Cloning And Expression Of Aspergillus Niger β-glucosidase Gene

P-Glucosidase is an enzyme which can hydrolyze theβ-1,4-D-indican linkages, It belongs to the enzyme family of cellulase, can hydrolyze cellobiose to glucose.β-Glucosidase is widely existent in various living things, and has important research value. It has a broad application in textile and oil industry, pharmacy, feed and food-making, papermaking, dyeing and printing as well as in biological researchs. The object of the present study is to construct the recombinant yeast for the high-level expression of theβ-Glucosidase, in order to provide an efficient way to solve the problems such as low production and high cost, and also we can get high-qualityβ-Glucosidase in the end.In this paper, the enzyme produce Aspergillus niger AS3.796 was used as material. Theβ-Glucosidase Gene cDNA of Aspergillus niger was amplified with RT-PCR by use the special primers, The result of sequence analysis showed that the cDNA fragment was 2583bp, the sequence coded 360 amino acid. It was submitted to GenBank. According to the amino acids sequence analysis by bioinformatics database and softwares, this P-Glucosidase was a member of family3 of hydrolase, and amino acids sequence homology of bgl genes among other Aspergillus could reach 96%.In this paper, the Pichia Pastoris GS115 was used to expressβ-Glucosidase by secretion. The bgl gene was cloned and inserted into the plasmid pPICZaA to construct recombinant plasmid pPICZaA-bgl. Then it was transformed into the host Pichia pastoris GS115 by homologous recombination between the transforming DNA and regions of homology within the genome, and this gene insert in the region of the AOXl promoter or the AOXl transcription termination region. The transforments were plated on medium cantaining Zeocin, and 6 colonies had been screened. Then, the colonies were cultured in methanol inducement medium in order to produce enzyme. According to the SDS-PAGE analysis of fermentation supernatant, the molecular weight of protein was about 97kD, the expression level was about 0.2-0.3mg/ml. The activity of recombinantβ-Glucosidase was 0.184-0.448 IU/ml. it shows that the optimal condition for enzyme reaction are pH5.5 and 50℃.The target protein was expressed in Pichia Pastoris GS115 successfully, but the expression level and enzyme activity ofβ-Glucosidase is not very good.