α－环糊精葡萄糖基转移酶的高效表达及酶法制备AA－2G条件优化

摘要

目的：构建分泌表达α－环糊精葡萄糖基转移酶（α－Cyclodextrin Glycosyltransferase ，α－CGTase）的重组菌株，并利用该酶液制备AA－2G。方法经PCR扩增浸麻芽孢杆菌154基因组获得α－CGTase基因序列，将该基因序列和pET26b载体分别经Nco I、Xho I双酶切后用T4 DNA连接酶连接，并转化大肠杆菌BL21。利用发酵培养基分泌表达α－CGTase，利用该酶和淀粉糊精、维生素C反应制备AA－2G，并进行制备条件优化。结果在大肠杆菌中实现了α－CGTase的胞外分泌表达，胞外环化酶活力达到120 U／mL，利用该酶的转糖基反应将淀粉糊精、维生素C转化为AA－2G，经HPLC检测正确。结论利用自制α－CGTase得到了AA－2G，通过制备条件优化AA－2G产量为17．46 g／L，转化率达到58．2％（mg／mg）。%Objective To construct a prokaryotic expression vector in BL21 to secretorily expressα-Cyclodextrin Glycosyltransferase(α-CGTase). Methods α-CGT gene was amplified from Bacillus macerens genome by PCR.pET26b and α-CGT gene were connected after digested with Nco I, Xho I respectivly, and then transformed into Escherichia coli BL21 strain.α-CGTase was expressed in fermentation culture medium and AA-2G was prepared by using α-CGTase, VC and starch.Results α-CGTase was expressed secretorily and the enzyme activity was up to 120 U/mL.AA-2G was prepared by the biotransformation of VC and starch using α-CGTase which proved to be correct by HPLC.Conclusion AA-2G was prepared by using self-madeα-CGTase, after optimized the preparation conditions the yield of AA-2G was 17.46 g/L, and the conversion rate reached 58.2%(mg/mg).