枯草芽孢杆菌寡聚-1,6-葡萄糖苷酶基因在毕赤酵母中的表达研究

The expression of oligo-1,6-glucosidase from Bacillus subtilis in Pichia pastoris

根据枯草芽孢杆菌(Bacillus subtilis)寡聚-1,6-葡萄糖苷酶基因序列设计引物,以pHBM003为模板,扩增得到寡聚-1,6-葡萄糖苷酶基因,克隆至毕氏酵母(Pichia pastoris)表达载体pHBM905上,获得重组毕氏酵母表达载体pHBM9053.将此质粒分别转化毕氏酵母GS115、KM71和SMD1168菌株,筛选获得重组毕赤酵母GS115(pHBM9053)、KM71(pHBM9053)和SMD1168(pHBM9053);然后进行摇瓶诱导培养,这3株毕氏酵母分别在诱导培养60 h、48 h和24 h后,酶活力达到最高,对应为2.233 u/mL、0.34 u/mL和1.235 u/mL;GS115(pHBM9053)所产寡聚-1,6-葡萄糖苷酶的最适反应温度为75 ℃,最适反应pH值为6,在30～75 ℃、pH 8～9范围内较稳定.

According to the published sequence of oligo-1,6-glucosidase gene from Bacillus subtilis,a pair of primers was designed to amplify the oligo-1,6-glucosidase gene.The PCR fragment was treated by T4 DNA polymerase and dTTP.Then ligated with the expression vector pHBM905 which has digested by CpoI and Notl.A recombinant vector pHBM9053 was acquired and transformed into Pichia pastoris GS115,KM71,SMD1168 respectively.Oligo-1,6-glucosidase was expressed in all this three strains. The recombinant Pichia pastoris GS115 （pHBM9053） has the highest level of expression oligo-1,6-glucosidase of 2.223u/ml,compared with other two recombinant strains KM71 and SMD1168.The oligo-1,6-glucosidase expressed by GS115（pHB9053） has a pH optimum of 6 and a temperature optimum of 75℃, and are aclive at pH ranged from 8 to 11 and temperature ranged from 30℃ to 75℃.