Studies On The Cloning And Expression Of β-Glucosidase Gene

Cellulose is the most abundant renewable resource on the earth. It can be hydrolyzed to glucose, oligosaccharide and a variety of active aglycone by cellulase. Its hydrolysate can be applied to textiles, paper, food, animal feed, fuel, chemicals, and pharmaceutical industry, etc. Cellobiose can be hydrolyzed into two molecules of glucose by the β-glucosidase, which is the rate limiting enzyme in the whole cellulose hydrolysis reaction and determines the overall activity of cellulose.The aim of this paper is to improve the activity and expression of β-glucosidase from Aspergillus niger strain by gene cloning and heterologous expression. The cDNA sequence (2583 bp) and the DNA sequence (2934 bp) of the β-glucosidase were amplified by RT-PCR and PCR, respectively. The obtained cDNA sequence was cloned into pET28a, pET32a, and pPIC9K vector and expressed heterologously in E. coli and Pichia pastor is, respectively. The obtained DNA sequence was cloned into pPICZaA vector and expressed heterologously in Pichia pastoris.The result showed that the gene of β-glucosidase can be expressed in high level in E. coli but the product of expression does not have β-glucosidase hydrolysis activity. It was found that the recombinant of pET28a may be the inclusion body and it has not the β-glucosidase hydrolysis activity by denaturation and renaturation. The phenotype of pPIC9K recombinant transformed into Pichia pastoris GS115 were identified as type of Muts on the MD and MM culture medium. It was expressed by methanol induction and showed lower β-glucosidase activity using esculin and pNPG as substrate. The phenotype of pPICZaA recombinant transformed into Pichia pastoris GS115 was identified as type of Mut+by PCR. It was also expressed by methanol induction and showed higher β-glucosidase activity (0.274 U/mL) at 30℃ and pH 6.0 using pNPG as substrate.The produced P-glucosidase was purified by ammonium sulfate precipitation and Ni-affinity chromatography. The SDS-PAGE analysis of purified products showed that there were still many impurities protein. The purification of produced (3-glucosidase should be further investigated.