A family 2a carbohydrate-binding module suitable as an affinity tag for proteins produced in Pichia pastoris

The family 2a carbohydrate-binding module (CBM), Cel5ACBM2a, from the C-terminus of Cel5A from Cellulomonas fimi, and Xyn10ACBM2a, the family 2a CBM from the C-terminus of Xyn10A from C. fimi, were compared as fusion partners for proteins produced in the methylotrophic yeast Pichia pastoris. Gene fusions of murine stem-cell factor (SCF) with both CBMs were expressed in P. pastoris. The secreted SCF-Xyn10ACBM2a polypeptides were highly glycosylated and bound poorly to cellulose. In contrast, fusion of SCF to Cel5ACBM2a, which lacks potential N-linked glycosylation sites, resulted in the production of polypeptides which bound tightly to cellulose. Cloning and expression of these CBM2a in P. pastoris without a fusion partner confirmed that N-linked glycosylation at several sites was responsible for the poor cellulose binding. The nonglycosylated CBMs produced in E. coli had very similar cellulose-binding properties.     

Recognition and hydrolysis of noncrystalline cellulose

Cellulase Cel5A from alkalophilic Bacillus sp. 1139 contains a family 17 carbohydrate-binding module (BspCBM17) and a family 28 CBM (BspCBM28) in tandem. The two modules have significantly similar amino acid sequences, but amino acid residues essential for binding are not conserved. BspCBM28 was obtained as a discrete polypeptide by engineering the cel5A gene. BspCBM17 could not be obtained as a discrete polypeptide, so a family 17 CBM from endoglucanase Cel5A of Clostridium cellulovorans, CcCBM17, was used to compare the binding characteristics of the two families of CBM. Both CcCBM17 and BspCBM28 recognized two classes of binding sites on amorphous cellulose: a high affinity site (K(a) approximately 1 x 10(6) M(-1)) and a low affinity site (K(a) approximately 2 x 10(4) M(-1)). They did not compete for binding to the high affinity sites, suggesting that they bound at different sites on the cellulose. A polypeptide, BspCBM17/CBM28, comprising the tandem CBMs from Cel5A, bound to amorphous cellulose with a significantly higher affinity than the sum of the affinities of CcCBM17 and BspCBM28, indicating cooperativity between the linked CBMs. Cel5A mutants were constructed that were defective in one or both of the CBMs. The mutants differed from the wild-type enzyme in the amounts and sizes of the soluble products produced from amorphous cellulose. This suggests that either the CBMs can modify the action of the catalytic module of Cel5A or that they target the enzyme to areas of the cellulose that differ in susceptibility to hydrolysis.     

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.     

The Contribution of Non-catalytic Carbohydrate Binding Modules to the Activity of Lytic Polysaccharide Monooxygenases

Lignocellulosic biomass is a sustainable industrial substrate. Copper-dependent lytic polysaccharide monooxygenases (LPMOs) contribute to the degradation of lignocellulose and increase the efficiency of biofuel production. LPMOs can contain non-catalytic carbohydrate binding modules (CBMs), but their role in the activity of these enzymes is poorly understood. Here we explored the importance of CBMs in LPMO function. The family 2a CBMs of two monooxygenases, CfLPMO10 and TbLPMO10 from Cellulomonas fimi and Thermobispora bispora, respectively, were deleted and/or replaced with CBMs from other proteins. The data showed that the CBMs could potentiate and, surprisingly, inhibit LPMO activity, and that these effects were both enzyme-specific and substrate-specific. Removing the natural CBM or introducing CtCBM3a, from the Clostridium thermocellum cellulosome scaffoldin CipA, almost abolished the catalytic activity of the LPMOs against the cellulosic substrates. The deleterious effect of CBM removal likely reflects the importance of prolonged presentation of the enzyme on the surface of the substrate for efficient catalytic activity, as only LPMOs appended to CBMs bound tightly to cellulose. The negative impact of CtCBM3a is in sharp contrast with the capacity of this binding module to potentiate the activity of a range of glycoside hydrolases including cellulases. The deletion of the endogenous CBM from CfLPMO10 or the introduction of a family 10 CBM from Cellvibrio japonicus LPMO10B into TbLPMO10 influenced the quantity of non-oxidized products generated, demonstrating that CBMs can modulate the mode of action of LPMOs. This study demonstrates that engineered LPMO-CBM hybrids can display enhanced industrially relevant oxygenations.     

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.     

Fusion of a family 9 cellulose-binding module improves catalytic potential of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> cellodextrin phosphorylase on insoluble cellulose

Clostridium thermocellum cellodextrin phosphorylase (CtCDP), a single-module protein without an apparent carbohydrate-binding module, has reported activities on soluble cellodextrin with a degree of polymerization (DP) from two to five. In this study, CtCDP was first discovered to have weak activities on weakly water-soluble celloheptaose and insoluble regenerated amorphous cellulose (RAC). To enhance its activity on solid cellulosic materials, four cellulose binding modules, e.g., CBM3 (type A) from C. thermocellum CbhA, CBM4-2 (type B) from Rhodothermus marinus Xyn10A, CBM6 (type B) from Cellvibrio mixtus Cel5B, and CBM9-2 (type C) from Thermotoga maritima Xyn10A, were fused to the C terminus of CtCDP. Fusion of any selected CBM with CtCDP did not influence its kinetic parameters on cellobiose but affected the binding and catalytic properties on celloheptaose and RAC differently. Among them, addition of CBM9 to CtCDP resulted in a 2.7-fold increase of catalytic efficiency for degrading celloheptaose. CtCDP-CBM9 exhibited enhanced specific activities over 20% on the short-chain RAC (DP = 14) and more than 50% on the long-chain RAC (DP = 164). The chimeric protein CtCDP-CBM9 would be the first step to construct a cellulose phosphorylase for in vitro hydrogen production from cellulose by synthetic pathway biotransformation (SyPaB).     

Contribution of glutamate decarboxylase in Lactobacillus reuteri to acid resistance and persistence in sourdough fermentation

Background

The gene encoding an atypical multi-modular glycoside hydrolase family 45 endoglucanase bearing five different family 1 carbohydrate binding modules (CBM1), designated PpCel45A, was identified in the Pichia pastoris GS115 genome.

Results

PpCel45A (full-length open reading frame), and three derived constructs comprising (i) the catalytic module with its proximal CBM1, (ii) the catalytic module only, and (iii) the five CBM1 modules without catalytic module, were successfully expressed to high yields (up to 2 grams per litre of culture) in P. pastoris X33. Although the constructs containing the catalytic module displayed similar activities towards a range of glucans, comparison of their biochemical characteristics revealed striking differences. We observed a high thermostability of PpCel45A (Half life time of 6 h at 80°C), which decreased with the removal of CBMs and glycosylated linkers. However, both binding to crystalline cellulose and hydrolysis of crystalline cellulose and cellohexaose were substantially boosted by the presence of one CBM rather than five.

Conclusions

The present study has revealed the specific features of the first characterized endo β-1,4 glucanase from yeast, whose thermostability is promising for biotechnological applications related to the saccharification of lignocellulosic biomass such as consolidated bioprocessing.     

Expression of family 3 cellulose-binding module (CBM3) as an affinity tag for recombinant proteins in yeast

Easy and low-cost protein purification methods for the mass production of commonly used enzymes that play important roles in biotechnology are in high demand. In this study, we developed a fast, low-cost recombinant protein purification system in the methylotrophic yeast Pichia pastoris using the family 3 cellulose-binding module (CBM3)-based affinity tag. The codon of the cbm3 gene from Clostridium thermocellum was optimized based on the codon usage of P. pastoris. The CBM3 tag was then fused with enhanced green fluorescent protein (CBM3-EGFP) or with inulinase and expressed in P. pastoris to demonstrate its ability to function as an affinity tag in a yeast expression system. We also examined the effects of glycosylation on the secreted CBM3-tag. The secreted wild-type CBM3-EGFP was glycosylated; however, this had little influence on the adsorption of the fusion protein to the regenerated amorphous cellulose (RAC; maximum adsorption capacity of 319 mg/g). Two CBM3-EGFP mutants lacking glycosylation sites were also constructed. The three CBM3-EGFPs expressed in P. pastoris and the CBM3-EGFP expressed in Escherichia coli all had similar RAC adsorption capacity. To construct a tag-free recombinant protein purification system based on CBM3, a CBM3-intein-EGFP fusion protein was expressed in P. pastoris. This fusion protein was stably expressed and the self-cleavage of intein was efficiently induced by DTT or l-cysteine. In this study, we were able to purify the recombinant fusion protein with high efficiency using both intein and direct fusion-based strategies.     

Engineering chimeric thermostable GH7 cellobiohydrolases in Saccharomyces cerevisiae

We report here the effect of adding different types of carbohydrate-binding modules (CBM) to a single-module GH7 family cellobiohydrolase Cel7A from a thermophilic fungus Talaromyces emersonii (TeCel7A). Both bacterial and fungal CBMs derived from families 1, 2 and 3, all reported to bind to crystalline cellulose, were used. Chimeric cellobiohydrolases with an additional S–S bridge in the catalytic module of TeCel7A were also made. All the fusion proteins were secreted in active form and in good yields by Saccharomyces cerevisiae. The purified chimeric enzymes bound to cellulose clearly better than the catalytic module alone and demonstrated high thermal stability, having unfolding temperatures (T _m) ranging from 72 °C to 77 °C. The highest activity enhancement on microcrystalline cellulose could be gained by a fusion with a bacterial CBM3 derived from Clostridium thermocellum cellulosomal-scaffolding protein CipA. The two CBM3 fusion enzymes tested were more active than the reference enzyme Trichoderma reesei Cel7A both at moderate (45 °C and 55 °C) and at high temperatures (60 °C and 65 °C), the hydrolysis yields being two- to three-fold better at 60 °C, and six- to seven-fold better at 65 °C. The best enzyme variant was also tested on a lignocellulosic feedstock hydrolysis, which demonstrated its potency in biomass hydrolysis even at 70 °C.     

Binding mechanisms for Thermobifida fusca Cel5A,Cel6B,and Cel48A cellulose-binding modules on bacterial microcrystalline cellulose

The family II cellulose-binding modules (CBM) from Thermobifida fusca Cel5A and Cel48A were cloned in the Escherichia coli/Streptomyces shuttle vector pD730, and the plasmids were transformed into Streptomyces lividans TKM31. CBM(Cel5A), and CBM(Cel48A), CBM(Cel6B) were expressed and purified from S. lividans. The molecular masses were determined by mass spectrometry, and the values were 10595 +/- 2, 10915 +/- 2, and 11291 +/- 2 Da for CBM(Cel5A), CBM(Cel6B), and CBM(Cel48A), respectively. Three different binding models (Langmuir, Interstice Penetration, and Interstice Saturation) were tested to describe the binding isotherms of these CBMs on bacterial microcrystalline cellulose (BMCC). The experimental binding isotherms of T. fusca family II CBMs on BMCC are best modeled by the Interstice Saturation model, which includes binding to the constrained interstice surface of BMCC as well as traditional Langmuir binding on the freely accessible surface. The Interstice Saturation model consists of three different steps (Langmuir binding, interstice binding, and interstice saturation). Full reversibility only occurred in the Langmuir region. The irreversibility in the interstice binding and saturation regions probably was caused by interstice entrapment. Temperature shift experiments in different binding regions support the interstice entrapment assumption. There was no systematic difference in binding between the two types of exocellulase CBMs--one that hydrolyzes cellulose from the nonreducing (CBM(Cel6B)) end and one that hydrolyzes cellulose from the reducing end (CBM(Cel48A)).     

Carbohydrate-binding modules influence substrate specificity of an endoglucanase from Clostridium thermocellum

Most cellulases contain carbohydrate-binding modules (CBMs) that largely contribute to their activity for insoluble substrates. Clostridium thermocellum Cel5E is an endoglucanase having xylanolytic activity. The Cel5E originally has a family 11 CBM preferentially binding to β-1,4- and β-1,3-1,4-mixed linkage glucans. In this study, we replaced the CBM with a different type of CBM, either a family 3 microcrystalline cellulose-directed CBM from Clostridium josui scaffoldin, or a family 6 xylan-directed CBM from Clostridium stercorarium xylanase 11A. Chimeric endoglucanases showed enhanced activity that was affected by CBM binding specificity. These chimeric enzymes could efficiently degrade milled lignocellulosic materials, such as corn hulls, because of heterologous components in the plant cell wall, indicating that diverse CBMs play roles in degradation of lignocellulosic materials.     

Identification and characterization of novel cellulolytic and hemicellulolytic genes and enzymes derived from German grassland soil metagenomes

Soil metagenomes represent an unlimited resource for the discovery of novel biocatalysts from soil microorganisms. Three large-inserts metagenomic DNA libraries were constructed from different grassland soil samples and screened for genes conferring cellulase or xylanase activity. Function-driven screening identified a novel cellulase-encoding gene (cel01) and two xylanase-encoding genes (xyn01 and xyn02). From sequence and protein domain analyses, Cel01 (831 amino acids) belongs to glycoside hydrolase family 9 whereas Xyn01 (170 amino acids) and Xyn02 (255 amino acids) are members of glycoside hydrolase family 11. Cel01 harbors a family 9 carbohydrate-binding module, previously found only in xylanases. Both Xyn01 and Xyn02 were most active at 60°C with high activities from 4 to 10 and optimal at pH 7 (Xyn01) and pH 6 (Xyn02). The cellulase gene, cel01, was expressed in E. coli BL21 and the recombinant enzyme (91.9 kDa) was purified. Cel01 exhibited high activity with soluble cellulose substrates containing β-1,4-linkages. Activity with microcrystalline cellulose was not detected. These data, together with the analysis of the degradation profiles of carboxymethyl cellulose and barley glucan indicated that Cel01 is an endo 1,4-β-glucanase. Cel01 showed optimal activity at 50°C and pH 7 being highly active from pH range 5 to 9 and possesses remarkable halotolerance.     

Extra tyrosine in the carbohydrate-binding module of Irpex lacteus Xyn10B enhances its cellulose-binding ability

The xylanase (Xyn10B) that strongly adsorbs on microcrystalline cellulose was isolated from Driselase. The Xyn10B contains a Carbohydrate-binding module family 1 (CBM1) (IrpCBM_Xyn10B) at N-terminus. The canonical essential aromatic residues required for cellulose binding were conserved in IrpCBM_Xyn10B; however, its adsorption ability was markedly higher than that typically observed for the CBM1 of an endoglucanase from Trametes hirsuta (ThCBM_EG1). An analysis of the CBM-GFP fusion proteins revealed that the binding capacity to cellulose (7.8 μmol/g) and distribution coefficient (2.0 L/μmol) of IrpCBM_Xyn10B-GFP were twofold higher than those of ThCBM_EG1-GFP (3.4 μmol/g and 1.2 L/μmol, respectively), used as a reference structure. Besides the canonical aromatic residues (W24-Y50-Y51) of typical CBM1-containing proteins, IrpCBM_Xyn10B had an additional aromatic residue (Y52). The mutation of Y52 to Ser (IrpCBM_Y52S-GFP) reduced these adsorption parameters to 4.4 μmol/g and 1.5 L/μmol, which were similar to those of ThCBM_EG1-GFP. These results indicate that Y52 plays a crucial role in strong cellulose binding.     

Cloning, Sequencing, and Expression of a Eubacterium cellulosolvens 5 Gene Encoding an Endoglucanase (Cel5A) with Novel Carbohydrate-Binding Modules, and Properties of Cel5A

A novel Eubacterium cellulosolvens 5 gene encoding an endoglucanase (Cel5A) was cloned and expressed in Escherichia coli, and its enzymatic properties were characterized. The cel5A gene consists of a 3,444-bp open reading frame and encodes a 1,148-amino-acid protein with a molecular mass of 127,047 Da. Cel5A is a modular enzyme consisting of an N-terminal signal peptide, two glycosyl hydrolase family 5 catalytic modules, two novel carbohydrate-binding modules (CBMs), two linker sequences, and a C-terminal sequence with an unknown function. The amino acid sequences of the two catalytic modules and the two CBMs are 94% and 73% identical to each other, respectively. Two regions that consisted of one CBM and one catalytic module were tandemly connected via a linker sequence. The CBMs did not exhibit significant sequence similarity with any other CBMs. Analyses of the hydrolytic activity of the recombinant Cel5A (rCel5A) comprising the CBMs and the catalytic modules showed that the enzyme is an endoglucanase with activities with carboxymethyl cellulose, lichenan, acid-swollen cellulose, and oat spelt xylan. To investigate the functions of the CBMs and the catalytic modules, truncated derivatives of rCel5A were constructed and characterized. There were no differences in the hydrolytic activities with various polysaccharides or in the hydrolytic products obtained from cellooligosaccharides between the two catalytic modules. Both CBMs had the same substrate affinity with intact rCel5A. Removal of the CBMs from rCel5A reduced the catalytic activities with various polysaccharides remarkably. These observations show that CBMs play an important role in the catalytic function of the enzyme.     

Understanding the biological rationale for the diversity of cellulose-directed carbohydrate-binding modules in prokaryotic enzymes

Plant cell walls are degraded by glycoside hydrolases that often contain noncatalytic carbohydrate-binding modules (CBMs), which potentiate degradation. There are currently 11 sequence-based cellulose-directed CBM families; however, the biological significance of the structural diversity displayed by these protein modules is uncertain. Here we interrogate the capacity of eight cellulose-binding CBMs to bind to cell walls. These modules target crystalline cellulose (type A) and are located in families 1, 2a, 3a, and 10 (CBM1, CBM2a, CBM3a, and CBM10, respectively); internal regions of amorphous cellulose (type B; CBM4-1, CBM17, CBM28); and the ends of cellulose chains (type C; CBM9-2). Type A CBMs bound particularly effectively to secondary cell walls, although they also recognized primary cell walls. Type A CBM2a and CBM10, derived from the same enzyme, displayed differential binding to cell walls depending upon cell type, tissue, and taxon of origin. Type B CBMs and the type C CBM displayed much weaker binding to cell walls than type A CBMs. CBM17 bound more extensively to cell walls than CBM4-1, even though these type B modules display similar binding to amorphous cellulose in vitro. The thickened primary cell walls of celery collenchyma showed significant binding by some type B modules, indicating that in these walls the cellulose chains do not form highly ordered crystalline structures. Pectate lyase treatment of sections resulted in an increased binding of cellulose-directed CBMs, demonstrating that decloaking cellulose microfibrils of pectic polymers can increase CBM access. The differential recognition of cell walls of diverse origin provides a biological rationale for the diversity of cellulose-directed CBMs that occur in cell wall hydrolases and conversely reveals the variety of cellulose microstructures in primary and secondary cell walls.     

Fusion of Carbohydrate Binding Modules to Bifunctional Cellulase to Enhance Binding Affinity and Cellulolytic Activity

Bifunctional cellulase (glycoside hydrolase 5, GH5) from Bacillus sp. D04 having both endo- and exoglucanase activities was fused with two types of carbohydrate binding modules (CBMs). CBM3 from Bacillus sp. D04 and CBM9 from Thermotoga maritima Xyn10A were added to GH5 to hydrolyze microcrystalline cellulose (Avicel) as well as water-soluble cellulose (carboxymethyl cellulose, CMC). The optimum temperature of GH5 was 50oC, while it increased to 60oC for the fusion GH5-CBM3 and GH5-CBM9, indicating that addition of CBM increased the thermostability of the enzyme. Addition of CBM3 and CBM9 enhanced the GH5 affinity (K_M), for which K_M decreased from 104 to 33.9 ~ 35.1 mg/mL for CMC, and from 115 to 55.5 ~ 80.3 mg/mL for Avicel, respectively. The catalytic efficiency (k_cat/K_M) also increased from 4.80 to 5.36 ~ 6.46 (mL/mg)/sec for CMC, and from 1.77 to 2.40 ~ 4.45 (mL/mg)/sec for Avicel, respectively, by addition of CBM3 and CBM9.     

Engineering of Family-5 Glycoside Hydrolase (Cel5A) from an Uncultured Bacterium for Efficient Hydrolysis of Cellulosic Substrates

Cel5A, an endoglucanase, was derived from the metagenomic library of vermicompost. The deduced amino acid sequence of Cel5A shows high sequence homology with family-5 glycoside hydrolases, which contain a single catalytic domain but no distinct cellulose-binding domain. Random mutagenesis and cellulose-binding module (CBM) fusion approaches were successfully applied to obtain properties required for cellulose hydrolysis. After two rounds of error-prone PCR and screening of 3,000 mutants, amino acid substitutions were identified at various positions in thermotolerant mutants. The most heat-tolerant mutant, Cel5A_2R2, showed a 7-fold increase in thermostability. To enhance the affinity and hydrolytic activity of Cel5A on cellulose substrates, the family-6 CBM from Saccharophagus degradans was fused to the C-terminus of the Cel5A_2R2 mutant using overlap PCR. The Cel5A_2R2-CBM6 fusion protein showed 7-fold higher activity than the native Cel5A on Avicel and filter paper. Cellobiose was a major product obtained from the hydrolysis of cellulosic substrates by the fusion enzyme, which was identified by using thin layer chromatography analysis.     

Molecular and Biochemical Analyses of CbCel9A/Cel48A,a Highly Secreted Multi-Modular Cellulase by Caldicellulosiruptor bescii during Growth on Crystalline Cellulose

During growth on crystalline cellulose, the thermophilic bacterium Caldicellulosiruptor bescii secretes several cellulose-degrading enzymes. Among these enzymes is CelA (CbCel9A/Cel48A), which is reported as the most highly secreted cellulolytic enzyme in this bacterium. CbCel9A/Cel48A is a large multi-modular polypeptide, composed of an N-terminal catalytic glycoside hydrolase family 9 (GH9) module and a C-terminal GH48 catalytic module that are separated by a family 3c carbohydrate-binding module (CBM3c) and two identical CBM3bs. The wild-type CbCel9A/Cel48A and its truncational mutants were expressed in Bacillus megaterium and Escherichia coli, respectively. The wild-type polypeptide released twice the amount of glucose equivalents from Avicel than its truncational mutant that lacks the GH48 catalytic module. The truncational mutant harboring the GH9 module and the CBM3c was more thermostable than the wild-type protein, likely due to its compact structure. The main hydrolytic activity was present in the GH9 catalytic module, while the truncational mutant containing the GH48 module and the three CBMs was ineffective in degradation of either crystalline or amorphous cellulose. Interestingly, the GH9 and/or GH48 catalytic modules containing the CBM3bs form low-density particles during hydrolysis of crystalline cellulose. Moreover, TM3 (GH9/CBM3c) and TM2 (GH48 with three CBM3 modules) synergistically hydrolyze crystalline cellulose. Deletion of the CBM3bs or mutations that compromised their binding activity suggested that these CBMs are important during hydrolysis of crystalline cellulose. In agreement with this observation, seven of nine genes in a C. bescii gene cluster predicted to encode cellulose-degrading enzymes harbor CBM3bs. Based on our results, we hypothesize that C. bescii uses the GH48 module and the CBM3bs in CbCel9A/Cel48A to destabilize certain regions of crystalline cellulose for attack by the highly active GH9 module and other endoglucanases produced by this hyperthermophilic bacterium.     

Proteomic identification of CBM37-containing cellulases produced by the rumen cellulolytic bacterium <Emphasis Type="Italic">Ruminococcus albus</Emphasis> 20 and their putative involvement in bacterial adhesion to cellulose

The objective of this study was to identify and characterize other proteins than fimbrial proteins potentially involved in R. albus 20 adhesion to cellulose using an adhesion-related antiserum preparation (i.e. anti-Adh serum). From protein fractions of R. albus 20 grown on cellulose, the serum recognized at least 10 cellulose-binding proteins (CBPs), among which homologs of glycoside hydrolases (family 5, 9 and 48) of R. albus 8 (i.e. Cel5G, Cel9B and Cel48A) were identified by a proteomic approach. In strain 20, Cel9B and Cel48A were identified as two major CBPs and as bacterial cell-associated proteins. The anti-Adh serum was also shown to target the C-terminal family 37 carbohydrate-binding module (CBM37) of Cel9B and Cel48A, indicating that this module, unique to R. albus, may play a significant role in bacterial adhesion to cellulose as suggested previously for R. albus 8. Overall, our results support the hypothesis of an adhesion mechanism involving the CBM37 of Cel9B and Cel48A. This adhesion mechanism may not be restricted to these two enzymes but may also involve other CBM37-containing proteins such as Cel5G and the other uncharacterised proteins recognized by the anti-Adh serum. The EMBL accession numbers for the sequences reported in this paper are FM872295 for Cel9B and FM872296 for Cel48A.     

O-glycosylation of a recombinant carbohydrate-binding module mutant secreted by Pichia pastoris

Carbohydrate-binding modules (CBMs; previously called cellulose-binding domains) make excellent fusion partners for the immobilization or purification of polypeptides. However, their use in eukaryotic hosts has been limited by glycosylation, which interferes with the ability of the CBM to bind to cellulose. We have engineered the C-terminal carbohydrate-binding module from Cellulomonas fimi xylanase 10A such that it lacks N-glycosylation sites. This variant, called CBM2aNgly-, was produced and secreted by the methylotrophic yeast Pichia pastoris and found to be O-glycosylated. The O-linked glycans were composed entirely of mannose in a ratio of 1 mol of mannose to 4 mol of protein. The overall distribution of mannose on the O-glycosylated CBM mutant ranged from 1 to 9 mannose residues with the oligosaccharide sizes ranging from Man(1) to Man(4). MALDI-TOF (all matrix-assisted-laser-desorption time of flight) mass spectrometry (MS) was used to map the O-glycosylation to three regions of the polypeptide, each region having a maximum of 4 mannose residues attached to each. Glycans chemically released from CBM2aNgly- and analyzed by fluorophore-assisted carbohydrate electrophoresis were found to contain alpha-1,2-, alpha-1,3-, and alpha-1,6-linkages. Significantly, the O-glycosylation did not influence binding, making CBM2aNgly- a suitable fusion partner for polypeptides produced in P. pastoris and other eukaryotic hosts.