摘要
用PCR的方法从克鲁维酵母(Kluyveromyces marxianus)ATCC12424中克隆出外切菊粉酶基因.将该基因克隆到毕赤酵母表达载体pHBM905C、pHBM906上,构建了外切菊粉酶毕赤酵母表达载体pHBM1200、pHBM1201.将两者转化毕赤酵母GS115,得到重组菌株GS115(pHBM1200)、GS115(pHBM1201),将这两种重组菌株进行摇瓶发酵,测得带α-信号肽的菌株GS115(pHBM1200)表达的外切菊粉酶最高酶活为89.43 U/mL,带自身信号肽的菌株GS115(pHBM1201)表达的外切菊粉酶最高酶活为14.828 U/mL.SDS-PAGE电泳表明,外切菊粉酶的表观分子量为90 kD.将外切菊粉酶用去糖基化酶endoH处理后,SDS-PAGE电泳表明,分子量为60 kD,和预计的分子量一致.
The exoinulinase gene was cloned from Kluyveromyces marxianus by PCR, and cloned to the expression vectors pHBM 905C ,pHBM 906. The recombinant vectors pHBM 1200,pHBM 1201 were acquired. The two recombinant vectors were transformed into Pichia pastoris GS115, and the recombinant strain GS115（pHBM 1200）, GS115 （pHBM 1201）were fermented in flask. Enzyme activity analysis showed that the strain GS115（pHBM 1200） which contained a - signal peptide reached higher enzyme activity （89.43 U/mL） than the strain GS115（pHBM 1201）which contained signal peptide of it＇s own （14. 828 U/mL ）. The exoinulinase＇ s molecular weight was estimated to be about 90 kD by SDS - PAGE, and it reduced to 60 kD when the enzyme was treated with deglycosylase （Endon）.
出处
《湖北大学学报（自然科学版）》
CAS
北大核心
2006年第4期385-388,共4页
Journal of Hubei University：Natural Science
基金
湖北省科技攻关项目(2003AA101C22)资助
