苏云金芽孢杆菌普鲁兰酶编码基因amyX的鉴定与重组酶性质研究

目的]研究苏云金芽孢杆菌konkukian亚种amyX基因在大肠杆菌中的表达及表达产物的性质。方法]以苏云金芽孢杆菌(Bacillus thuringiensis)染色体DNA为模板,PCR扩增得到amyX普鲁兰酶结构基因,再与大肠杆菌表达载体pET28a连接,构建能表达普鲁兰酶的重组大肠杆菌。重组菌经IPTG诱导,表达有活性的普鲁兰酶,初步分析重组普鲁兰酶的酶学性质。结果]结果表明,苏云金芽孢杆菌基因组序列有两条基因(pulA和amyX)可能编码普鲁兰酶。温度为50℃,pH为6.0时,重组酶不能水解淀粉,属于I型普鲁兰酶。结论]amyX基因能在大肠杆菌细胞内成功表达,表达产物具有典型的I型普鲁兰酶的特性。

Identification of the Pullulanase Encoding Gene amyX of Bacillus thuringiensis and Charaterization of the Recombinant Enzyme

Objective] The aim of this paper was to study the expression of Bacillus thuringiensis Konkukian amyX gene in Backwoods coli and the character of expression production.Method] Taking Bacillus thuringiensis chromosome DNA as the template,amyX pullulanase genes were got by PCR amplification,and then connected with Backwoods coli carrier pET28a and established recombinant Backwoods coli that could express pullulanase.Recombinant fungus were induced by the IPTG to express active pullulanase and analyse the enzymology character of the recombinant pullulanase.Result] The results showed that genome sequence of Bacillus thurinbiensis revealed the two genes(pulA and amyX) that could code the pullulanase.The optimal conditions that recombinant enzyme could′t hydrolyze the amylum were the temperature of 50 ℃,PH value of 6.0.This enzyme belonged to I type pullulanase.Conclusion] AmyX gene could be successfully expressed in the Backwoods coli,and expression producetion had the typical I type pullulanase character.