重组α-环糊精葡萄糖基转移酶表达条件的优化及其产物专一性的分析

Optimization expression of recombinant α-cyclodextrin glucanotransferase and product specificity analysis

将来源于Bacillus sp 602-1的α-环糊精葡萄糖基转移酶(α-CGT)基因(cgt)插入到表达载体PQE30中,构建重组质粒PQE30/cgt,成功转化宿主菌E.coli M15后,得到重组菌株E.coli M15(PQE30/cgt)。在IPTG的诱导下得到酶表达的最适条件:TB培养基,0.01 mmol/L IPTG,诱导温度16℃,胞内酶比活力最高可达5 209 U/mL;加入IPTG 24 h后,添加甘氨酸和甘露醇会促使酶向胞外分泌。酶蛋白自诱导表达的适宜条件为在TB培养基中添加乳糖3.0 g/L,葡萄糖1.2 g/L,16℃培养96 h,酶比活力达到8 635 U/mL,明显高于IPTG诱导的效果。通过SDS-PAGE验证了上述结论。酶催化转化实验表明:重组酶转化质量分数为1%可溶淀粉24 h后,α-环糊精(α-CD)转化率可达38.2%,α和β的峰面积比约为3.4∶1,α-CD具有较高的专一性,因此该重组α-CGT酶具有较好的工业化应用前景。

The gene of α-cyclodextrin glucanotransferase（α-CGTase） was amplified by colony PCR from Bacillus sp 602-1 to insert into expression vector PQE30,and then constructed a recombinant strain E.coli M15（PQE30/cgt）.The optimal conditions for the expression of α-CGTase by the IPTG induction were as follows： TB medium,0.01 mmol/L IPTG,16 ℃,with the activity of 5 209 U/mL culture.In addition,glycine and mannitol were found to be able to help secretion of the α-CGTase.The activity in an optimized auto-inducing medium,in which TB medium containing 1.2 g/L glucose and 3.0 g/L lactose,achieved 8 635 U/mL.The enzyme catalytic tests demonstrated that the yield of α-CD reached 38.2% after 24 h incubation with an α∶β ratio of 3.4∶ 1.Thus,the recombinant α-CGTase showed better industrial perspective in the α-CD production.