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β-glucosidase (BGL) is a class of hydrolytic enzyme capable of hydrolyzing the β-1 ,4-D-glycosidic bond endo are cellulose enzymes, it is able to degrade the cellobiose to glucose. beta-glucosidase widely exist in a variety of plants, animals and micro-organisms in vivo has important research value, in recent years through the research and development of a variety of sources of beta-glucosidase, these enzymes in the pharmaceutical, food, feed, paper making, printing and dyeing, textile, oil, mining and biotechnology research aspects have been widely used. The main purpose of this study was obtained from Aspergillus niger beta-glucosidase gene, and to high-level expression of β-glucosidase, beta-glucosidase production low cost of building engineering yeast, but also for The cellulose degradation efficient, the production of fuel ethanol to provide an effective solution. In this study, Aspergillus niger, Aspergillus niger 3.796 for the material, the full sequence of the beta-glucosidase gene cDNA was cloned into the basis for other Aspergillus niger beta-glucosidase the bgl gene sequence specificity of primers by RT-PCR method. Sequence analysis showed that the cDNA sequence of full-length 2583bp, encoding 860 amino acids, the gene has been registered GenBank, and the use of bioinformatics databases and software to analyze and predict the gene and its encoded protein. The analysis showed that the β-glucosidase 3 members belong to the family of hydrolytic enzymes, compared to its amino acid sequence with other Aspergillus up to 96% homology. This study uses the the Pichia yeast Pichia Pastoris GS115 secretion expression of β-glucosidase. Beta-glucosidase gene was cloned into the secretion expression vector pPICZaA to construct recombinant the plasmid pPICZaA-bgl. After homologous recombination integrated into Pichia pastoris GS115 chromosome recombination sites in the AOX1 gene promoter and transcription terminator between. Zeocin resistance screening and PCR positive clones. Positive clones were screened again for methanol-induced enzyme production culture, expression product by SDS-PAGE electrophoresis analysis, the relative molecular weight of about 97kD, expression levels of 0.2-0.3 mg / ml. The beta-glucosidase activity of the recombinant strain measurement results for 0.184-0.448 IU / ml. Enzymatic Properties enzyme reaction optimum pH 5.5, the optimum temperature of 50 ° C. According to SDS-PAGE electrophoresis and enzyme activity analysis to prove that the target protein has been achieve expression in Pichia pastorisGS115 successfully be improved, but the expression levels and enzyme activity.
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