Processive Endoglucanase Research Articles

Improving the Catalytic Efficiency of a GH5 Processive Endoglucanase by a Combinatorial Strategy Using Consensus Mutagenesis and Loop Engineering

Improving the Catalytic Efficiency of a GH5 Processive Endoglucanase by a Combinatorial Strategy Using Consensus Mutagenesis and Loop Engineering

Immobilization and Characterization of a Processive Endoglucanase EG5C-1 from Bacillus subtilis on Melamine-Glutaraldehyde Dendrimer-Functionalized Magnetic Nanoparticles.

Exploring an appropriate immobilization approach to enhance catalytic activity and reusability of cellulase is of great importance to reduce the price of enzymes and promote the industrialization of cellulose-derived biochemicals. In this study, Fe3O4 magnetic nanoparticles (MNPs) were functionalized with meso-2,3-dimercaptosuccinic acid to introduce carboxyl groups on the surface (DMNPs). Then, melamine-glutaraldehyde dendrimer-like polymers were grafted on DMNPs to increase protein binding sites for the immobilization of processive endoglucanase EG5C-1. Moreover, this dendrimer-like structure was beneficial to protect the conformation of EG5C-1 and facilitate the interaction between substrate and active center. The loading capacity of the functionalized copolymers (MG-DMNPs) for EG5C-1 was about 195 mg/g, where more than 90% of the activity was recovered. Immobilized EG5C-1 exhibited improved thermal stability and increased tolerability over a broad pH range compared with the free one. Additionally, MG-DMNP/EG5C-1 biocomposite maintained approximately 80% of its initial hydrolysis productivity after five cycles of usage using filter paper as the substrate. Our results provided a promising approach for the functionalization of MNPs, enabling the immobilization of cellulases with a high loading capacity and excellent activity recovery.

A Role of Cellulose Binding Module of the Thermophilic Endoglucanase TbCel12A

Endoglucanases are enzymes that play an important role in hydrolysis of lignocellulose by attacking glycosidic linkages in cellulose fibers and other glucans. The cellulose binding module (CBM) is responsible for binding the enzyme to the substrate. However, CBMs in certain enzymes interfere with substrate hydrolysis resulting in moderate or low activity. In a previous study, the processive endoglucanase TbCel12A including its CBM had low activity towards carboxymethyl cellulose (CMC). To assess the effect of the CBM, the catalytic domain of TbCel12A was produced without the CBM. The TbCel12A catalytic domain without the CBM hydrolyzed CMC 23 times more rapidly, while the pH and temperature optima and thermotolerance remained unchanged compared to full-length TbCel12A. Therefore, TbCel12A does not require the CBM for CMC hydrolysis and its application may be improved without it.

Processive binding mechanism of Cel9G from Clostridium cellulovorans: molecular dynamics and free energy landscape investigations.

The degradation of recalcitrant polysaccharides such as cellulose and chitin requires the synergistic functionality of processive glycosidase (GH) cocktails. Understanding the fundamental phenomenon of processivity is of biological and economic importance for the conversion of biomass into biofuel. In this work, cellulase family 9 from Clostridium cellulovorans (Cel9G), which is a processive endoglucanase, was used to elucidate the processive binding mechanism with respect to polysaccharides, since it exhibits a multimodular crystallographic structure. Metadynamics and molecular dynamics simulations were performed to explore the dynamics of cellulose chain binding to Cel9G via processive motion. The processive movement of the cellulose chain towards the catalytic domain may exhibit several local minima, which are related to strong CH/π interactions between the sugar rings and the aromatic residues distributed at the active site. For the binding of the G6 and G12 molecules, the energy barriers were determined to be 4.8 and 7.4 kcal mol-1, respectively. Based on the site-directed mutagenesis simulations of Y520A, it was found that the existence of Y520 is critical for processive binding. It is likely that Y520 and H125/Y416 form two anchor points to facilitate processive binding to polysaccharides. More importantly, the straight-line morphology of the substrate could be observed after the formation of the so-called slide mode, which is different from the V-shaped Michaelis complex structure revealed by quantum mechanics/molecular mechanics simulations. This indicates that an additional step, namely, catalytic activation, probably exists between processive binding and the hydrolysis reaction. Finally, a four-step catalytic cycle was proposed for Cel9G. Our work provides novel molecular-level insights into the structure-function relationship for the processive enzyme Cel9G and should aid the development of improved GH cocktails for the efficient cleavage of glycosidic linkages.

A thermo-alkali stable and detergent compatible processive β-1,4-glucanase from Himalayan Bacillus sp. PCH94.

Present study reports a novel and robust GH9 processive endoglucanase β-1,4-glucanase from Bacillus sp. PCH94 (EGaseBL) with thermo-alkali stable properties. The EGaseBL gene was cloned in pET-28b(+) and expressed in Escherichia coli BL21(DE3) cells. The recombinant protein was purified 94-fold with a yield of 67.8%. The biochemical characterization revealed an active enzyme at a wide pH (4.0-10.0) and temperature (4-100°C). It showed a Km and Vmax of 1.10 mg/ml and 208.24 IU/mg, respectively, using β-glucan as a substrate. The EGaseBL showed dual activities for endoglucanase (134.17 IU/mg) and exoglucanase (28.76 IU/mg), assayed using substrates β-glucan and Avicel, respectively. The enzyme is highly stable in neutral and alkaline pH and showed a half-life of 11.29 h, and 8.31 h in pH 7.0 and 9.0, respectively. The enzyme is also compatible with commercial detergents (Tide, Surf, Ghadi, Raj, and Healing tree) of the Indian market and retained > 85% enzyme activity. Concisely, robustness, extreme functionality, and detergent compatibility endorse EGaseBL as a potential bioresource for the detergent industry, in addition to its implications for the bioethanol industry. Highlights - Cloning, expression, and purification of putative novel GH9 family β-1,4-glucanase.- Processive endoglucanase with CBM3 domain and bi-functional (endo/exo) activity.- Broad pH-temperature active and stable enzyme.- Compatible with commercial detergent powders.

167 Monomodular and Multi-Functional Processive Endocellulases for Improving Fibre Utilization and Reducing Environmental Footprint in Pigs

Abstract Global emission of the major greenhouse gases (GHG) from food animal production accounts for 15% of the total human-induced GHG with pork production being a significant contributor. Poor efficiency of dietary fibre utilization not only limits global pork production profit margins but also adversely affects dietary crude protein and amino acid utilization; and these collectively lead to excessive manure fibre and nitrogenous compounds, resulting in anaerobic biogenesis and emission of the major GHG from swine manure slurry storage. Two unique glycosyl hydrolase family-5 (GH5) processive endoglucanases, referred to as GH5-tCel5A1 and GH5-, have been reported. The GH5-tCel5A1 was originally identified via targeted genome sequencing of the extremely thermophilic Thermotoga maritima while the GH5-p4818Cel5_2A was screened from the porcine hindgut microbial metagenomic expression library. Both GH5-tCel5A1 and GH5-p4818_2A have been characterized to be monomodular, act as processive β-1,4-endoglucanases and have multi-functionality towards hydrolyzing dietary natural crystalline cellulose and pre-treated cellulosic substrates as well as several hemicelluloses including β-glucans, xylan, xylogulcans, mannans, galactomannans and glucomannans. We have recently overexpressed both GH5-tCel5A1 and GH5-p4818Cel5_2A in the ClearColiBL21(DE3) cell and investigated the susceptibility of both these cellulases to auto-oxidation by air-borne O2. Furthermore, we have also examined these endoglucanases’ stability under the mimicked in vitro porcine gastric and the small intestinal pH and proteases’ conditions. Further pig nutrition and growth performance studies are being conducted to investigate the in vivo efficacy of these two processive endoglucanases as newly emerging potential exogenous fibre enzymes.

A processive GH9 family endoglucanase of Bacillus licheniformis and the role of its carbohydrate-binding domain.

One of the critical steps in lignocellulosic deconstruction is the hydrolysis of crystalline cellulose by cellulases. Endoglucanases initially facilitate the breakdown of cellulose in lignocellulosic biomass and are further aided by other cellulases to produce fermentable sugars. Furthermore, if the endoglucanase is processive, it can adsorb to the smooth surface of crystalline cellulose and release soluble sugars during repeated cycles of catalysis before dissociating. Most glycoside hydrolase family 9 (GH9) endoglucanases have catalytic domains linked to a CBM (carbohydrate-binding module) (mostly CBM3) and present the second-largest cellulase family after GH5. GH9 endoglucanases are relatively less characterized. Bacillus licheniformis is a mesophilic soil bacterium containing many glycoside hydrolase (GH) enzymes. We identified an endoglucanase gene, gh9A, encoding the GH9 family enzyme H1AD14 in B. licheniformis and cloned and overexpressed H1AD14 in Escherichia coli. The purified H1AD14 exhibited very high enzymatic activity on endoglucanase substrates, such as β-glucan, lichenan, Avicel, CMC-Na (sodium carboxymethyl cellulose) and PASC (phosphoric acid swollen cellulose), across a wide pH range. The enzyme is tolerant to 2 M sodium chloride and retains 74% specific activity on CMC after 10 days, the highest amongst the reported GH9 endoglucanases. The full-length H1AD14 is a processive endoglucanase and efficiently saccharified sugarcane bagasse. The deletion of the CBM reduces the catalytic activity and processivity. The results add to the sparse knowledge of GH9 endoglucanases and offer the possibility of characterizing and engineering additional enzymes from B. licheniformis toward developing a cellulase cocktail for improved biomass deconstruction. KEY POINTS: • H1AD14 is a highly active and processive GH9 endoglucanase from B. licheniformis. • H1AD14 is thermostable and has a very long half-life. • H1AD14 showed higher saccharification efficiency than commercial endoglucanase.

Characterization of in vitro stability for two processive endoglucanases as exogenous fibre biocatalysts in pig nutrition

Development of highly efficacious exogenous fibre degradation enzymes can enhance efficiency of dietary fibre utilization and sustainability of global pork production. The objectives of this study were to investigate in vitro stability for two processive endoglucanases, referred to as GH5-tCel5A1 and GH5-p4818Cel5_2A that were overexpressed in CLEARCOLIBL21(DE3). Three-dimensional models predicted presence of Cys residues on the catalytic site surfaces of GH5-tCel5A1 and GH5-p4818Cel5_2A; and time course experimental results shown that both cellulases were susceptible to auto-oxidation by airborne O2 and were unstable. Furthermore, we examined these endoglucanases’ stability under the mimicked in vitro porcine gastric and the small intestinal pH and proteases’ conditions. Eadie-Hofstee inhibition kinetic analyses showed that GH5-tCel5A1 and GH5-p4818Cel5_2A respectively lost 18 and 68% of their initial activities after 2-h incubations under the gastric conditions and then lost more than 90% of their initial activities after 2–3 h of incubations under the small intestinal conditions. Therefore, further enzyme protein engineering to improve resistance and alternatively post-fermentation enzyme processing such as coating to bypass the gastric-small intestinal environment will be required to enable these two processive endoglucanases as efficacious exogenous fibre enzymes in pig nutrition application.

Enzymatic generation of short chain cello-oligosaccharides from Miscanthus using different pretreatments

Enzyme combinations producing short-chain cello-oligosaccharides (COS) as major bio-products from cellulose of Miscanthus Mx2779 accessed through different pretreatment methods were compared. Over short hydrolysis times, processive endoglucanase TfCel9a produced a high percentage of cellotetraose and cellopentaose and is synergistic with endoglucanase CcCel9m for producing short oligomers from amorphous cellulose but had low activity on untreated Miscanthus. Hydrolysis of the latter improved when these were combined with a mutant cellobio/triohydrolase OsCelC7(−105) and a lytic polysaccharide monooxygenase TrCel61a, a combination which also produced the highest COS yields from phosphoric acid swollen cellulose. Steam explosion pretreatment of Miscanthus increased COS yields, with/without phosphoric acid swelling, while increased swelling time (from 20 to 45 min) also increased yields but decreased the need for TrCel61a. The highest COS yields (933 mg/g glucan) and most stable product profile were obtained using ionic liquid [C2mim][OAc] pretreatment and the three enzyme mixture TfCel9a, Cel9m and OsCel7a(−105).

Coevolutionary analysis reveals a distal amino acid residue pair affecting the catalytic activity of GH5 processive endoglucanase from Bacillus subtilis BS-5.

EG5C-1, processive endoglucanase from Bacillus subtilis, is a typical bifunctional cellulase with endoglucanase and exoglucanase activities. The engineering of processive endoglucanase focuses on the catalytic pocket or carbohydrate-binding module tailoring based on sequence/structure information. Herein, a computational strategy was applied to identify the desired mutants in the enzyme molecule by evolutionary-coupling analysis; subsequently, four residue pairs were selected as evolutionary mutational hotspots. Based on iterative-saturation mutagenesis and subsequent enzymatic activity analysis, a superior mutant K51T/L93T has been identified away from the active center. This variant had increased specific activity from 4170 U/µmol of wild-type (WT) to 5678 U/µmol towards carboxymethyl cellulose-Na and an increase towards the substrate Avicel from 320 U/µmol in WT to 521 U/µmol. In addition, kinetic measurements suggested that superior mutant K51T/L93T had a high substrate affinity (Km ) and a remarkable improvement in catalytic efficiency (kcat /Km ). Furthermore, molecular dynamics simulations revealed that the K51T/L93T mutation altered the spatial conformation at the active site cleft, enhancing the interaction frequency between active site residues and substrate, and improving catalytic efficiency and substrate affinity. The current studies provided some perspectives on the effects of distal residue substitution, which might assist in the engineering of processive endoglucanase or other glycoside hydrolases.

Highly efficient, processive and multifunctional recombinant endoglucanase RfGH5_4 from Ruminococcus flavefaciens FD-1 v3 for recycling lignocellulosic plant biomasses

Gene encoding endoglucanase, RfGH5_4 from R. flavefaciens FD-1 v3 was cloned, expressed in Escherichia coli BL21(DE3) cells and purified. RfGH5_4 showed molecular size 41 kDa and maximum activity at pH 5.5 and 55 °C. It was stable between pH 5.0–8.0, retaining 85% activity and between 5 °C–45 °C, retaining 75% activity, after 60 min. RfGH5_4 displayed maximum activity (U/mg) against barley β-D-glucan (665) followed by carboxymethyl cellulose (450), xyloglucan (343), konjac glucomannan (285), phosphoric acid swollen cellulose (86), beechwood xylan (21.7) and carob galactomannan (16), thereby displaying the multi-functionality. Catalytic efficiency (mL.mg−1 s−1) of RfGH5_4 against carboxymethyl cellulose (146) and konjac glucomannan (529) was significantly high. TLC and MALDI-TOF-MS analyses of RfGH5_4 treated hydrolysates of cellulosic and hemicellulosic polysaccharides displayed oligosaccharides of degree of polymerization (DP) between DP2-DP11. TLC, HPLC and Processivity-Index analyses revealed RfGH5_4 to be a processive endoglucanase as initially, for 30 min it hydrolysed cellulose to cellotetraose followed by persistent release of cellotriose and cellobiose. RfGH5_4 yielded sufficiently high Total Reducing Sugar (TRS, mg/g) from saccharification of alkali pre-treated sorghum (72), finger millet (62), sugarcane bagasse (38) and cotton (27) in a 48 h saccharification reaction. Thus, RfGH5_4 can be considered as a potential endoglucanase for renewable energy applications.

QM/MM investigation of the catalytic mechanism of processive endoglucanase Cel9G from Clostridium cellulovorans.

Carbohydrate degradation catalyzed by glucoside hydrolases (GHs) is a major mechanism in biomass conversion. GH family 9 endoglucanase (Cel9G) from Clostridium cellulovorans, a typical multimodular enzyme, contains a catalytic domain closely linked to a family 3c carbohydrate-binding module (CBM3c). Unlike the conventional behavior proposed for other carbohydrate-binding modules, CBM3c has a direct impact on catalytic activity. In this work, extensive molecular dynamics (MD) simulations were employed to clarify the functional role of CBM3c. Furthermore, the detailed catalytic mechanism of Cel9G was investigated at the atomistic level using the combined quantum mechanical and molecular mechanical (QM/MM) method. Based on these simulations, owing to the rigidity of the peptide linker, CBM3c may affect the enzymatic activity via direct interactions with alpha helix 4 of GH9, especially with the K123 and H125 residues. In addition, using cellohexaose as a substrate, the QM/MM MD simulations confirmed that this enzyme can cleave the β-1,4-glycosidic linkage via an inverting mechanism. An oxocarbenium ion-like transition state was located with a barrier height of 19.6 kcal mol-1. Furthermore, the G(-1) pyranose unit preferentially adopted a distorted 1S5/4H5 conformer in the enzyme-substrate complex. For the cleavage of the glycosidic bond, we were able to identify a plausible route (1S5/4H5 → [4H5/4E]# → 4C1) from the reactant to the product at the G(-1) site.

PSI-6 Characterization of in vitro stability for two multi-functional processive endoglucanases as exogenous biocatalysts in pig nutrition

Abstract Poor efficiency of dietary fibre utilization limits global pork production profit margins and mitigation of footprint on environment. The objective of this study was to characterize in vitro stability for two unique processive endoglucanases of tCel5A1 and p4818Cel5_2A that are reported to hydrolyze natural cellulose and have multi-functionality towards hemicelluloses (Basit and Akhtar, Biotechnology and Bioengineering, 2018, 115:1675; Wang et al., Scientific Reports 2019, 9:13630). Both tCel5A1 and p4818Cel5_2A were modelled with the SWISS-MODEL online server and analyzed and visualized by PyMOL2.4.1. These two cellulases were overexpressed in ClearColi®BL21(DE3). Their endoglucanase activities were determined using 0.70 % sodium carboxymethyl cellulose and 5 mM dithiothreitol (DTT) with or without N2 gas purging; and incubated at pH 6.5–7.0 and 37 °C for 15 min. The three-dimensional models showed 1 and 4 Cys residues on the surfaces of tCel5A1 and p4818Cel5_2A, respectively, suggesting their susceptibility to auto-oxidation by air-borne O2. This prediction was tested by comparing the activities of N2-purged and non-purged enzyme preparations as well as their processing and incubation handling. Both tCel5A1 and p4818Cel5_2A activities were enhanced (P < 0.01) by 50% when 5-mM DTT and N2-purging were adopted. Furthermore, after incubating both tCel5A1 and p4818Cel5_2A enzyme preparations under the porcine gastric pH (3.5) and pepsin (274 U/mL) as well as intestinal trypsin (78 U/mL) and chymotrypsin (20 U/mL) activities at pH 6.5 during 0–5 h, Eadie-Hofstee inhibition kinetic analyses showed that tCel5A1 and p4818Cel5_2A respectively lost 18 and 68% (P < 0.01) of their initial activities after 2 h under the gastric conditions and more than 90% (P < 0.01) of their initial activities after 2–3 h under the intestinal conditions. Therefore, further enzyme protein engineering and/or post-fermentation treatments, such as coating for by-passing the gastric-intestinal environment, will be required to enable these two processive endoglucanases as efficacious exogenous fibre enzymes.

Metal Affinity Immobilization of the Processive Endoglucanase EG5C-1 from Bacillus subtilis on a Recyclable pH-Responsive Polymer

Establishing a suitable immobilization strategy to improve the accessibility of immobilized cellulase for insoluble cellulose and subsequently recover the enzyme from the remaining substrate is crucial to promote the industrialization of biofuels. Here, using iminodiacetic acid (IDA) and Ni2+ ions, a novel metal-chelated copolymer of methacrylic acid and methyl methacrylate was prepared for the immobilization of His-tagged processive endoglucanase EG5C-1 via affinity interaction between polyhistidine tag and Ni2+. The optimum loading capacity of the functionalized polymer (Eud-IDA-Ni2+) for EG5C-1 was about 280 mg/g, where more than 80% of the activity was recovered. Immobilized EG5C-1 exhibited improved thermal and pH stability and better reusability than the free one, and after five cycles of usage, the hydrolysis productivity remained above 70% of the initial value. The Eud-IDA-Ni2+/EG5C-1 biocomposite displayed reversibly soluble–insoluble characteristics with pH change, which was in the soluble state during the enzyme reaction process but could be recovered in an insoluble form by lowering the pH after the reaction. Thus, the yield obtained from the hydrolysis of an insoluble phosphoric acid-swollen cellulose substrate was similar for the free and immobilized form of EG5C-1. Our combined results suggested that a metal-ion-chelated, pH-responsive polymer had the potential for His-tagged cellulase immobilization to improve both the operational efficiency and economic benefit of the cellulosic biorefinery industry.

Metagenomic Discovery and Characterization of Multi-Functional and Monomodular Processive Endoglucanases as Biocatalysts

Biomass includes cellulose, hemicelluloses, pectin and lignin; constitutes the components of dietary fibre of plant and alge origins in animals and humans; and can potentially provide inexhaustible basic monomer compounds for developing sustainable biofuels and biomaterials for the world. Development of efficacious cellulases is the key to unlock the biomass polymer and unleash its potential applications in society. Upon reviewing the current literature of cellulase research, two characterized and/or engineered glycosyl hydrolase family-5 (GH5) cellulases have displayed unique properties of processive endoglucanases, including GH5-tCel5A1 that was engineered and was originally identified via targeted genome sequencing of the extremely thermophilic Thermotoga maritima and GH5-p4818Cel5_2A that was screened out of the porcine hindgut microbial metagenomic expression library. Both GH5-tCel5A1 and GH5-p4818Cel5_2A have been characterized as having small molecular weights with an estimated spherical diameter at or < 4.6 nm; being monomodular without a required carbohydrate-binding domain; and acting as processive β-1,4-endoglucanases. These two unique GH5-tCel5A1 and GH5-p4818Cel5_2A processive endocellulases are active in hydrolyzing natural crystalline and pre-treated cellulosic substrates and have multi-functionality towards several hemicelluloses including β-glucans, xylan, xylogulcans, mannans, galactomannans and glucomannans. Therefore, these two multifunctional and monomodular GH5-tCel5A1 and GH5-p4818Cel5_2A endocellulases already have promising structural and functional properties for further optimization and industrial applications.

Impacts of cotton linter pulp characteristics on the processivity of glycoside hydrolase family 5 endoglucanase from Volvariella Volvacea

EG1 from Volvariella volvacea is a processive endoglucanase belonging to glycoside hydrolase family 5. The impacts of cotton linter pulp characteristics, such as degree of polymerization (DP), crystallinity, and the initial cellulose-reducing ends, on the processivity of EG1 were investigated. Three commercial cotton linter pulp with different DP were used in present study. Ball milling was used to alter the crystallinity and DP of cellulose. The results indicate that the crystallinity has the most significant impact on enzyme processivity followed by initial cellulose-reducing ends. Whereas the DP indirectly affects the enzymatic hydrolysis and influenced by the pulp preparation method and conditions. The initial cellulose-reducing ends also affect enzyme adsorption but their impact is not obvious when the crystallinity is very low. These results also demonstrate the endo- and exo-action are both exist for EG1. The processive exo-action can start from the newly created cellulose-reducing ends by endo-action as well as the initial cellulose-reducing ends. The contribution of initial cellulose-reducing ends is affected by its quantity and cellulose crystallinity. A plausible action mode for EG1 is also proposed.

Enhancing the catalytic activity of a GH5 processive endoglucanase from Bacillus subtilis BS-5 by site-directed mutagenesis

Processive endoglucanases possess both endo- and exoglucanase activity, making them attractive discovery and engineering targets. Here, a processive endoglucanase EG5C-1 from Bacillus subtilis was employed as the starting point for enzyme engineering. Referring to the complex structure information of EG5C-1 and cellohexaose, the amino acid residues in the active site architecture were identified and subjected to alanine scanning mutagenesis. The residues were chosen for a saturation mutagenesis since their variants showed similar activities to EG5C-1. Variants D70Q and S235W showed increased activity towards the substrates CMC and Avicel, an increase was further enhanced in D70Q/S235W double mutant, which displayed a 2.1- and 1.7-fold improvement in the hydrolytic activity towards CMC and Avicel, respectively. In addition, kinetic measurements showed that double mutant had higher substrate affinity (Km) and a significantly higher catalytic efficiency (kcat/Km). The binding isotherms of wild-type EG5C-1 and double mutant D70Q/S235W suggested that the binding capability of EG5C-1 for the insoluble substrate was weaker than that of D70Q/S235W. Molecular dynamics simulations suggested that the collaborative substitutions of D70Q and S235W altered the hydrogen bonding network within the active site architecture and introduced new hydrogen bonds between the enzyme and cellohexaose, thus enhancing both substrate affinity and catalytic efficiency.

Changes in supramolecular structure and improvement in reactivity of dissolving pulp via enzymatic pretreatment with processive endoglucanase EG1 from Volvaria volvacea

Processive endoglucanase EG1 and its core domain, EG1(CD), were used to pretreat the commercial dissolving pulp to improve cellulose reactivity. The Fock reactivity of the pulp which was treated with EG1 and EG1 (CD) at 50 U/g enzyme loading increased from 74.3% of the control to 90.6% and 88.4%, respectively. Refining also improved the Fock reactivity of the pulp, but not as effective as EG1 or EG1(CD) treatment. Refining prior to EG1 or EG1(CD) treatment could slightly further improve the Fock reactivity, to 91.6% and 90.0%, respectively. After enzymatic treatment and (or) refining, the water retention value, differential scanning calorimetry and alkaline solubility analysis indicated that enzyme treatment, especially by EG1, significantly increased the accessibility of fibers to reaction reagents. Combined with the characteristics of soluble reducing sugar produced by EG1 treatment and the changes of degree of polymerization, it is inferred that a small fraction of cellulose crystallization regions are destroyed in the enzymatic hydrolysis process due to the processive acting ability of EG1, and some microchannels in the fiber cell wall were created, which is similar to the effect of “drilling holes”, so that the reaction reagent can reach the inside of the cell wall evenly, thus obviously improving the reactivity of the dissolved pulp.

A processive endoglucanase with multi-substrate specificity is characterized from porcine gut microbiota

Cellulases play important roles in the dietary fibre digestion in pigs, and have multiple industrial applications. The porcine intestinal microbiota display a unique feature in rapid cellulose digestion. Herein, we have expressed a cellulase gene, p4818Cel5_2A, which singly encoded a catalytic domain belonging to glycoside hydrolase family 5 subfamily 2, and was previously identified from a metagenomic expression library constructed from porcine gut microbiome after feeding grower pigs with a cellulose-supplemented diet. The activity of purified p4818Cel5_2A was maximal at pH 6.0 and 50 °C and displayed resistance to trypsin digestion. This enzyme exhibited activities towards a wide variety of plant polysaccharides, including cellulosic substrates of avicel and solka-Floc®, and the hemicelluloses of β-(1 → 4)/(1 → 3)-glucans, xyloglucan, glucomannan and galactomannan. Viscosity, reducing sugar distribution and hydrolysis product analyses further revealed that this enzyme was a processive endo-β-(1 → 4)-glucanase capable of hydrolyzing cellulose into cellobiose and cellotriose as the primary end products. These catalytic features of p4818Cel5_2A were further explored in the context of a three-dimensional homology model. Altogether, results of this study report a microbial processive endoglucanase identified from the porcine gut microbiome, and it may be tailored as an efficient biocatalyst candidate for potential industrial applications.

Role of carbohydrate binding module (CBM3c) of GH9 β-1,4 endoglucanase (Cel9W) from Hungateiclostridium thermocellum ATCC 27405 in catalysis

The function of CBM3c in the enzymatic catalysis varies among the members of family 9 Glycoside Hydrolases (GH). A new member of family 9 GH (Cel9W) from thermophilic anaerobic bacterium, Hungateiclostridium thermocellum was explored for elucidation of the role of CBM3c in catalysis by GH9. Cel9W is a multimodular theme B1, cellulase enzyme comprising a catalytic module of family 9 glycoside hydrolase (HtGH9t) at N-terminal, a family 3c carbohydrate binding module (HtCBM3c) and a dockerin domain at the C-terminal. The ORF of Cel9W encoding full length β-1,4-glucanase, HtGH9 (containing both GH9 and CBM3c modules), the truncated GH9 catalytic module (HtGH9t) and module CBM3c (HtCBM3c) were cloned and over-expressed using E. coli BL21 cells. HtGH9 showed maximum activity at pH 6.5 and 90 °C. It displayed highest activity of 64 U/mg against lichenan followed by 44.6 U/mg (β-glucan) and 22.3 U/mg (Carboxymethyl cellulose). HtGH9 showed stability in the pH ranging from 5.0 to 9.0 and thermal stability up to 70 °C for 1.0 h. The presence of EDTA and EGTA decreased the activity of HtGH9 and also shifted the melting curve peak from 93 °C to 88 °C indicating that the enzyme inherently possesses metal ions, which play role in catalysis and structural stability. The TLC analysis of HtGH9 hydrolysed Avicel showed the presence cellotetraose indicating the processive endoglucanase activity in HtGH9. The module, HtGH9t alone exhibited very low level of activity (1.22 U/mg) against lichenan. It partially recovered (51%) the enzyme activity in presence of equimolar concentration of HtCBM3c. Non-denaturing gel electrophoresis revealed a non-covalent binding interaction between the catalytic HtGH9t module with HtCBM3c module and their physical association showed the partial recovery of its endoglucanase activity. This study confirmed that the physical association of the catalytic module HtGH9t with HtCBM3c is necessary for HtGH9 to efficiently hydrolyze the cellulosic substrates.