Functions of family-22 carbohydrate-binding modules in Clostridium josui Xyn10A

Clostridium josui xylanase Xyn10A is a modular enzyme comprising two family-22 carbohydrate-binding modules (CBMs), a family-10 catalytic module (CM), a family-9 CBM, and two S-layer homologous modules, consecutively from the N-terminus. To study the functions of the family-22 CBMs, truncated derivatives of Xyn10A were constructed: a recombinant CM polypeptide (rCM), a family-22 CBM polypeptide (rCBM), and a polypeptide composed of the family-22 CBMs and CM (rCBM-CM). Recombinant proteins were characterized by enzyme and binding assays. rCBM-CM showed the highest activity toward xylan and weak activity toward some polysaccharides such as barley beta-glucan and carboxymethyl-cellulose. Although rCBM showed an affinity for insoluble and soluble xylan as well as barley beta-glucan and Avicel in qualitative binding assays, removal of the CBMs negligibly affected the catalytic activity and thermostability of the CM.     

Function of the family-9 and family-22 carbohydrate-binding modules in a modular beta-1,3-1,4-glucanase/xylanase derived from Clostridium stercorarium Xyn10B

Clostridium stercorarium Xyn10B having hydrolytic activities on xylan and beta-1,3-1,4-glucan is a modular enzyme composed of two family-22 carbohydrate-binding modules (CBMs), a family-10 catalytic module of the glycoside hydrolases, a family-9 CBM, and two S-layer homologous modules, consecutively from the N-terminus. We investigated the function of family-9 and family-22 CBMs in a modular enzyme by comparing the enzymatic properties of a truncated enzyme composed of two family-22 CBMs and the catalytic module (rCBM22-CM), an enzyme composed of the catalytic module and family-9 CBM (rCM-CBM9), an enzyme composed of two family-22 CBMs, the catalytic module, and family-9 CBM (rCBM22-CM-CBM9), and the catalytic module polypeptide (rCM). Although the addition of family-9 CBM to rCM and rCBM22-CM did not significantly change catalytic activity toward xylan and beta-1,3-1,4-glucan, the addition of family-22 CBM to rCM and rCM-CBM9 drastically enhanced catalytic activity toward xylan and especially beta-1,3-1,4-glucan. Furthermore, the addition of family-22 CBM to rCM and rCM-CBM9 shifted the optimum temperature from 65 degrees C to 75 degrees C, but that of family-9 CBM to rCM and rCBM22-CM did not affect the optimum temperature. These facts suggest that the enzyme properties of Xyn10B were mainly dependent on the presence of the family-22 CBMs but not family-9 CBM.     

The role of carbohydrate-binding module (CBM) repeat of a multimodular xylanase (XynX) from Clostridium thermocellum in cellulose and xylan binding

A non-cellulosomal xylanase from Clostridium thermocellum, XynX, consists of a family-22 carbohydratebinding module (CBM22), a family-10 glycoside hydrolase (GH10) catalytic module, two family-9 carbohydrate-binding modules (CBM9-I and CBM9-II), and an S-layer homology (SLH) module. E. coli BL21(DE3) (pKM29), a transformant carrying xynX', produced several truncated forms of the enzyme. Among them, three major active species were purified by SDS-PAGE, activity staining, gel-slicing, and diffusion from the gel. The truncated xylanases were different from each other only in their C-terminal regions. In addition to the CBM22 and GH10 catalytic modules, XynX(1) had the CBM9-I and most of the CBM9-II, XynX(2) had the CBM9-I and about 40% of the CBM9-II, and XynX(3) had about 75% of the CBM9-I. The truncated xylanases showed higher binding capacities toward Avicel than those toward insoluble xylan. XynX(1) showed a higher affinity toward Avicel (70.5%) than XynX(2) (46.0%) and XynX(3) (42.1%); however, there were no significant differences in the affinities toward insoluble xylan. It is suggested that the CBM9 repeat, especially CBM9-II, of XynX plays a role in xylan degradation in nature by strengthening cellulose binding rather than by enhancing xylan binding.     

Importance of the carbohydrate-binding module of Clostridium stercorarium Xyn10B to xylan hydrolysis

The Clostridium stercorarium xylanase Xyn10B is a modular enzyme comprising two thermostabilizing domains, a family 10 catalytic domain of glycosyl hydrolases, a family 9 carbohydrate-binding module (CBM), and two S-layer homologous (SLH) domains [Biosci. Biotechnol. Biochem., 63, 1596-1604 (1999)]. To investigate the role of this CBM, we constructed two derivatives of Xyn10B and compared their hydrolytic activity toward xylan and some preparations of plant cell walls; Xyn10BdeltaCBM consists of a catalytic domain only, and Xyn10B-CBM comprises a catalytic domain and a CBM. Xyn10B-CBM bound to various insoluble polysaccharides including Avicel, acid-swollen cellulose, ball-milled chitin, Sephadex G-25, and amylose-resin. A cellulose binding assay in the presence of soluble saccharides suggested that the CBM of Xyn10B had an affinity for even monosaccharides such as glucose, galactose, xylose, mannose and ribose. Removal of the CBM from the enzyme negated its cellulose- and xylan-binding abilities and severely reduced its enzyme activity toward insoluble xylan and plant cell walls but not soluble xylan. These findings clearly indicated that the CBM of Xyn10B is important in the hydrolysis of insoluble xylan. This is the first report of a family 9 CBM with an affinity for insoluble xylan in addition to crystalline cellulose and the ability to increase hydrolytic activity toward insoluble xylan.     

Characterization of Xyn30A and Axh43A of Bacillus licheniformis SVD1 identified by its genomic analysis

The genome sequence of Bacillus licheniformis SVD1, that produces a cellulolytic and hemi-cellulolytic multienzyme complex, was partially determined, indicating that the glycoside hydrolase system of this strain is highly similar to that of B. licheniformis ATCC14580. All of the fifty-six genes encoding glycoside hydrolases identified in B. licheniformis ATCC14580 were conserved in strain SVD1. In addition, two new genes, xyn30A and axh43A, were identified in the B. licheniformis SVD1 genome. The xyn30A gene was highly similar to Bacillus subtilis subsp. subtilis 168 xynC encoding for a glucuronoarabinoxylan endo-1,4-β-xylanase. Xyn30A, produced by a recombinant Escherichia coli, had high activity toward 4-O-methyl-d-glucurono-d-xylan but showed definite activity toward oat-spelt xylan and unsubstituted xylooligosaccharides. Recombinant Axh43A, consisting of a family-43 catalytic module of the glycoside hydrolases and a family-6 carbohydrate-binding module (CBM), was an arabinoxylan arabinofuranohydrolase (α-l-arabinofuranosidase) classified as AXH-m23 and capable of releasing arabinosyl residues, which are linked to the C-2 or C-3 position of singly substituted xylose residues in arabinoxylan or arabinoxylan oligomers. The isolated CBM polypeptide had an affinity for soluble and insoluble xylans and removal of the CBM from Axh43A abolished the catalytic activity of the enzyme, indicating that the CBM plays an essential role in hydrolysis of arabinoxylan.     

Characterization of Paenibacillus curdlanolyticus B-6 Xyn10D, a xylanase that contains a family 3 carbohydrate-binding module

Paenibacillus curdlanolyticus B-6 Xyn10D is a xylanase containing a family 3 carbohydrate-binding module (CBM3). Biochemical analyses using recombinant proteins derived from Xyn10D suggested that the CBM3 polypeptide has an affinity for cellulose and xylan and that CBM3 in Xyn10D is important for hydrolysis of insoluble arabinoxylan and natural biomass.     

Cloning, sequencing, and expression of the gene encoding a cell-bound multi-domain xylanase from Clostridium josui, and characterization of the translated product

The nucleotide sequence of the Clostridium josui FERM P-9684 xyn10A gene, encoding a xylanase Xyn10A, consists of 3,150 bp and encodes 1,050 amino acids with a molecular weight of 115,564. Xyn10A is a multidomain enzyme composed of an N-terminal signal peptide and six domains in the following order: two thermostabilizing domains, a family 10 xylanase domain, a family 9 carbohydrate-binding module (CBM), and two S-layer homologous (SLH) domains. Immunological analysis indicated the presence of Xyn10A in the culture supernatant of C. josui FERM P-9684 and on the cell surface. The full-length Xyn10A expressed in a recombinant Escherichia coli strain bound to ball-milled cellulose (BMC) and the cell wall fragments of C. josui, indicating that both the CBM and the SLH domains are fully functional in the recombinant enzyme. An 85-kDa xylanase species derived from Xyn10A by partial proteolysis at the C-terminal side, most likely at the internal region of the CBM, retained the ability to bind to BMC. This observation suggests that the catalytic domain or the thermostabilizing domains are responsible for binding of the enzyme to BMC. Xyn10A-II, the 100-kDa derivative of Xyn10A, was purified from the recombinant E. coli strain and characterized. The enzyme was highly active toward xylan but not toward p-nitrophenyl-beta-D-xylopyranoside, p-nitrophenyl-beta-D-cellobioside, or carboxymethylcellulose.     

Molecular and biochemical characterization of <Emphasis Type="Italic">Ba</Emphasis>-EGA,a cellulase secreted by <Emphasis Type="Italic">Bacillus</Emphasis> sp. AC-1 from <Emphasis Type="Italic">Ampullaria crosseans</Emphasis>

A novel gene (Ba-ega) of Bacillus sp. AC-1, encoding an endoglucanase (Ba-EGA), was cloned and expressed in Escherichia coli. Ba-ega, containing a 1,980-bp open reading frame (ORF), encoded a protein of 659 amino acids and had a molecular mass of 74.87 kDa. Ba-EGA was a modular enzyme composed of a family-9 glycosyl hydrolase catalytic module (CM9) and a family-3 carbohydrate-binding module (CBM3). To investigate the functions of the CBM3 and CM9, a number of truncated derivatives of Ba-EGA were constructed, and all were active. The catalytic module (rCM9) alone was less stable at high temperature than the recombinant Ba-EGA (rBa-EGA). The temperature stability for the complex of rCM9 and rCBM3 was still lower than rBa-EGA, but higher than rCM9 alone. These observations indicated the existence of a non-covalent interaction between CM9 and CBM3 that might strengthen the stability of CM9. However, this interaction is not strong enough to mimic the protective effect of the CBM in the wild-type enzyme.     

Modular glucuronoxylan-specific xylanase with a family CBM35 carbohydrate-binding module

Xyn30D from the xylanolytic strain Paenibacillus barcinonensis has been identified and characterized. The enzyme shows a modular structure comprising a catalytic module family 30 (GH30) and a carbohydrate-binding module family 35 (CBM35). Like GH30 xylanases, recombinant Xyn30D efficiently hydrolyzed glucuronoxylans and methyl-glucuronic acid branched xylooligosaccharides but showed no catalytic activity on arabinose-substituted xylans. Kinetic parameters of Xyn30D were determined on beechwood xylan, showing a K(m) of 14.72 mg/ml and a k(cat) value of 1,510 min(-1). The multidomain structure of Xyn30D clearly distinguishes it from the GH30 xylanases characterized to date, which are single-domain enzymes. The modules of the enzyme were individually expressed in a recombinant host and characterized. The isolated GH30 catalytic module showed specific activity, mode of action on xylan, and kinetic parameters that were similar to those of the full-length enzyme. Computer modeling of the three-dimensional structure of Xyn30D showed that the catalytic module is comprised of a common (β/α)(8) barrel linked to a side-associated β-structure. Several derivatives of the catalytic module with decreasing deletions of this associated structure were constructed. None of them showed catalytic activity, indicating the importance of the side β-structure in the catalysis of Xyn30D. Binding properties of the isolated carbohydrate-binding module were analyzed by affinity gel electrophoresis, which showed that the CBM35 of the enzyme binds to soluble glucuronoxylans and arabinoxylans. Analysis by isothermal titration calorimetry showed that CBM35 binds to glucuronic acid and requires calcium ions for binding. Occurrence of a CBM35 in a glucuronoxylan-specific xylanase is a differential trait of the enzyme characterized.     

Influence of a mannan binding family 32 carbohydrate binding module on the activity of the appended mannanase

In general, cellulases and hemicellulases are modular enzymes in which the catalytic domain is appended to one or more noncatalytic carbohydrate binding modules (CBMs). CBMs, by concentrating the parental enzyme at their target polysaccharide, increase the capacity of the catalytic module to bind the substrate, leading to a potentiation in catalysis. Clostridium thermocellum hypothetical protein Cthe_0821, defined here as C. thermocellum Man5A, is a modular protein comprising an N-terminal signal peptide, a family 5 glycoside hydrolase (GH5) catalytic module, a family 32 CBM (CBM32), and a C-terminal type I dockerin module. Recent proteomic studies revealed that Cthe_0821 is one of the major cellulosomal enzymes when C. thermocellum is cultured on cellulose. Here we show that the GH5 catalytic module of Cthe_0821 displays endomannanase activity. C. thermocellum Man5A hydrolyzes soluble konjac glucomannan, soluble carob galactomannan, and insoluble ivory nut mannan but does not attack the highly galactosylated mannan from guar gum, suggesting that the enzyme prefers unsubstituted β-1,4-mannoside linkages. The CBM32 of C. thermocellum Man5A displays a preference for the nonreducing ends of mannooligosaccharides, although the protein module exhibits measurable affinity for the termini of β-1,4-linked glucooligosaccharides such as cellobiose. CBM32 potentiates the activity of C. thermocellum Man5A against insoluble mannans but has no significant effect on the capacity of the enzyme to hydrolyze soluble galactomannans and glucomannans. The product profile of C. thermocellum Man5A is affected by the presence of CBM32.     

The N-terminal family 22 carbohydrate-binding module of xylanase 10B of Clostridium themocellum is not a thermostabilizing domain

Xylanase Xyn10B from Clostridium thermocellum is a modular enzyme that contains two family 22 carbohydrate binding modules N- (CBM22-1) and C- (CBM22-2) terminal of the family 10 glycoside hydrolase catalytic domain (GH10). In a previous study, we showed that removal of CBM22-1 reduces the resistance to thermoinactivation of the enzyme suggesting that this module is a thermostabilizing domain. Here, we show that it is the module border on the N-terminal side of GH10 that confers resistance to thermoinactivation and to proteolysis. Therefore, CBM22-1 does not function as a thermostabilizing domain and the role for this apparently non-functional CBM remains elusive.     

Exploring Multimodularity in Plant Cell Wall Deconstruction: STRUCTURAL AND FUNCTIONAL ANALYSIS OF Xyn10C CONTAINING THE CBM22-1–CBM22-2 TANDEM*

Elucidating the molecular mechanisms regulating multimodularity is a challenging task. Paenibacillus barcinonensis Xyn10C is a 120-kDa modular enzyme that presents the CBM22/GH10/CBM9 architecture found in a subset of large xylanases. We report here the three-dimensional structure of the Xyn10C N-terminal region, containing the xylan-binding CBM22-1–CBM22-2 tandem (Xyn10C-XBD), which represents the first solved crystal structure of two contiguous CBM22 modules. Xyn10C-XBD is folded into two separate CBM22 modules linked by a flexible segment that endows the tandem with extraordinary plasticity. Each isolated domain has been expressed and crystallized, and their binding abilities have been investigated. Both domains contain the R(W/Y)YYE motif required for xylan binding. However, crystallographic analysis of CBM22-2 complexes shows Trp-308 as an additional binding determinant. The long loop containing Trp-308 creates a platform that possibly contributes to the recognition of precise decorations at subsite S2. CBM22-2 may thus define a subset of xylan-binding CBM22 modules directed to particular regions of the polysaccharide. Affinity electrophoresis reveals that Xyn10C-XBD binds arabinoxylans more tightly, which is more apparent when CBM22-2 is tested against highly substituted xylan. The crystal structure of the catalytic domain, also reported, shows the capacity of the active site to accommodate xylan substitutions at almost all subsites. The structural differences found at both Xyn10C-XBD domains are consistent with the isothermal titration calorimetry experiments showing two sites with different affinities in the tandem. On the basis of the distinct characteristics of CBM22, a delivery strategy of Xyn10C mediated by Xyn10C-XBD is proposed.     

Evidence for synergy between family 2b carbohydrate binding modules in Cellulomonas fimi xylanase 11A

Glycoside hydrolases often contain multiple copies of noncatalytic carbohydrate binding modules (CBMs) from the same or different families. Currently, the functional importance of this complex molecular architecture is unclear. To investigate the role of multiple CBMs in plant cell wall hydrolases, we have determined the polysaccharide binding properties of wild type and various derivatives of Cellulomonas fimi xylanase 11A (Cf Xyn11A). This protein, which binds to both cellulose and xylan, contains two family 2b CBMs that exhibit 70% sequence identity, one internal (CBM2b-1), which has previously been shown to bind specifically to xylan and the other at the C-terminus (CBM2b-2). Biochemical characterization of CBM2b-2 showed that the module bound to insoluble and soluble oat spelt xylan and xylohexaose with K(a) values of 5.6 x 10(4), 1.2 x 10(4), and 4.8 x 10(3) M(-1), respectively, but exhibited extremely weak affinity for cellohexaose (<10(2) M(-1)), and its interaction with insoluble cellulose was too weak to quantify. The CBM did not interact with soluble forms of other plant cell wall polysaccharides. The three-dimensional structure of CBM2b-2 was determined by NMR spectroscopy. The module has a twisted "beta-sandwich" architecture, and the two surface exposed tryptophans, Trp 570 and Trp 602, which are in a perpendicular orientation with each other, were shown to be essential for ligand binding. In addition, changing Arg 573 to glycine altered the polysaccharide binding specificity of the module from xylan to cellulose. These data demonstrate that the biochemical properties and tertiary structure of CBM2b-2 and CBM2b-1 are extremely similar. When CBM2b-1 and CBM2b-2 were incorporated into a single polypeptide chain, either in the full-length enzyme or an artificial construct comprising both CBM2bs covalently joined via a flexible linker, there was an approximate 18-20-fold increase in the affinity of the protein for soluble and insoluble xylan, as compared to the individual modules, and a measurable interaction with insoluble acid-swollen cellulose, although the K(a) (approximately 6.0 x 10(4) M(-1)) was still much lower than for insoluble xylan (K(a) = approximately 1.0 x 10(6) M(-1)). These data demonstrate that the two family 2b CBMs of Cf Xyn11A act in synergy to bind acid swollen cellulose and xylan. We propose that the increased affinity of glycoside hydrolases for polysaccharides, through the synergistic interactions of CBMs, provides an explanation for the duplication of CBMs from the same family in some prokaryotic cellulases and xylanases.     

Cloning,Sequencing, and Expression of the Gene Encoding a Cell-bound Multi-domain Xylanase from Clostridium josui,and Characterization of the Translated Product

The nucleotide sequence of the Clostridium josui FERM P-9684 xyn10A gene, encoding a xylanase Xyn10A, consists of 3,150 bp and encodes 1,050 amino acids with a molecular weight of 115,564. Xyn10A is a multidomain enzyme composed of an N-terminal signal peptide and six domains in the following order: two thermostabilizing domains, a family 10 xylanase domain, a family 9 carbohydrate-binding module (CBM), and two S-layer homologous (SLH) domains. Immunological analysis indicated the presence of Xyn10A in the culture supernatant of C. josui FERM P-9684 and on the cell surface. The full-length Xyn10A expressed in a recombinant Escherichia coli strain bound to ball-milled cellulose (BMC) and the cell wall fragments of C. josui, indicating that both the CBM and the SLH domains are fully functional in the recombinant enzyme. An 85-kDa xylanase species derived from Xyn10A by partial proteolysis at the C-terminal side, most likely at the internal region of the CBM, retained the ability to bind to BMC. This observation suggests that the catalytic domain or the thermostabilizing domains are responsible for binding of the enzyme to BMC. Xyn10A-II, the 100-kDa derivative of Xyn10A, was purified from the recombinant E. coli strain and characterized. The enzyme was highly active toward xylan but not toward p-nitrophenyl-β-D-xylopyranoside, p-nitrophenyl-β-D-cellobioside, or carboxymethylcellulose.     

Characterization of a cellulase containing a family 30 carbohydrate-binding module (CBM) derived from Clostridium thermocellum CelJ: importance of the CBM to cellulose hydrolysis

Clostridium thermocellum CelJ is a modular enzyme containing a family 30 carbohydrate-binding module (CBM) and a family 9 catalytic module at its N-terminal moiety. To investigate the functions of the CBM and the catalytic module, truncated derivatives of CelJ were constructed and characterized. Isothermal titration calorimetric studies showed that the association constants (K(a)) of the CBM polypeptide (CBM30) for the binding of cellopentaose and cellohexaose were 1.2 x 10(4) and 6.4 x 10(4) M(-1), respectively, and that the binding of CBM30 to these ligands is enthalpically driven. Qualitative analyses showed that CBM30 had strong affinity for cellulose and beta-1,3-1,4-mixed glucan such as barley beta-glucan and lichenan. Analyses of the hydrolytic action of the enzyme comprising the CBM and the catalytic module showed that the enzyme is a processive endoglucanse with strong activity towards carboxymethylcellulose, barley beta-glucan and lichenan. By contrast, the catalytic module polypeptide devoid of the CBM showed negligible activity toward these substrates. These observations suggest that the CBM is extremely important not only because it mediates the binding of the enzyme to the substrates but also because it participates in the catalytic function of the enzyme or contributes to maintaining the correct tertiary structure of the family 9 catalytic module for expressing enzyme activity.     

Activity and thermostability increase of xylanase following transplantation with modules sub-divided from hyper-thermophilic CBM9_1-2

Transplantation is useful for elucidating the functions of structural modules and for engineering enzyme properties. Unexpectedly, transplanting a hyper-thermophilic carbohydrate-binding module, CBM9_1-2, into the mesophilic Aspergillus niger GH11 xylanase (Xyn) slightly decreased the thermal inactivation half-life of Xyn. This effect was further investigated by dividing the CBM9_1-2 module into two smaller parts, C1 and C2, which were transplanted into Xyn to create the chimeras Xyn-C1 and Xyn-C2. Both chimeras exhibited higher catalytic activities on xylan than native Xyn. Xyn-C2 exhibited higher binding affinities for both oat spelt and birch wood xylans, and its thermal inactivation half-life (69.3 min) was 4 or 5 times longer than that of Xyn (17.6 min), Xyn-C1 (13.4 min), and the original chimera containing CBM9_1-2 (13.8 min). In contrast, Xyn-C1 exhibited higher binding affinity for oat spelt xylan, but not for birch wood xylan. Through this rational engineering of the fungal xylanase, the C2 sub-module was shown to have a different thermostabilizing effect than the C1 sub-module. The different functions of the smaller parts of a large module can play pivotal roles in transplantation.     

The C-terminal region of xylanase domain in Xyn11A from Paenibacillus curdlanolyticus B-6 plays an important role in structural stability

Paenibacillus curdlanolyticus B-6 produces an extracellular multienzyme complex containing a major xylanase subunit, designated Xyn11A, which includes two functional domains belonging to glycosyl hydrolase family-11 (GH11) and carbohydrate binding module family-36 (CBM36) and possesses a glycine and asparagine-rich linker (linker). To clarify the roles of each functional domain, recombinant proteins XynXL and XynX (CBM36 deleted and CBM36 and linker deleted, respectively) were constructed. Their xylanase activities were similar toward soluble xylan, whereas XynXL showed decreased hydrolysis activity toward insoluble xylan while XynX had no xylanase activity. To determine the significance of the linker and its neighbor region, XynX was subjected to secondary structural alignments using circular dichroism (CD) spectroscopy and three-dimensional (3D) structural analysis. A seven amino acid (NTITIGG) neighbor linker sequence was highly conserved among GH11 xylanases of Paenibacillus species. Although XynX exhibited a typical GH11 xylanase structure, conformational gaps were observed in the β6- and β12-sheets and in CD spectra. Flipping of the Arg163 side chains in the subsite was also observed upon analysis of superimposed models. Docking analysis using xylohexaose indicated that flipping of the Arg163 side chains markedly affected substrate binding in the subsite. To identify the amino acids related to stabilizing the substrate binding site, XynX with an extended C-terminal region was designed. At least seven amino acids were necessary to recover substrate binding and xylanase activity. These results indicated that the seven amino acid neighbor Xyn11A linker plays an important role in the activity and conformational stability of the xylanase domain.     

Enzymatic properties of two catalytic modules of Clostridium stercorarium pectate lyase Pel9A

Clostridium stercorarium F-9 pectate lyase Pel9A is a modular enzyme composed of two hypothetical family-9 catalytic modules of the polysaccharide lyases, CM9-1 and CM9-2, in order from the N terminus. In this study, we constructed and characterized CM9-1 and CM9-2 polypeptides as rCM9-1 and rCM9-2 respectively. Both of them, like the full-length Pel9A, required the Ca2+ ion for their enzyme activities and showed high activity toward polygalacturonic acid but lower activity toward pectin. The specific activity of rCM9-2 was three times higher than that of rCM9-1 and rCM9-2 by itself efficiently catalyzed the depolymerization reaction of polygalacturonic acid into monosaccharide as the major product. It was found that rCM9-1 and rCM9-2 adsorbed to polygalacturonic acid and pectin on native affinity PAGE analysis, suggesting that they contain an independent carbohydrate-binding module separable from a catalytic module or consist of a catalytic module with a binding affinity for pectic substrates.     

Novel architecture of family-9 glycoside hydrolases identified in cellulosomal enzymes of Acetivibrio cellulolyticus and Clostridium thermocellum

We have sequenced a new gene, cel9B, encoding a family-9 cellulase from a cellulosome-producing bacterium, Acetivibrio cellulolyticus. The gene includes a signal peptide, a family-9 glycoside hydrolases (GH9) catalytic module, two family-3 carbohydrate-binding modules (CBM3c-CBM3b tandem dyad) and a C-terminal dockerin module. An identical modular arrangement exists in two putative GH9 genes from the draft sequence of the Clostridium thermocellum genome. The three homologous CBM3b modules from A. cellulolyticus and C. thermocellum were overexpressed, but, surprisingly, none bound cellulosic substrates. The results raise fundamental questions concerning the possible role(s) of the newly described CBMs. Phylogenetic analysis and preliminary site-directed mutagenesis studies suggest that the catalytic module and the CBM3 dyad are distinctive in their sequences and are proposed to constitute a new GH9 architectural theme.     

Thermostable xylanases, Xyn10A and Xyn11A, from the actinomycete Nonomuraea flexuosa: isolation of the genes and characterization of recombinant Xyn11A polypeptides produced in Trichoderma reesei

Two endoxylanases, Nf Xyn11A and Nf Xyn10A, were cloned from a Nonomuraea flexuosa (previously Actinomadura flexuosa) DSM43186 genomic expression library in Escherichia coli. The coding sequences of xyn11A and xyn10A consist of 344 and 492 amino acids, respectively. The catalytic domains belong to family 11 and family 10 of glycoside hydrolases. The C-termini share strong amino acid sequence similarity to carbohydrate-binding module (CBM) families CBM2 and CBM13, respectively. Native Nf Xyn11A, and recombinant Xyn11A expressed in the filamentous fungus Trichoderma reesei, were purified from cultivation media and characterized. The molecular masses of the full-length enzymes determined by mass spectrometry were 32.9 kDa and 33.4 kDa, the recombinant enzyme having higher molecular mass due to glycosylation. In addition, shorter polypeptides with molecular masses of 23.8 kDa and 22.0 kDa were characterized from the T. reesei culture medium, both lacking the C-terminal CBM and the 22.0 kDa polypeptide also lacking most of the linker region. The recombinant polypeptides were similar to each other in terms of specific activity, pH and temperature dependence. However, the 23.8 kDa and 22.0 kDa polypeptides were more thermostable at 80°C than the full-length enzyme. All polypeptide forms were effective in pretreatment of softwood kraft pulp at 80°C.