β-葡萄糖苷酶高产菌株的筛选及其基因的克隆与表达

摘要

Immobilization of α-amylase with ethyl cellulose was studied,the immobilization conditions were optimized,and the performances of immobilized enzyme and free enzyme were compared. The results showed that the immobilized α-amylase had the best repeat-operation stability at 4℃ with α-amylase concentration 4 g ·L-1 ,mass fraction of ethyl cellulose 0.50%. The optimum reaction pH value and reaction temperature of immobilized α-amylase was 7. 0 and 60℃ ,respectively,and it had good thrernostability,reusability and storage stability. The immobilization method was simple, convenient, reducing enzyme denaturation, remaining on maximum enzyme activity.%利用栀子苷培养基从滨海新区盐碱地土样中筛选得到一株高产β-葡萄糖苷酶菌株,酶活力达到14.82U·mL-1,经16S rDNA鉴定,命名为短小芽孢杆菌B-4.克隆获得B-4 β-葡萄糖苷酶基因,测序结果表明,其大小为1437bp,与GenBank中短小芽孢杆菌SAFR-032 β-葡萄糖苷酶基因YP_001488769.1序列比对,核苷酸序列同源性达97％,氨基酸序列同源性达99％.进一步利用表达载体pET-22b(+),实现β-葡萄糖苷酶基因bglB在大肠杆菌BL21(DE3)中的高效表达,酶活力达46.85U·mL-1.