga, R.K. (2000) Structure of a twoU resolution. domain chitotriosidase from Serratia marcescens at 1.9-A Proc. Natl. Acad. Sci. USA 97, 5842^5847. [5] Yanisch, P.C., Vieira, J. and Messing, J. (1985) Improved M13 phage cloning vectors and host strains : nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103^119. [6] Sambrook, J. and Russel, D.W. (2001) Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, Plainview, NY. [7] Higuchi, R., Krummel, B. and Saiki, R.K. (1988) A general method of in vitro preparation and speci¢c mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 16, 7351^ 7367. [8] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277, 680^685. [9] Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248^254. [10] Shimahara, K. and Takiguchi, Y. (1988) Preparation of crustacean chitin. Methods Enzymol. 161, 417^423. [11] Linder, M., Lindeberg, G., Reinikainen, T., Teeri, T.T. and Pettersson, G. (1995) The di¡erence in a⁄nity between two fungal cellulosebinding domain is dominated by a single amino acid substitution. FEBS Lett. 372, 96^98. [12] Itoh, Y., Kawase, T., Nikaidou, N., Fukada, H., Mitsutomi, M., Watanabe, T. and Itoh, Y. (2002) Functional analysis of the chitinbinding domain of a family 19 chitinase from Streptomyces griseus HUT6037: substrate-binding a⁄nity and cis-dominant increase of antifungal function. Biosci. Biotechnol. Biochem. 66, 1084^1092. [13] Tsujibo, H., Orikoshi, H., Shiotani, K., Hayashi, M., Umeda, J., Miyamoto, K., Imada, C., Okami, Y. and Inamori, Y. (1998) Characterization of chitinase C from a marine bacterium, Alteromonas sp. strain O-7, and its corresponding gene and domain structure. Appl. Environ. Microbiol. 64, 472^478.
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[14] Hashimoto, M., Ikegami, T., Seino, S., Ohuchi, N., Fukada, H., Sugiyama, J., Shirakawa, M. and Watanabe, T. (2000) Expression and characterization of the Chitin-binding domain of chitinase A1 from Bacillus circulans WL-12. J. Bacteriol. 182, 3045^3054. [15] Linder, M., Salovuori, I., Ruohonen, L. and Teeri, T.T. (1996) Characterization of a double cellulose-binding domain. Synergistic high
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a⁄nity binding to crystalline cellulose. J. Biol. Chem. 271, 21268^ 21272. [16] Helen, D.S. and Frederic, B. (1999) Functional analysis of the carbohydrate-binding domains of Erwinia chrysanthemi Cel5 (Endoglucanase Z) and an Escherichia coli putative chitinase. J. Bacteriol. 181, 4611^4616.
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