扣囊复膜孢酵母β-葡萄糖苷酶基因在毕赤酵母中的克隆与表达

Expression of β-Glucosidase gene of Sacchromycopsis fibuligera in Pichia pastoris

利用PCR技术,从扣囊复膜孢酵母的总DNA中扩增得到β-葡萄糖苷酶(β-Glucosidase)基因(BGL1),长度为2596bp,连接到pGEM-T载体上,用限制性内切酶切下目的基因,插入到巴斯德毕赤酵母表达载体pPIC9K中,使之位于α-因子信号肽下游,且与之同框,构建成重组质粒pSHL9K。通过电转化将重组质粒pSHL9K插入到PichiapastorisGS115菌株染色体中,获得高效表达BGL1基因的毕赤酵母重组工程菌株。重组酶的最适温度为50℃,最适pH为5.4。培养基中β-葡萄糖苷酶活性最高可达47U/mL。

A β-Glucosidase gene （ BGL 1 ） was amplified with PCR from the total DNA of Sacchromycopsis fibuligera, and was linked with pGEM-T vector. After cut down by restriction enzyme from pGEM-T vector, BGL1 was inserted into the expression vector pPIC9K of Pichia pastoris in reading frame with a-factor secreting signal peptide sequence to construct the recombinant plasmid pSHL9K. The recombinant plasmid pSHL9K was transformed into Pichia pastoris GS115 with electroporation. The recombinant Pichia pastoris strains which could efficiently secret recombinant β-Glucosidase were selected. The optimum temperature of the recombinant β-Glucosidase was 50℃, and the optimum pH was 5.4. The activity of β-Glucosidase could reach to 47U/mL in the culture medium.