Enhancement of acetyl xylan esterase activity on cellulose acetate through fusion to a family 3 cellulose binding module

The current study investigates the potential to increase the activity of a family 1 carbohydrate esterase on cellulose acetate through fusion to a family 3 carbohydrate binding module (CBM). Specifically, CtCBM3 from Clostridium thermocellum was fused to the carboxyl terminus of the acetyl xylan esterase (AnAXE) from Aspergillus nidulans, and active forms of both AnAXE and AnAXE–CtCBM3 were produced in Pichia pastoris. CtCBM3 fusion had negligible impact on the thermostability or regioselectivity of AnAXE; activities towards acetylated corncob xylan, 4-methylumbelliferyl acetate, p-nitrophenyl acetate, and cellobiose octaacetate were also unchanged. By contrast, the activity of AnAXE–CtCBM3 on cellulose acetate increased by two to four times over 24 h, with greater differences observed at earlier time points. Binding studies using microcrystalline cellulose (Avicel) and a commercial source of cellulose acetate confirmed functional production of the CtCBM3 domain; affinity gel electrophoresis using acetylated xylan also verified the selectivity of CtCBM3 binding to cellulose. Notably, gains in enzyme activity on cellulose acetate appeared to exceed gains in substrate binding, suggesting that fusion to CtCBM3 increases functional associations between the enzyme and insoluble, high molecular weight cellulosic substrates.     

Enhanced Polysaccharide Binding and Activity on Linear β-Glucans through Addition of Carbohydrate-Binding Modules to Either Terminus of a Glucooligosaccharide Oxidase

The gluco-oligosaccharide oxidase from Sarocladium strictum CBS 346.70 (GOOX) is a single domain flavoenzyme that favourably oxidizes gluco- and xylo- oligosaccharides. In the present study, GOOX was shown to also oxidize plant polysaccharides, including cellulose, glucomannan, β-(1→3,1→4)-glucan, and xyloglucan, albeit to a lesser extent than oligomeric substrates. To improve GOOX activity on polymeric substrates, three carbohydrate binding modules (CBMs) from Clostridium thermocellum, namely CtCBM3 (type A), CtCBM11 (type B), and CtCBM44 (type B), were separately appended to the amino and carboxy termini of the enzyme, generating six fusion proteins. With the exception of GOOX-CtCBM3 and GOOX-CtCBM44, fusion of the selected CBMs increased the catalytic activity of the enzyme (kcat) on cellotetraose by up to 50%. All CBM fusions selectively enhanced GOOX binding to soluble and insoluble polysaccharides, and the immobilized enzyme on a solid cellulose surface remained stable and active. In addition, the CBM fusions increased the activity of GOOX on soluble glucomannan by up to 30 % and on insoluble crystalline as well as amorphous cellulose by over 50 %.     

Functional and structural characterization of family 6 carbohydrate-binding module (CtCBM6A) of Clostridium thermocellum α-L-arabinofuranosidase

The gene encoding the family 6 carbohydrate-binding module (CtCBM6A) from Clostridium thermocellum, cloned in pET-21a(+) expression vector, was overexpressed using Escherichia coli BL-21(DE3) cells and purified by immobilized metal-ion affinity chromatography. SDS-PAGE analysis of the recombinant CtCBM6A showed molecular size of approximately 15 kDa. Ligand-binding analysis of CtCBM6A with rye arabinoxylan and oat spelt xylan by affinity gel electrophoresis showed low affinity for these ligands (K _a of 40 and 26 liter/g, respectively), and analysis by fluorescence spectroscopy (K _a of 33 and 15 liter/g, respectively) corroborated lower binding affinity with the above soluble ligands. However, CtCBM6A displayed significantly higher ligand-binding affinity with insoluble wheat arabinoxylan with equilibrium association constant K _a of 230 M^?1 and binding capacity (N ₀) of 11 μmole/g. The protein melting curve of CtCBM6A displayed a peak shift from 53 to 58°C in the presence of Ca²⁺, indicating that Ca²⁺ imparts thermal stability to the CtCBM6A structure. Homology modeling of CtCBM6A revealed a characteristic β-sandwich core structure. The Ramachandran plot of CtCBM6A showed 89% of the residues in the most favorable region, 10% in additionally favored region, and 1% in generously allowed region, indicating that CtCBM6A has a stable conformation.     

Family 42 carbohydrate-binding modules display multiple arabinoxylan-binding interfaces presenting different ligand affinities

Enzymes that degrade plant cell wall polysaccharides display a modular architecture comprising a catalytic domain bound to one or more non-catalytic carbohydrate-binding modules (CBMs). CBMs display considerable variation in primary structure and are grouped into 59 sequence-based families organized in the Carbohydrate-Active enZYme (CAZy) database. Here we report the crystal structure of CtCBM42A together with the biochemical characterization of two other members of family 42 CBMs from Clostridium thermocellum. CtCBM42A, CtCBM42B and CtCBM42C bind specifically to the arabinose side-chains of arabinoxylans and arabinan, suggesting that various cellulosomal components are targeted to these regions of the plant cell wall. The structure of CtCBM42A displays a beta-trefoil fold, which comprises 3 sub-domains designated as α, β and γ. Each one of the three sub-domains presents a putative carbohydrate-binding pocket where an aspartate residue located in a central position dominates ligand recognition. Intriguingly, the γ sub-domain of CtCBM42A is pivotal for arabinoxylan binding, while the concerted action of β and γ sub-domains of CtCBM42B and CtCBM42C is apparently required for ligand sequestration. Thus, this work reveals that the binding mechanism of CBM42 members is in contrast with that of homologous CBM13s where recognition of complex polysaccharides results from the cooperative action of three protein sub-domains presenting similar affinities.     

Structure and functional investigation of ligand binding by a family 35 carbohydrate binding module (CtCBM35) of β-mannanase of family 26 glycoside hydrolase from Clostridium thermocellum

Functional attributes of recombinant CtCBM35 (family 35 carbohydrate binding module) of β-mannanase of family 26 Glycoside Hydrolase from Clostridium thermocellum were deduced by biochemical and in silico approaches. Ligand-binding analysis of expressed CtCBM35 analyzed by affinity-gel electrophoresis and fluorescence spectroscopy exhibited association constants K _a ～ 1.2·10⁵ and 3.0·10⁵ M^?1 with locust bean galactomannan and mannotriose, respectively. However, CtCBM35 showed low ligand-binding affinity with insoluble ivory nut mannan with K _a of 5.0·10^?5 M^?1. Unfolding transition analysis by fluorescence spectroscopy explained the conformational changes of CtCBM35 in the presence of guanidine hydrochloride (5 M) and urea (6.25 M). This explained that CtCBM35 has good conformational stability and requires higher free energy of denaturation to invoke unfolding. The three-dimensional (3-D) model of CtCBM35 from C. thermocellum generated by Modeller9v8 displayed predominance of β-sheets arranged as β-jelly-roll fold. The secondary structure of CtCBM35 by PredictProtein showed the presence of two α-helices (3%), 12 β-sheets (45%), and 15 random coils (52%). Secondary structural element analysis of cloned, expressed, and purified recombinant CtCBM35 by circular dichroism also corroborated the in silico predicted secondary structure. Multiple sequence alignment of CtCBM35 showed conserved residues (Tyr123, Gly124, and Phe125), which are commonly observed in mannan specific CBMs. Docking analysis of CtCBM35 with manno-oligosaccharide displayed the involvement of Tyr26, Gln29, Asn43, Trp66, Tyr68, Leu69, Arg76, and Leu127 residues, making polar contact with the ligand molecules. Ligand docking analysis of CtCBM35 exhibiting higher binding affinity with mannotriose and galactomannan (Man-Gal-Man moiety) substantiated the affinity binding and fluorescence results, displaying similar values of K _a.     

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).     

Functional characterization and mutation analysis of family 11, Carbohydrate-Binding Module (<Emphasis Type="Italic">Ct</Emphasis>CBM11) of cellulosomal bifunctional cellulase from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

The non-catalytic, family 11 carbohydrate binding module (CtCBM11) belonging to a bifunctional cellulosomal cellulase from Clostridium thermocellum was hyper-expressed in E. coli and functionally characterized. Affinity electrophoresis of CtCBM11 on nondenaturing PAGE containing cellulosic polysaccharides showed binding with β-glucan, lichenan, hydroxyethyl cellulose and carboxymethyl cellulose. In order to elucidate the involvement of conserved aromatic residues Tyr 22, Trp 65 and Tyr 129 in the polysaccharide binding, site-directed mutagenesis was carried out and the residues were changed to alanine. The results of affinity electrophoresis and binding adsorption isotherms showed that of the three mutants Y22A, W65A and Y129A of CtCBM11, two mutants Y22A and Y129A showed no or reduced binding affinity with polysaccharides. These results showed that tyrosine residue 22 and 129 are involved in the polysaccharide binding. These residues are present in the putative binding cleft and play a critical role in the recognition of all the ligands recognized by the protein.     

Essential role of a family-32 carbohydrate-binding module in substrate recognition by Clostridium thermocellum mannanase CtMan5A

The family-5 glycoside hydrolase domain (GH5) and the family-32 carbohydrate-binding module (CBM32) of Clostridium thermocellum mannanase CtMan5A, along with their genetically inactivated derivatives, were collectively or separately expressed. Their catalytic and substrate-binding abilities were measured to investigate importance of CBM32 in substrate recognition by CtMan5A. Characterization of the truncated derivatives of CtMan5A and isothermal calorimetry analysis of the interaction between the inactivated proteins and mannooligosaccharides suggested that GH5 and CBM32 collectively formed a substrate-binding site capable of accommodating a mannotetraose unit in CtMan5A. This suggested that CBM32 directly participated in the substrate recognition required for catalytic action.     

Mannan specific family 35 carbohydrate-binding module (CtCBM35) of Clostridium thermocellum: structure analysis and ligand binding

The three-dimensional model of the CtCBM35 (Cthe 2811), i.e. the family 35 carbohydrate binding module (CBM) from the Clostridium thermocellum family 26 glycoside hydrolase (GH) β-mannanase, generated by Modeller9v8 displayed predominance of β-sheets arranged as β-sandwich fold. Multiple sequence alignment of CtCBM35 with other CBM35s showed a conserved signature sequence motif Trp-Gly-Tyr, which is probably a specific determinant for mannan binding. Cloned CtCBM35 from Clostridium thermocellum ATCC 27405 was a homogenous, soluble 16 kDa protein. Ligand binding analysis of CtCBM35 by affinity electrophoresis displayed higher binding affinity against konjac glucomannan (K _a = 2.5 × 10⁵ M^?1) than carob galactomannan (K _a = 1.4 × 10⁵ M^?1). The presence of Ca²⁺ ions imparted slightly higher binding affinity of CtCBM35 against carob galactomannan and konjac glucomannan than without Ca²⁺ ion additive. However, CtCBM35 exhibited a low ligand-binding affinity K _a = 2.5 × 10^?5 M^?1 with insoluble ivory nut mannan. Ligand binding study by fluorescence spectroscopy showed K _a against konjac glucomannan and carob galactomannan, 2.4 × 10⁵ M^?1 and 1.44 × 10⁵ M^?1, and ΔG of binding ?27.0 and ?25.0 kJ/mol, respectively, substantiating the findings of affinity electrophoresis. Ca²⁺ ions escalated the thermostability of CtCBM35 and its melting temperature was shifted to 70°C from initial 55°C. Therefore thermostable CtCBM35 targets more β-(1,4)-manno-configured ligands from plant cell wall hemicellulosic reservoir. Thus a non-catalytic CtCBM35 of multienzyme cellulosomal enzymes may gain interest in the biofuel and food industry in the form of released sugars by targeting plant cell wall polysaccharides.     

The family 6 carbohydrate-binding module (CtCBM6B) of Clostridium thermocellum alpha-L-arabinofuranosidase binds xylans and thermally stabilized by Ca2+ ions

Abstract

The gene encoding CtCBM6B of Clostridium thermocellum α-L-arabinofuranosidase (Ct43Araf) was cloned in pET-21a(+) vector, over-expressed using Escherichia coli BL-21(DE3) cells and purified by immobilized metal-ion affinity chromatography (IMAC). The recombinant CtCBM6B showed a molecular size close to 15 kDa by SDS-PAGE analysis, which was close to the expected size of 14.74 kDa. The ligand-binding affinity of CtCBM6B was assessed against ligands for which the catalytic enzyme, Ct43Araf showed maximum activity. The affinity-gel electrophoresis of CtCBM6B with rye arabinoxylan showed lower equilibrium association constant (K_a, 4.0% C^{? 1}), whereas, it exhibited higher affinity (K_a, 19.6% C^{? 1}) with oat spelt xylan. The ligand-binding analysis of CtCBM6B by fluorescence spectroscopy also revealed similar results with low K_a (3.26% C^{? 1}) with rye arabinoxylan and higher affinity for oat spelt xylan (K_a, 17.9% C^{? 1}) which was corroborated by greater blue-shift in case of oat spelt xylan binding. The CtCBM6B binding with insoluble wheat arabinoxylan by adsorption isotherm analysis showed significant binding affinity as reflected by the equilibrium association constant (K_a), 9.4 × 10³ M^{? 1}. The qualitative analysis by SDS-PAGE also corroborated the CtCBM6B binding with insoluble wheat arabinoxylan. The protein-melting curve of CtCBM6B displayed the peak shift from 53°C to 59°C in the presence of Ca²⁺ ions indicating that Ca²⁺ ions impart thermal stability to the CtCBM6B structure.     

Display of both N- and C-terminal target fusion proteins on the Aspergillus oryzae cell surface using a chitin-binding module

A novel cell surface display system in Aspergillus oryzae was established by using a chitin-binding module (CBM) from Saccharomyces cerevisiae as an anchor protein. CBM was fused to the N or C terminus of green fluorescent protein (GFP) and the fusion proteins (GFP-CBM and CBM-GFP) were expressed using A. oryzae as a host. Western blotting and fluorescence microscopy analysis showed that both GFP-CBM and CBM-GFP were successfully expressed on the cell surface. In addition, cell surface display of triacylglycerol lipase from A. oryzae (tglA), while retaining its activity, was also successfully demonstrated using CBM as an anchor protein. The activity of tglA was significantly higher when tglA was fused to the C terminus than N terminus of CBM. Together, these results show that CBM used as a first anchor protein enables the fusion of both the N and/or C terminus of a target protein.     

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.     

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.     

Binding characteristics of a bacterial expansin (BsEXLX1) for various types of pretreated lignocellulose

BsEXLX1 from Bacillus subtilis is the first discovered bacterial expansin as a structural homolog of a plant expansin, and it exhibited synergism with cellulase on the cellulose hydrolysis in a previous study. In this study, binding characteristics of BsEXLX1 were investigated using pretreated and untreated Miscanthus x giganteus in comparison with those of CtCBD3, a cellulose-binding domain from Clostridium thermocellum. The amounts of BsEXLX1 bound to cellulose-rich substrates were significantly lower than those of CtCBD3. However, the amounts of BsEXLX1 bound to lignin-rich substrates were much higher than those of CtCBD3. A binding competition assay between BsEXLX1 and CtCBD3 revealed that binding of BsEXLX1 to alkali lignin was not affected by the presence of CtCBD3. This preferential binding of BsEXLX1 to lignin could be related to root colonization in plants by bacteria, and the bacterial expansin could be used as a lignin blocker in the enzymatic hydrolysis of lignocellulose.     

Multifunctional cellulase catalysis targeted by fusion to different carbohydrate-binding modules

Background

Carbohydrate binding modules (CBMs) bind polysaccharides and help target glycoside hydrolases catalytic domains to their appropriate carbohydrate substrates. To better understand how CBMs can improve cellulolytic enzyme reactivity, representatives from each of the 18 families of CBM found in Ruminoclostridium thermocellum were fused to the multifunctional GH5 catalytic domain of CelE (Cthe_0797, CelEcc), which can hydrolyze numerous types of polysaccharides including cellulose, mannan, and xylan. Since CelE is a cellulosomal enzyme, none of these fusions to a CBM previously existed.

Results

CelEcc_CBM fusions were assayed for their ability to hydrolyze cellulose, lichenan, xylan, and mannan. Several CelEcc_CBM fusions showed enhanced hydrolytic activity with different substrates relative to the fusion to CBM3a from the cellulosome scaffoldin, which has high affinity for binding to crystalline cellulose. Additional binding studies and quantitative catalysis studies using nanostructure-initiator mass spectrometry (NIMS) were carried out with the CBM3a, CBM6, CBM30, and CBM44 fusion enzymes. In general, and consistent with observations of others, enhanced enzyme reactivity was correlated with moderate binding affinity of the CBM. Numerical analysis of reaction time courses showed that CelEcc_CBM44, a combination of a multifunctional enzyme domain with a CBM having broad binding specificity, gave the fastest rates for hydrolysis of both the hexose and pentose fractions of ionic-liquid pretreated switchgrass.

Conclusion

We have shown that fusions of different CBMs to a single multifunctional GH5 catalytic domain can increase its rate of reaction with different pure polysaccharides and with pretreated biomass. This fusion approach, incorporating domains with broad specificity for binding and catalysis, provides a new avenue to improve reactivity of simple combinations of enzymes within the complexity of plant biomass.

     

In silico structural characterization and molecular docking studies of first glucuronoxylan-xylanohydrolase (Xyn30A) from family 30 glycosyl hydrolase (GH30) from Clostridium thermocellum

CtXynGH30 is a carbohydrate active modular enzyme and component of cellulosome of Clostridium thermocellum. The full length CtXynGH30 contains an N-terminal catalytic module named as Xyn30A and a family 6 carbohydrate binding module (CBM6) at C-terminus. Xyn30A was modeled by computer program Modeller9v8 taking crystal structure of XynC from B. subtilis as a template to generate the molecular model. Model refinement was done using energy minimization by implementing steepest descent algorithm with GROMOS96 43a1 force field. Quality assessment by Ramachandran plot showed that 91% amino acids lie in most favourable region and 9% in additional allowed region. Structural analysis depicted that Xyn30A has a (β/α)₈ barrel fold. Additionally, it had a β-strand rich structure called ‘side β-structure’ attached with main catalytic core. Structural superimposition reflected that Glu136 act as a catalytic acid/base while Glu225 act as a catalytic nucleophile. Multiple sequence alignment showed that these catalytic residues are conserved within the family. The docking results showed that these residues display polar interaction with linear and substituted xylo-oligosaccharides. The binding interaction of ligands depicted that aromatic amino acids Trp81, Tyr139, Trp143, Phe172, His198, Tyr200, Tyr227, Trp264 and Tyr265 create binding site pocket around the active site. We report overall structural feature, conserved active site residues and enzyme-ligand docking of first glucuronoxylan-xylanohydrolase (Xyn30A) of family 30 glycosyl hydrolase (GH30) from Clostridium thermocellum.     

Isolation and characterization of isochorismate synthase and cinnamate 4-hydroxylase during salinity stress,wounding, and salicylic acid treatment in Carthamus tinctorius

Salicylic acid (SA) is a prominent signaling molecule during biotic and abiotic stresses in plants biosynthesized via cinnamate and isochorismate pathways. Cinnamate 4-hydroxylase (C4H) and isochorismate synthase (ICS) are the main enzymes in phenylpropanoid and isochorismate pathways, respectively. To investigate the actual roles of these genes in resistance mechanism to environmental stresses, here, the coding sequences of these enzymes in safflower (Carthamus tinctorius), as an oilseed industrial medicinal plant, were partially isolated and their expression profiles during salinity stress, wounding, and salicylic acid treatment were monitored. As a result, safflower ICS (CtICS) and C4H (CtC4H) were induced in early time points after wounding (3–6 h). Upon salinity stress, CtICS and CtC4H were highly expressed for the periods of 6–24 h and 3–6 h after treatment, respectively. It seems evident that ICS expression level is SA concentration dependent as if safflower treatment with 1 mM SA could induce ICS much stronger than that with 0.1 mM, while C4H is less likely to be so. Based on phylogenetic analysis, safflower ICS has maximum similarity to its ortholog in Vitis vinifera up to 69%, while C4H shows the highest similarity to its ortholog in Echinacea angustifolia up to 96%. Overall, the isolated genes of CtICS and CtC4H in safflower could be considered in plant breeding programs for salinity tolerance as well as for pathogen resistance.     

Characterization of a novel theme C glycoside hydrolase family 9 cellulase and its CBM-chimeric enzymes

In bacterial cellulase systems, glycoside hydrolase family 9 (GH9) cellulases are generally regarded as the major cellulose-degrading factors besides GH48 exoglucanase. In this study, umcel9A, which was cloned from uncultured microorganisms from compost, with the encoded protein being theme C GH9 cellulase, was heterologously expressed in Escherichia coli, and the biochemical properties of the purified enzyme were characterized. Hydrolysis of carboxylmethylcellulose (CMC) by Umcel9A led to the decreased viscosity of CMC solution and production of reducing sugars. Interestingly, cellobiose was the major product when cellulosic materials were hydrolyzed by Umcel9A. Six representative carbohydrate-binding modules (CBMs) from different CBM families (CBM1, CBM2, CBM3, CBM4, CBM10, and CBM72) were fused with Umcel9A at the natural terminal position, resulting in significant enhancement of the binding capacity of the chimeric enzymes toward four different insoluble celluloses as compared with that of Umcel9A. Catalytic activity of the chimeric enzymes against insoluble celluloses, including phosphoric acid-swollen cellulose (PASC), alkali-pretreated sugarcane bagasse (ASB), filter paper powder (FPP), and Avicel, was higher than that of Umcel9A, except for Umcel9A-CBM3. In these chimeric enzymes, CBM4-Umcel9A exhibited the highest activity toward the four tested insoluble celluloses and displayed 4.2-, 3.0-, 2.4-, and 6.6-fold enhanced activity toward PASC, ASB, FPP, and Avicel, respectively, when compared with that of Umcel9A. CBM4-Umcel9A also showed highest V _max and catalytic efficiency (k _cat/K _M) against PASC. Construction of chimeric enzymes may have potential applications in biocatalytic processes and provides insight into the evolution of the molecular architecture of catalytic module and CBM in GH9 cellulases.     

Fusion of cellulose binding domain from Trichoderma reesei CBHI to Cryptococcus sp. S-2 cellulase enhances its binding affinity and its cellulolytic activity to insoluble cellulosic substrates

Cryptococcus sp. S-2 carboxymethyl cellulase (CSCMCase) is active in the acidic pH and lacks a binding domain. The absence of the binding domain makes the enzyme inefficient against insoluble cellulosic substrates. To enhance its binding affinity and its cellulolytic activity to insoluble cellulosic substrates, cellulose binding domain (CBD) of cellobiohydrolase I (CBHI) from Trichoderma reesei belonging to carbohydrate binding module (CBM) family 1 was fused at the C-terminus of CSCMCase. The constructed fusion enzymes (CSCMCase-CBD and CSCMCase-2CBD) were expressed in a newly recombinant expression system of Cryptococcus sp. S-2, purified to homogeneity, and then subject to detailed characterization. The recombinant fusion enzymes displayed optimal pH similar to those of the native enzyme. Compared with rCSCMCase, the recombinant fusion enzymes had acquired an increased binding affinity to insoluble cellulose and the cellulolytic activity toward insoluble cellulosic substrates (SIGMACELL^® and Avicel) was higher than that of native enzyme, confirming the presence of CBDs improve the binding and the cellulolytic activity of CSCMCase on insoluble substrates. This attribute should make CSCMCase an attractive applicant for various application.     

Molecular Cloning,Expression and Characterization of Pectin Methylesterase (<Emphasis Type="Italic">Ct</Emphasis>PME) from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Many phytopathogenic micro-organisms such as bacteria and fungi produce pectin methylesterases (PME) during plant invasion. Plants and insects also produce PME to degrade plant cell wall. In the present study, a thermostable pectin methylesterase (CtPME) from Clostridium thermocellum belonging to family 8 carbohydrate esterase (CE8) was cloned, expressed and purified. The amino acid sequence of CtPME exhibited similarity with pectin methylesterase from Erwinia chrysanthemi with 38% identity. The gene encoding CtPME was cloned into pET28a(+) vector and expressed using Escherichia coli BL21(DE3) cells. The recombinant CtPME expressed as a soluble protein and exhibited a single band of molecular mass approximately 35.2 kDa on SDS-PAGE gels. The molecular mass, 35.5 kDa of the enzyme, was also confirmed by MALDI-TOF MS analysis. Notably, highest protein concentration (11.4 mg/mL) of CtPME was achieved in auto-induction medium, as compared with LB medium (1.5 mg/mL). CtPME showed maximum activity (18.1 U/mg) against citrus pectin with >85% methyl esterification. The optimum pH and temperature for activity of CtPME were 8.5 and 50 °C, respectively. The enzyme was stable in pH range 8.0–9.0 and thermostable between 45 and 70 °C. CtPME activity was increased by 40% by 5 mM Ca²⁺ or Mg²⁺ ions. Protein melting curve of CtPME gave a peak at 80 °C. The peak was shifted to 85 °C in the presence of 5 mM Ca²⁺ ions, and the addition of 5 mM EDTA shifted back the melting peak to 80 °C. CtPME can be potentially used in food and textile industry applications.