利用重组大肠杆菌生产α-环糊精葡萄糖基转移酶  

Production of α-cyclodextrin glycosyltransferase in recombinant Escherichia coli

作　　者：成成[1,2] 李兆丰[1,2] 李彬[1,2] 刘花[1,2] 陈坚[1,2] 吴敬[1,2] 

机构地区：[1]江南大学食品科学与技术国家重点实验室,无锡214122 [2]江南大学生物工程学院,工业生物技术教育部重点实验室,无锡214122

出　　处：《生物加工过程》2009年第3期56-63,共8页Chinese Journal of Bioprocess Engineering

基　　金：国家杰出青年基金资助项目(20625619);食品科学与技术国家重点实验室科研基金资助项目(SKLF-MB-200802);江苏省自然科学基金资助项目(BK2007019)

摘　　要：将来源于软化类芽孢杆菌(Paenibacillus macerans)的α-环糊精葡萄糖基转移酶(α-CGT)基因插入含pelB信号肽的质粒pET-20b(+)中,构建了表达载体pET-20b(+)/cgt,并将其转化表达宿主E.coliBL21(DE3)。对重组菌E.coliBL21/pET-cgt进行摇瓶发酵条件的优化,确定了其胞外表达α-CGT酶的最适条件:葡萄糖8 g/L,乳糖0.5 g/L,蛋白胨12 g/L,酵母膏24 g/L,K2HPO472 mmol/L,KH2PO417 mmol/L,CaC l22.5 mmol/L;初始pH为7.0,诱导温度为25℃。在该条件下培养90 h后最终α-CGT酶的胞外比活达到22.1 U/mL,与来源菌P.macerans所产天然酶比活相比提高了42倍,实现了α-CGT酶的高效生产。将基因工程菌在上述条件下于3 L发酵罐中发酵,90 h后胞外酶比活达到22.6 U/mL,证实了工业化放大的可能性。α-Cyclodextrin glycosyltransferase （α-CGTase） genefrom Paenibacillus macerans was cloned and inserted into the expression plasmid pET 20b（＋） containing a secretive pelB as signal peptide. The constructed recombinant pET-20b（＋）/cgt was transformed into E. coli BL21 （DE3） for the overexpression. The optimum conditions for the extracellular production of α-CGTase in shaking flask were determined as follows： 8 g/L glucose, 0. 5 g/L lactose, 12 g/L peptone, 24 g/L yeast extract, 72 mmol/L KEHPO4, 17 mmol/L KH2PO4, 2.5 mmol/L CaCl2, initial pH 7.0 and culture temperature was 25 ℃. With these conditions, the activity of recombinant α-CGTase of culture supernatant achieved 22. 1 U/mL after 90 h, it was about forty-two times higher than that in the parent strain P. macerans. When the engineered strain was cultivated in 3 L fermentor, α-CGTase activity in culture media reached 22. 6 U/mL, thus providing a possibility for further industrial production of α-CGTase.

关 键 词：Α-环糊精葡萄糖基转移酶 cgt基因 大肠杆菌 克隆 

分 类 号：Q555.4[生物学—生物化学]