| β-glucosidase (EC3.2.1.21; β-D-glucosidase) can hydrolyze the β-1,4glycosidic linkage in cellobiose. And it is impact to efficiency of the cellulose degradation, β-glucosidase has been applied in the wine, tea, beverage industry and has made great economic benefits. In addition, the application of P-glucosidase in the hydrolysis of resveratrol and soy isoflavones has increasingly be concerned in recent years.A fosmid library of Paenibacillus cookii GX-4containing about6,000clones was constructed successfully. Four positive clones of β-glucosidase were obtained on the selective plates with sammonium ferric citrate-esculin.The two positive clones were subcloned, posessing the same ORF. The protein encoded by the ORF was analyzed, indicating that the protein belong to the β-glucosidase from the glycosyl hydrolase family3. The ORF was named Pbgl. The gene Pbgl was amplified by PCR and cloned into expression vector pSE380.The recombinant Pbgl was expressed in the E.coli and purified by nickel affinity chromatography. The purified Pbgl, with size of approximately84kDa, was monomeric form on the polyacrylamide gel electrophoresis. As pNPG as substrate, optimal temperature and pH of the Pbgl were50℃and8.0, respectively. Km and Vmax of Pbgl were0.1754±0.0194mM and149±3.4U/mg.The glucose inhibition constants of IC50and Ki were177.1mM and34.65mM, respectively. Pbgl is able to hydrolyze cellobiose and lactose.The amino acids related to glucose tolerance of Pbgl were studied. The six forward mutants were obtained, named Rpbgl, W60L, W386A, W386C, W386F, W386L, respectively. The six mutants were characterized. Compared with the Pbgl, the the enzymatic activity of the other five mutants are decreased in varying degrees except for Rpbgl. The optimum temperature of the four mutants W386A, W386C, W386F, W386L were reduced from50℃to40℃. The results showed the glucose tolerance of the six mutants were higher than wild-type Pbgl. The glucose tolerance of W386A increased11.88times than Pbgl. |
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