| Cellulose is the most abundant carbohydrate in nature, but much of it is not effectively utilized, while cellulase can degrade cellulose into oligosaccharides or glucose monomers, allowing it to be utilized. Therefore, using cellulase to convert the wasted cellulose in nature into an energy source represents a possible solution to energy shortages, environmental contamination and other problems. Additionally, cellulase could be widely used in food and fodder processing, pharmaceuticals, brewing, and papermaking. For these reasons, the purpose of this study have focused our researches on improving specific properties of cellulase for industrial use.A strain of Paenibacillus alvei YD236 was isolated from Bos frontalis feces samples collected from Nujiang Lisu Autonomous Prefecture, Yunnan Province for this study. Using Mi Seq high-throughput second generation whole-genome sequencing analysis, enzyme gene fragments were obtained. Comparative analysis of the nucleic acid sequence results yielded around 100 genes from about 50 families, including 6 cellulase genes.The six cellulase genes were cloned into the expression vector p EASY-E2 to generate a recombinant plasmid, which was transfected into Escherichia coli BL21(DE3) for expression. The active recombinant enzymes Pglu E3, a GH3 family β-glucosidase, and Pglu E8, a GH8 family endo-1,4-β-glucanase, were successfully expressed and purified by Ni2+-NTA metal chelating affinity chromatography. The purified enzymes were then subjected to characterization and catalytic activity assays, with the following results.(1) Recombinant Pglu E3 showed an optimum p H of 7.0 and is relatively stable between p H 5.0–9.0. Its optimum temperature is 45 oC; it retained 17.0% of its enzymatic activity at 0 oC, and had good thermal stability at 37 oC and 45 oC. It was resistant against trypsin and ethanol to a certain extent, and could hydrolyze lactose and modified starch to a certain extent.(2) Recombinant Pglu E8 showed an optimum p H of 5.5 and was relatively stable between p H 3.0–10.0. Its optimum temperature is 50 oC; it retained 78.6%, 41.6%, and 34.5% of its enzymatic activity at 20 oC, 10 oC, and 0 oC, respectively. It had good thermal stability at 37 oC and 50 oC. Treatment with 10 m M β-mercaptoethanol enhanced its function. It exhibited resistance against trypsin and proteinase K, and could hydrolyze CMC-Na, soluble starch, barley β-glucan, fucoidan, and yeast glucan. It had relatively strong tolerance to salt, and it could hydrolyze barley β-glucan into cellotriose, cellotetraose, and cellopentaose, and other oligosaccharides.(3) Sequence alignment and homology modeling analyses employing the point mutation method confirmed that Asp277 was the catalytic residue of Pglu E3 and that Glu55 and Asp116 were the catalytic residues of Pglu E8. Catalytic activity increased to varying degrees if residues Cys23 and Cys364 of making up disulfide bond were mutated. This result advances the understanding of the role of the disulfide bond in this class of enzyme, and the mechanism by which β-mercaptoethanol increases catalytic activity.In summary, this study showed that a β-glucosidase with low-temperature activity, hydrolysis of lactose and modified starch, and an endo-1,4-β-glucanase with low-temperature activity, stability at wide p H range and tolerance to salt. They likely have broad potential applications in pharmaceuticals, food and feed processing, and brewing, and also provided insight on cellulase structure and its relationship to its mechanism of action. |
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