β-1,4-内切葡聚糖酶基因在毕赤酵母中的表达研究

Expression of endo-1,4-β-glucanasegene from Bacillus sp in Pichia pastoris

根据芽孢杆菌β-1,4-内切葡聚糖酶基因序列设计引物,以pHBM102为模板,扩增得到β-1,4-内切葡聚糖酶GluD基因片段,克隆至毕赤酵母(Pichia pastoris)表达载体pHBM905上,获得重组毕赤酵母表达载体pHBM905D,将此质粒分别转化毕赤酵母GS115,KM71和SMD1168菌株,筛选获得GS115(pHBM905D),KM71(pHBM905D),和SMD1168(pHBM905D),平板诱导培养表明,GS115(pHBM905D)所产生的水解圈最大,酶活力最高.摇瓶诱导培养,表达β-1,4-内切葡聚糖酶,此酶的最适反应温度为65℃,最适反应pH值6.4,在诱导8 d后酶活力达到最高为0.185 8 U/mL.

The endo-1,4-β-glucanase GluD gene from Bacillus sp pHBM102 was cloned into the Pichia pastoris expression vector pHBM905, the combinant plasmid was named pHBM905D. The digested recombinant plasmid was transformed into Pichia astoris KM71, GS115, SMD1168, respectively. There combinant Pichia pastoris KM71 （pHBM905D） GS11 （pHBM905D）, SMD1168 （pHBM905D） secreted functional endo-1,4-β- glucanase, and the strain GS115 （pHBM905D）, which produces the maximum hydrolysis halos was screened out . The endo-1,4-β-glucanase expressed by GS115（pHBM905D）has a optimum of 6.4 and a temperature optimum of 65℃. It has the highest level of expression endo-1,4-β-glucanase of 0. 185 8 U/mL.