Bacillus clarkii 7364 γ-环糊精葡萄糖基转移酶的可溶性表达及其催化特性分析

摘要

按照大肠杆菌偏爱密码子对Bacillus clarkii 7364来源的γ-环糊精葡萄糖基转移酶(γ-CGTase)进行优化,构建γ-CGTase原核表达菌株,摸索γ-CGTase的可溶性表达条件及纯化条件,并对其催化特性进行研究.结果表明:在28 ℃条件下实现了γ-CGTase的高效可溶性表达,可溶性蛋白占总蛋白表达量的63%,酶活可达3 830 U/mL.经(NH4)2SO4沉淀和α-CD-Sepharose 6B亲和柱纯化后,酶蛋白纯化了12.97倍,酶收率20.31%.使用该酶对木薯淀粉进行转化,转化产物中γ-环糊精(γ-CD)的比例可达90.9%,几乎无α-环糊精(α-CD),与天然酶的79%相比提高了15%.将该基因工程菌在20 L发酵罐中发酵,10 h后酶活达到4 375 U/mL,证实了其工业化放大的可能.该酶具有非常高的转化专一性,有非常好的工业化前景.%The γ-CGTase gene sequence from Bacillus clarkii 7364 was optimized according to the biased condons of E.coli and its prokaryotic expression strain was constructed.The condition of expression of γ-CGTase and the purification method of γ-CGTase was established,and the catalytic characterization of γ-CGTase was studied.The γ-CGTase achieved highly soluble expression at 28 ℃ and the soluble expression protein accounts for 63% of the total protein,the enzyme activity reached 3 830 U/mL in the periplasm.The recombinant γ-CGTase was purified by ammonia sulfate precipitation,followed by affinity chromatography on a α-CD-Sepharose 6B affinity column,and the purification fold and yield was 12.97 and 20.31%,separately.After incubation with cassava starch,the majority (90.9%) of product CDs was γ-CD that was about 15% higher than the wild γ-CGTase,and nearly no α-CD was detected.The engineering strain was cultivated in 20 L fermentor and γ-CGTase activity could reach 4 375 U/mL,which provided the possibility for further industrialization.The recombinant γ-CGTase showed very high specificity of transformation and would have better perspective in industrialization.