Expression Of β-glucosidase Gene From Aspergillus Oryzae Gif-10 And Characterization Of Recombinant Enzyme

There have an Aspergillus oryzae gif-10 in our laboratory. the genomic sequence of the β-glucosidase was cloned from it. In order to improve the β-glucosidase enzyme activity and enzymatic properties and base cellulase coexpressing the basic platform, this study will build a yeast engineered bacteria of β-glucosidase. The results of the experimental are as follows:1、We extracted the total RNA of Aspergillus oryzae gif-10 by TRIZOL method,and use it as a template, we get the cDNA sequence of the β-glucosidase. the total length of the cDNA sequence is 2586bp, the GC content of the gene sequence is 54.4%, encode 861 amino acids. Compared with the GenBank database analysis found that the β-glucosidase gene bgl with the β-glucosidase gene in Aspergillus oryzae RIB40 sequence homology is 99%.2、Adding the restriction sites Sac Ⅱ and Xba Ⅰ.Put the target gene fragment with restriction sites and the plasmid pPICZaA ligation, transformation, and sequence. Then electricity transferred into the yeast X33, and the yeast transformants screened, identified, selected six yeast transformants.The enzyme activity of them were 5.95,6.10,5.77,6.88,6.22,5.99. Select the No.4 Yeast Strain for the following experiments, and was named pPICZaA-bgl.3、Put the yeast engineered bacteria culture, the best time of induction is 96h,10 consecutive continuous culture induced the yeast engineered bacteria,the growth density and beta-glucosidase enzyme activity remained stable, it have a good genetic stability.4、We have a single protein of about 100KD by precipitation with ammonium sulfate, purified by nickel column and by SDS-PAGE detection, the size of the protein theoretical value is 94KD, because it have glycosylation sites, so the size is the correct one.Purified β-glucosidase activity is 17.19U/mL, the protein content is 0.286mg/mL; enzyme activity is 60.10U/mg.5、The enzymatic properties of β-glucosidase analysis showed that the optimum temperature is 50℃,in the Environment of 20℃ to 50℃,it has a good stability of the enzyme activity; the optimum pH is pH 5.0, in environmental of pH 4.0-6.0, its enzyme activity can hold the high level.β-glucosidase substrate specificity for salicin and cellobiose is strong; Different metal ions shows different effects on β-glucosidase activity., the activating effect is most obvious is MnCl2, inhibition of the most obvious is CuCl2.