Biosci. Biotechnol. Biochem., 69 (1), 160–165, 2005

Functions of Family-22 Carbohydrate-Binding Module in thermocellum Xyn10C

Ehsan ALI, Guangshan ZHAO, Makiko SAKKA, Tetsuya KIMURA, y Kunio OHMIYA,* and Kazuo SAKKA

Faculty of Bioresources, Mie University, Tsu 514-8507, Japan

Received September 1, 2004; Accepted October 20, 2004

Clostridium thermocellum xylanase Xyn10C (formerly of the glycoside hydrolases. XynC) is a modular enzyme, comprising a family-22 Family-22 CBMs are often found with a family-10 carbohydrate-binding module (CBM), a family-10 cata- CM in a modular enzyme, e.g., Caldibacillus cellulo- lytic module of the glycoside hydrolases, and a dockerin vorans XynA,3) Clostridium josui Xyn10A,4) Clostridi- module responsible for cellulosome assembly consec- um stercorarium Xyn10B,5) Clostridium thermocellum utively from the N-terminus. To study the functions of Xyn10B,6) Polyplastron multivesiculatum Xyn10B,7) or the CBM, truncated derivatives of Xyn10C were con- Thermotoga maritima XynA.8) Family-22 CBMs were structed: a recombinant catalytic module polypeptide originally identified and specified as thermostabilizing (rCM), a family-22 CBM polypeptide (rCBM), and a modules since their removal from thermophilic enzymes polypeptide composed of the family-22 CBM and CM resulted in a decrease in the thermostability and/or (rCBM–CM). The recombinant proteins were charac- optimal temperature of the enzymes.6,8,9) Later, how- terized by enzyme and binding assays. Although the ever, these modules were compiled and classified in catalytic activity of rCBM–CM toward insoluble xylan family 22 of CBMs since they were found to have was four times higher than that of rCM toward the same affinities for different types of substrates, e.g., soluble substrate, removal of the CBM did not severely affect xylan,10–13) barley -glucan11–13) or insoluble .7) catalytic activity toward soluble xylan or -1,3-1,4- Recently, we have found that a truncated derivative of glucan. rCBM showed an affinity for amorphous C. stercorarium Xyn10B, composed of two family-22 and insoluble and soluble xylan in qualitative CBMs and a CM, preferred -1,3-1,4-glucan such as binding assays. The optimum temperature of rCBM– barley -glucan and lichenan to xylan as a substrate and CM was 80 C and that of rCM was 60 C. These results removal of the CBMs from this enzymes drastically indicate that the family-22 CBM of C. thermocellum reduced cataytic activity toward -1,3-1,4-glucan but Xyn10C not only was responsible for the binding of the not toward xylan, suggesting that the family-22 CBMs enzyme to the substrates, but also contributes to the in C. stercorarium Xyn10B are essential for hydrolysis stability of the CM in the presence of the substrate at of -1,3-1,4-glucan.13) These observations suggest that high temperatures. functions of the family-22 CBMs are not necessarily common in different enzymes. Key words: xylanase; carbohydrate-binding module; C. thermocellum xylanase Xyn10C (formerly thermostabilizing module; Clostridium ther- XynC)14) has been found in the cellulosome (a highly mocellum organized enzyme complex containing and hemicellulases) of C. thermocellum as one of major Many glycoside hydrolases including xylanase consist catalytic components, although this bacterium cannot of two or more functional modules, such as a catalytic grow on xylan. Although Xyn10C is known to be a module (CM) and a carbohydrate-binding module modular enzyme, comprising a family-22 CBM, a (CBM). Based on amino acid sequence similarities, family-10 CM of the glycoside hydrolases, and a CMs of glycoside hydrolases can be classified into 97 dockerin module responsible for cellulosome assembly families and CBMs into 39 families (http://afmb.cnrs- consecutively from the N-terminus (Fig. 1), the function mrs.fr/CAZY/index.html).1,2) CMs of xylanases can be of the family-22 CBM has not yet been characterized. In divided into two substantial groups: families 10 and 11 this study, we constructed three truncated derivatives of

y To whom correspondence should be addressed. Tel: +81-59-231-9621; Fax: +81-59-231-9684; E-mail: [email protected] * Present address: Agricultural High-Tech Research Center, Faculty of Agriculture, Meijo University, Tenpaku, Nagoya 468-8502, Japan Abbreviations: ASC, acid-swollen cellulose; BMC, ball-milled cellulose; CBM, carbohydrate-binding module; CMC, carboxymethyl-cellulose; CD, catalytic module; FPLC, fast protein liquid chromatography; HMC, hydroxymethyl-cellulose; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; Tris, Tris(hydroxymethyl)aminomethane Family-22 CBM of C. thermocellum Xyn10C 161 agarose gel using GFX PCR DNA and Gel band purification kit (Amersham). These inserts were ligated between the BamH1 and SalI sites of pQE-30T and introduced into E. coli M15. The combinations of the primers were as follows: primers Xyn10C-N and Xyn10C-CBM to construct pCBM, yielding rCBM; primers Xyn10C-CM and Xyn10C-C to construct pCM, yielding rCM; primers Xyn10C-N and Xyn10C-C to construct pCBM–CM, yielding rCBM–CM. Schematic diagrams of these proteins and nucleotide sequences of the PCR primers are shown in Fig. 1.

Purification of rCBM, rCM and rCBM–CM. To produce the recombinant proteins, 1.5-liter cultures of E. coli recombinants were grown to mid-log phase (absorbance at 600 nm, 0.6) and then isopropyl--D- Fig. 1. Schematic Diagram of C. thermocellum Xyn10C and Its thiogalactopyranoside was added to the cultures to give Derivatives (A), and PCR Primers Used for the Construction of the Truncated Derivatives (B). a final concentration of 1 mM. After an additional The precursor form of Xyn10C comprises a signal peptide (sp), a incubation of 3 h, the cells were harvested, washed, family-22 CBM (CBM), a family-10 CM (CM), and a dockerin and disrupted by sonication. Cell debris was removed by module. The artificial BamHI and SalI sites in PCR primers are centrifugation, and the cell extracts thus obtained were underlined. used for purification of the recombinant proteins. These proteins were purified by HiTrap Chelating HP (Amer- Xyn10C, a recombinant CM polypeptide (rCM), a CBM sham) column chromatography. The 6xHis tag was polypeptide composed of the family-22 CBM (rCBM), removed from the recombinant proteins by thrombin and a polypeptide composed of the family-22 CBM and digestion and proteins that did not adsorb to the column the catalytic module (rCBM–CM) and evaluated the due to the removal of the affinity tag were collected and function of the family-22 CBM by measuring the further purified with a Resource Q column (Amersham) thermal stability, binding affinity for various polysac- by using FPLC. The purity of each fraction was charides, and substrate specificity of these polypeptides. analyzed by SDS–polyacrylamide gel electrophoresis (PAGE). Protein concentration was determined with Materials and Methods bovine serum albumin as the standard, using the Micro BCA protein assay reagent kit (Pierce). Strains and growth conditions. The Escherichia coli strains used in this study were XL1-Blue (Stratagene) Enzyme assays. Xylanase activity was measured by a and M15 (Qiagen). Recombinant E. coli strains were 10-min incubation at 60 C in Britton and Robinson’s cultured at 37 C in Luria broth supplemented with universal buffer (50 mM phosphoric acid, 50 mM boric ampicillin (50 mg/ml) or both ampicillin (50 mg/ml) and acid, and 50 mM acetic acid [the pH was adjusted to 2 to kanamycin (50 mg/ml). 12 with 1 M NaOH]). Reducing sugars released from birchwood xylan (Sigma Chemical) or oat-spelt xylan Plasmids and plasmid constructions. The plasmid (Fluka Ag, Buchs, Switzerland) were determined with pKS103 carrying the C. thermocellum xyn10C gene was the 3,6-dinitropthalic acid reagent, as described by described previously.14) The plasmid vectors used in this Miller.16) One unit of enzyme activity was defined as the study were pT7-Blue (Novagen) and pQE-30T.15) amount of enzyme releasing 1 mmol of xylose equivalent Plasmids used to produce the truncated derivatives of per min from xylan. Enzyme activities towards barley - Xyn10C were constructed as follows: DNA fragments glucan (Sigma), lichenan (Sigma), hydroxymethyl-cel- encoding the family-22 CBM region and/or the family- lulose (HMC; Fluka), carboxymethyl-cellulose (CMC; 10 CM were amplified by PCR from pKS103 containing Sigma), were assayed as described above, except that xyn10C with KOD DASH DNA polymerase (Toyobo) xylan and xylose were replaced by each substrate and and an appropriate combination of primers (Fig. 1) product. containing an artificial BamH1 site or a SalI restriction The optimum temperatures of rCM and rCBM–CM site for cloning the PCR fragments into the expression were determined by assaying their enzyme activities vector pQE-30T. The resulting PCR fragments were with oat-spelt xylan as the substrate at various temper- ligated into pT7-Blue and introduced into E. coli XL1- atures at pH 7.0 for 10 min. Optimum pH was measured Blue by a standard transformation protocol. Plasmids by assaying their enzyme activities at 60 C in Britton purified from recombinant clones were digested with and Robinson’s universal buffer solutions (pH 6–8.5). restriction endonucleases BamH1 and SalI and the Thermostability and pH stability of the purified enzyme inserted DNA fragments were recovered from an were measured by incubating the enzyme solution at 162 E. ALI et al. various temperatures (50 to 90 C) for 10 min at pH 7.0 or incubating the enzyme solution in Britton and Robinson’s buffer (pH 4–11) for 1 h at 60 C, followed by measurement of residual enzyme activities under the standard conditions.

Qualitative carbohydrate-binding assays. Binding of rCBM to insoluble polysaccharides was determined as follows: rCBM (40 mg) was mixed with insoluble polysaccharides (10 mg) in a 10 mM sodium phosphate buffer (pH 6.3) in a final volume of 0.2 ml and incubated Fig. 2. SDS–PAGE of the Purified Proteins. on ice for 1 h with occasional stirring. After centrifuga- The gel was stained with Coomasssie brilliant blue: lane 1, rCBM–CM; lane 2, rCM; lane 3, rCBM; lane M, protein molecular tion, the supernatant and the precipitated polysaccha- mass standard (molecular masses shown at the right). rides were analyzed by SDS–polyacrylamide gel elec- trophoresis (PAGE). The polysaccharides tested were Avicel (FMC), acid-swollen cellulose (ASC), ball- Family-22 CBMs were originally identified as ther- milled cellulose (BMC), the insoluble fraction of oat- mostabilizing modules, i.e., shifts (10–20 C) in opti- spelt xylan (Fluka), lichenan, and agar (Nacalai Tesque). mum temperature upon artificial removal of family-22 ASC and BMC were prepared in this laboratory from CBMs from thermophilic modular xylanases were KC Floc cellulose powder (Nippon Paper Chemicals), as observed in C. cellulovorans XynA (from 90 to described previously.17) 70 C),3) C. stercorarium Xyn10B (75 to 60 C),13) The affinities of these proteins for soluble polysac- C. thermocellum XynX (70 to 60 C)19) and XynY (75 charides, viz, CMC, HMC, barley -glucan (Sigma), and to 60 C),6) and T. saccharolyticum XynA (75 to 65 C)9) birchwood xylan (Sigma), were examined by native in the presence of the substrate. Furthermore, an affinity PAGE as described by Meissner et al.,12) with increase in thermostability in the absence of the some simplifications. We used the Laemmli system18) substrate was observed in C. cellulovorans XynA, for electrophoresis, excluding SDS from all solutions. C. thermocellum XynX, and C. thermocellum XynX The separating gel contained 10% acrylamide. The and XynY. Therefore, the effects of temperature and polysaccharides were incorporated into the gel at a pH were determined on the activity and stability of rCM concentration of 0.1% prior to polymerization. A control and rCBM–CM. When the xylanase activity of the gel without any polysaccharides was prepared and run truncated enzymes was determined by a 10-min incu- simultaneously. Protein samples were loaded onto gels bation at various temperatures, the optimum temperature in a standard loading buffer without SDS. Electro- of rCBM–CM was determined to be approximately phoresis was run at 4 C and 100 V for 2 h. Proteins were 80 C but that of rCM was approximately 60 C visualized by Coomassie blue staining. (Fig. 3A). This observation confirms the thermostabiliz- ing effect of family-22 CBMs in the presence of the Results and Discussion substrate. By contrast, no significant difference was observed between the thermal stability of rCBM–CM Construction and production of the Xyn10C deriva- and that of rCM (Fig. 3B) when they were heated in the tives absence of the substrate for 10 min. It is possible that the Truncated forms of Xyn10C were constructed as interaction between the CM and the CBM increases in described in ‘‘Materials and Methods’’. rCBM is the the presence of xylan, resulting in stabilization of the family-22 CBM polypeptide, rCM is the family-10 CM CM only in the presence of the substrate. As shown in polypeptide, and rCBM–CM is the polypeptide com- Fig. 3C and D, although the presence of the family-22 posed of the CBM and the CM collectively (Fig. 1). CBM adjacent to the CM negligibly affected the These proteins were purified from the whole-cell lysates optimum pH of the enzyme, rCBM–CM was slightly of E. coli recombinants using HiTrap Chelating HP more stable in acidic pH range and less stable in the column and Resource Q column at FPLC. Each of the alkaline pH range than rCM. In mesophilic xylanase purified preparations gave a single band on SDS–PAGE, Xyn10B from P. multivesiculatum, removal of the CBM and the molecular sizes of the purified rCBM–CM, rCM, did not affect its optimum temperature (40 C) but and rCBM were in good agreement with those deduced shifted its optimum pH from 7.0 to 8.0.7) from the nucleotide sequences (MW: 58,111 for rCBM– CM; 40,140 for rCM; and 18,134 for rCBM) (Fig. 2). Binding of rCBM to insoluble and soluble polysac- These purified proteins were used for enzyme and charides polysaccharide-binding assays. To investigate the function of the family-22 CBM of Xyn10C in hydrolytic action, we qualitatively examined Effects of temperature and pH on the activity and the ability of rCBM to bind insoluble polysaccharides by stability of rCM and rCBM–CM incubating the protein with the polysaccharides and Family-22 CBM of C. thermocellum Xyn10C 163

Fig. 3. Effects of Temperature on Activity (A) and Stability (B), and Effects of pH on Activity (C) and Stability (D) of rCBM–CM and rCM. Assay procedures are described in ‘‘Materials and Methods’’. Symbols: , rCBM–CM; , rCM.

Fig. 4. Adsorption of rCBM to Insoluble Polysaccharides. rCBM was incubated with insoluble polysaccharides including Avicel (2), ASC (3), BMC (4), the insoluble fraction of oat-spelt xylan (5), lichenan (6), and agar (7). After centrifugation, proteins in the precipitate (A) and in the supernatant (B) were analyzed by Fig. 5. Adsorption of rCBM to Soluble Polysaccharides. SDS–PAGE. Lane 1 contains the purified rCBM as control. The ffinities of rCBM for various soluble polysaccharides including birchwood xylan (b), barley -glucan (c), HMC (d), and CMC (e) were analyzed by native affinity PAGE. A gel without a comparing the protein concentrations in the supernatant polysaccharide served as a reference (a). Lanes 1 and M contain fraction (unbound protein) and in the precipitate fraction rCBM and bovine serum albumin as a control protein respectively. (bound protein) by SDS–PAGE. As shown in Fig. 4, rCBM showed relatively high affinity for BMC as xylan. By contrast, this CBM showed only a slight compared with Avicel, suggesting that this CBM affinity for barley -glucan and CMC and no affinity for polypeptide prefers amorphous cellulose to crystalline HMC. cellulose. rCBM was found to associate with the Although family-22 CBMs were specified at first as insoluble fraction of oat-spelt xylan. On the other hand, thermostabilizing modules, some of these modules were rCBM showed only low affinity for lichenan and found to have an affinity for soluble xylan and -1,3-1,4- negligible affinity for agar. glucan by qualitative and quantitative analyses and were To confirm the affinity of rCBM for soluble polysac- reassigned to family 22 of CBMs, e.g., the CBM of charides, this protein was subjected to native affinity C. thermocellum XynY showed a high affinity for rye PAGE analysis. As shown in Fig. 5, migration of the arabinoxylan (Ka, 1:1 105) and a low affinity for protein was strongly retarded in the gel containing barley -glucan (Ka, 7:8 102);11) one of two repeated birchwood xylan as compared to the control gel, CBMs of T. maritima XynA was shown by native indicating that it has a strong affinity for birchwood affinity PAGE to have an affinity for various kinds of 164 E. ALI et al. xylans such as oat-spelt xylan and birchwood xylan, Artificial connection of a family-6 CBM derived from barley -glucan, lichenan, hydroxyethyl-cellulose, and C. stercorarium XynA to Ruminococcus albus endoglu- methyl-cellulose but not for CMC;12) the CBM of canase EGVI improved the hydrolytic activity of the C. cellulovorans selectively bound to soluble xylan and R. albus enzyme toward insoluble cellulose but not weakly to hydroxyethyl-cellulose;3) and the CBMs of soluble cellulose.22) It was also found that the CBMs of C. stercorarium were qualitatively shown to have a high the Pseudomonas fluorescens subsp. cellulosa xylanase affinity for barley -glucan in addition to soluble XynA and arabinofuranosidase XylC potentiated cata- xylan.13) Although many family-22 CBMs showed lytic activity toward complex substrates.23,24) In these affinities only for soluble polysaccharides, it was found cases, it is likely that these CBMs contribute to the that the CBM of P. multivesiculatum bound strongly to enrichment of the enzymes on the surface of the celluloses of various crystallinities.7) The binding insoluble substrates, resulting in increased contact of specificity of C. thermocellum Xyn10C is similar to the enzymes with the substrates. that of P. multivesiculatum Xyn10B in that they have The CBM of Xyn10C showed an affinity not only for affinities for xylan and insoluble cellulose (Fig. 4) xylan but also for cellulose. The presence of the family- although the former appears to prefer amorphous 22 CBD in Xyn10C places the enzyme on cellulose in cellulose to crystalline cellulose. plant cell walls, resulting in an increase in substrate In the CBM of C. thermocellum XynY, Arg-583, Trp- concentration around the enzyme, since plant cell walls 611, Tyr-660, Tyr-136, and Glu-138 were identified as consist of cellulose, hemicellulose including xylan, and ligand-binding sites and/or critical residues for main- lignin. taining the structural integrity of the binding cleft,20) and Recently we found that the family-22 CBMs in these five residues were conserved in C. thermocellum C. stercorarium Xyn10B have an essential role in Xyn10C and the other related CBMs. hydrolysis of -1,3-1,4-glucan such as barley -glucan and lichenan. A truncated derivative of Xyn10B com- Substrate specificity of rCM and rCBM–CM prising two family-22 CBMs and a family-10 CM The substrate specificities of rCM and rCBM–CM showed stronger activity on -1,3-1,4-glucan than on were compared toward a series of different polysaccha- xylan although the family-10 of glycoside hydrolases is rides such as oat-spelt-xylan and barley -glucan. As well known as a group of xylanases, and removal of the shown in Fig. 6, the hydrolytic activity of rCBM–CM CBMs from this enzyme drastically reduced hydrolytic toward the insoluble fraction of oat-spelt xylan was activity toward -1,3-1,4-glucan but not toward xylan.13) about 4 times greater than that of rCM although that of This phenomenon was explained in the previous paper the former toward birchwood xylan was only 1.4 times as follows: the family-22 CBMs bind more strongly to higher than that of the latter, indicating that the presence glucan than to xylan, and hence, it is likely that the of the family-22 CBMs enhanced the catalytic activity CBMs act to direct its polysaccharide ligand into the of the CM toward the insoluble substrate but not the active site of the enzyme, thus creating a very large soluble substrate. Similar phenomena were reported for effective concentration of substrate at the active site. By C. stercorarium Xyn10B,21) i.e., removal of a family-9 contrast, as shown in Fig. 6, the hydrolytic activity of CBM from the truncated enzyme composed of a family- rCBM–CM toward barley -glucan and lichenan was 10 CM and the CBM negated its cellulose- and xylan- substantially lower than that toward oat-spelt xylan or binding abilities and severely reduced its enzyme birchwood xylan, and removal of the CBM from rCBM– activity towards insoluble xylan but not soluble xylan. CM did not strongly affect activity toward -1,3-1,4- glucan. Since rCBM showed a weaker binding affinity for -1,3-1,4-glucan than for xylan (Figs. 4 and 5), it is possible that the CBM cannot provide effectively the CM adjacent to the CBM with -1,3-1,4-glucan as the substrate. Alternatively, it is likely that C. thermocellum Xyn10C is an ordinary xylanolytic enzyme with sub- strate specificity restricted to xylan but that C. stercora- rium Xyn10B is adapted for hydrolysis of -1,3-1,4- glucan rather than xylan. This supposition is perhaps supported by the finding that the xylanase activity of rCBM–CM was about 10 times stronger than that of C. stercorarium Xyn10B.13) The results obtained for C. thermocellum Xyn10C and C. stercorarium Xyn10B suggest that the function and role of family-22 CBMs must be different in different enzymes. Fig. 6. Specific Activities of rCBM–CM and rCM toward Various Polysaccharides Such as Oat-Spelt Xylan (A), Birchwood Xylan (B), In this study, we found that family-22 CBM of Barley -Glucan (C), Lichenan (D), HMC (E), and CMC (F). C. thermocellum Xyn10C bound to soluble and insolu- Symbols: closed bar, rCBM–CM; open bar, rCM. ble xylan as well as to insoluble cellulose and that the Family-22 CBM of C. thermocellum Xyn10C 165 presence of the CBM enhanced the hydrolytic activity of thermostabilizing domain of the modular xylanase XynA rCBM–CM toward xylan but not toward -1,3-1,4- of Thermotoga maritima represents a novel type of glucan. binding domain with affinity for soluble xylan and mixed-linkage -1,3/-1,4-glucan. Mol. Microbiol., 36, 898–912 (2000). References 13) Araki, R., Ali, M. K., Sakka, M., Kimura, T., Sakka, K., and Ohmiya, K., Essential role of the family-22 1) Henrissat, B., and Bairoch, A., Updating the sequence- carbohydrate-binding modules for -1,3-1,4-glucanase based classification of glycosyl hydrolases. Biochem. J., activity of Clostridium stercorarium Xyn10B. FEBS 316, 695–696 (1996). Lett., 561, 155–158 (2004). 2) Boraston, A. B., Bolam, D. N., Gilbert, H. J., and Davies, 14) Hayashi, H., Takagi, K., Fukumura, M., Kimura, T., G. J., Carbohydrate-binding modules: fine tuning poly- Karita, S., Sakka, K., and Ohmiya, K., Sequence of xynC saccharide recognition. Biochem. J., Immediate Publica- and properties of XynC, a major component of the tion, doi:10.1042/BJ20040892. Clostridium thermocellum cellulosome. J. Bacteriol., 3) Sunna, A., Gibbs, M. D., and Bergquist, P. L., A novel 179, 4246–4253 (1997). thermostable multidomain 1,4--xylanase from ‘Caldi- 15) Ali, M. K., Hayashi, H., Karita, S., Goto, M., Kimura, T., bacillus cellulovorans’ and effect of its xylan-binding Sakka, K., and Ohmiya, K., Importance of the carbohy- domain on enzyme activity. Microbiology, 146, 2947– drate-binding module of Clostridium stercorarium 2955 (2000). Xyn10B to xylan hydrolysis. Biosci. Biotechnol. Bio- 4) Feng, J.-X., Karita, S., Fujino, E., Fujino, T., Kimura, T., chem., 65, 41–47 (2001). Sakka, K., and Ohmiya, K., Cloning, sequencing and 16) Miller, G. L., Use of dinitrosalicylic acid reagent for expression of the gene encoding a cell-bound multi- determination of reducing sugar. Anal. Chem., 31, 426– domain xylanase from Clostridium josui and character- 428 (1959). ization of the translated product. Biosci. Biotechnol. 17) Lamed, R., Kenig, R., Setter, E., and Bayer, E. A., Major Biochem., 64, 2614–2624 (2000). characteristics of the cellulolytic system of Clostridium 5) Ali, M. K., Fukumura, M., Sakano, K., Karita, S., thermocellum coincide with those of the purified Kimura, T., Sakka, K., and Ohmiya, K., Cloning, cellulosome. Enzyme Microb. Technol., 7, 37–41 (1985). sequencing, and expression of the gene encoding the 18) Laemmli, U. K., Cleavage of structural proteins during Clostridium stercorarium xylanase C in Escherichia coli. the assembly of the head of bacteriophage T4. Nature, Biosci. Biotechnol. Biochem., 63, 1596–1604 (1999). 227, 680–685 (1970). 6) Fontes, C. M. G. A., Hazlewood, G. P., Morag, E., Hall, 19) Kim, H., Jung, K. H., and Pack, M. Y., Molecular J., Hirst, B. H., and Gilbert, H. J., Evidence for a general characterization of xynX, a gene encoding a multidomain role for non-catalytic thermostabilizing domains in xylanase with a thermostabilizing domain from Clostri- xylanases from thermophilic . Biochem. J., 307, dium thermocellum. Appl. Microbiol. Biotechnol., 54, 151–158 (1995). 521–527 (2000). 7) Devillard, E., Bera-Maillet, C., Flint, H. J., Scott, K. P., 20) Xie, H., Gilbert, H. J., Charnock, S. J., Davies, G. J., Newbold, C. J., Wallace, R. J., Jouany, J. P., and Forano, Williamson, M. P., Simpson, P. J., Raghothama, S., E., Characterization of XYN10B, a modular xylanase Fontes, C. M. G. A., Dias, F. M. V., Ferreira, L. M. A., from the ruminal protozoan Polyplastron multivesicula- and Bolam, D. N., Clostridium thermocellum Xyn10B tum, with a family 22 carbohydrate-binding module that carbohydrate-binding module 22-2: the role of conserved binds to cellulose. Biochem. J., 373, 495–503 (2003). amino acids in ligand binding. Biochemistry, 40, 9167– 8) Winterhalter, C., Heinrich, P., Candussio, A., Wich, G., 9176 (2001). and Liebl, W., Identification of a novel cellulose-binding 21) Ali, M. K., Hayashi, H., Karita, S., Goto, M., Kimura, T., domain within the multidomain 120 kDa xylanase XynA Sakka, K., and Ohmiya, K., Importance of the carbohy- of the hyperthermophilic bacterium Thermotoga mariti- drate-binding module of Clostridium stercorarium ma. Mol. Microbiol., 15, 431–444 (1995). Xyn10B to xylan hydrolysis. Biosci. Biotechnol. Bio- 9) Lee, Y.-E., Lowe, S. L., Henrissat, B., and Zeikus, G., chem., 65, 41–47 (2001). Characterization of the active site and thermostability 22) Karita, S., Sakka, K., and Ohmiya, K., Cellulose-binding regions of endoxylanase from Thermoanaerobacterium domains confer an enhanced activity against insoluble saccharolyticum B6A-RI. J. Bacteriol., 175, 5890–5898 cellulose to Ruminococcus albus endoglucanase IV. J. (1993). Ferment. Bioeng., 81, 553–556 (1996). 10) Sunna, A., Gibbs, M. D., and Bergquist, P. L., The 23) Black, G. W., Rixon, J. E., Clarke, J. H., Hazlewood, G. thermostabilizing domain, XynA, of Caldibacillus cel- P., Theodorou, M. K., Morris, P., and Gilbert, H. J., lulovorans xylanase is a xylan binding domain. Biochem. Evidence that linker sequences and cellulose-binding J., 346, 583–586 (2000). domains enhance the activity of hemicellulases against 11) Charnock, S. J., Bolam, D. N., Turkenburg, J. P., Gilbert, complex substrates. Biochem. J., 319, 515–520 (1996). H. J., Ferreira, L. M., Davies, G. J., and Fontes, C. M. G. 24) Gill, J., Rixon, J. E., Bolam, D. N., McQueen-Mason, S., A., The X6 ‘‘thermostabilizing’’ domains of xylanases Simpson, P. J., Williamson, M. P., Hazlewood, G. P., are carbohydrate-binding modules: structure and bio- and Gilbert, H. J., The type II and X cellulose-binding chemistry of the Clostridium thermocellum X6b domain. domains of Pseudomonas xylanase A potentiate catalytic Biochemistry, 39, 5013–5021(2000). activity against complex substrates by a common 12) Meissner, K., Wassenberg, D., and Liebl, W., The mechanism. Biochem. J., 342, 473–480 (1999).