L-天冬氨酸α-脱羧酶工程菌株的构建及其培养条件研究

Construction and culture conditions of L-aspartate α-decarboxylase engineered strain

L-天冬氨酸α-脱羧酶以L-天冬氨酸为底物,脱去α-羧基后生成β-丙氨酸.利用PCR扩增技术,将Escherichia coliDH5α中的PanD基因克隆后连接到pET28c(+)载体上,先转化E.coliDH5α中,经过50μg/mL卡那霉素抗性筛选和酶切、测序验证后,转化表达菌株E.coliBL21(DE3),得到具有L-天冬氨酸α-脱羧酶活性的工程菌株E.coliBL21(DE3)/pET28c(+)-panD.经乳糖诱导该基因能有效表达C-末端具有组氨酸标签的L-天冬氨酸α-脱羧酶.通过对乳糖诱导时间、乳糖诱导质量浓度、诱导温度、诱导菌龄和培养基诱导初始pH值等发酵条件的优化,得到最佳培养条件为:乳糖诱导时间20 h,乳糖质量浓度12 g/L,诱导温度为35℃,诱导菌龄为3.5 h,诱导培养基初始pH 5.5,通过对转化产物β-丙氨酸的检测结果分析显示,工程菌株E.coliBL21(DE3)/pET28c(+)-panD在优化条件下,酶活力达186 U.

L-aspartate α-decarboxylase（PanD） is an enzyme catalyzing α-decarboxylation of L-Aspartate to β-Alanine.The PanD gene of Escherichia coli DH5α,encoding the L-aspartate α-decarboxylase,was amplified by PCR and cloned into the expression plasmid pET28c（＋）.The recombinant plasmid was then transformed into Escherichia coli DH5α and screened on the resistant plate with 50 μg/mL kanamycin.After the verification by restriction endonuclease assay and sequence analysis,the recombinant plasmid was transformed into E.coli BL21（DE3）.L-aspartate α-decarboxylase could be efficiently expressed in engineered strain E.coli BL21（DE3）/pET28c（＋）-panD by lactose induction and it had a His-tag at its C-terminus.Recombinant enzyme producing condition such as lactose concentration,induction time,induction temperature,induction cell age and pH value were studied.The optimal conditions for producing PanD were as follows： incubation at 30 ℃,12 g/L lactose induction after culture for 3.5 h,incubation time was 20 h,and the optimum initial induction pH value of LB media was 5.5.Under these condition,the recombination PanD activity was 186 U.