色氨酸酶重组基因表达及其酶法合成L-色氨酸  

Synthesis of L-tryptophan with Escherichia coli Whole Cells Expressing Tryptophanase from Enterobacter aerogenes

作　　者：高亮[1] 冯莹莹[1] 张宏娟[2] 刘茜[1] 焦庆才[1] 刘均忠[1] 

机构地区：[1]医药生物技术国家重点实验室南京大学生命科学学院,江苏南京210093 [2]南京医科大学药学院,江苏南京210029

出　　处：《精细化工》2016年第2期137-141,共5页Fine Chemicals

基　　金：国家自然科学基金青年科学基金项目(21302100)~~

摘　　要：为实现色氨酸酶高效、低成本催化合成L-色氨酸,利用p ET30a为载体在宿主细胞E.coli BL21(DE3)中重组表达了产气肠杆菌(Enterobacter aerogenes)来源的色氨酸酶,以丙酮酸、吲哚和氨为底物,探究其酶学性质,考察了反应温度、起始p H、底物摩尔比对酶促反应的影响,并利用丙酮酸发酵液为底物酶法合成L-色氨酸。结果表明,色氨酸酶重组表达成功,色氨酸酶最佳反应条件为:温度35℃,起始p H=9.0,底物摩尔比n(吲哚)∶n(丙酮酸)=0.6∶1,底物丙酮酸浓度为0.17 mol/L。利用重组色氨酸酶全细胞催化100 m L浓度为0.57 mol/L丙酮酸发酵液,流加浓度为4.27 mol/L吲哚酒精溶液6.5 m L,反应28 h后,L-色氨酸浓度达0.25 mol/L,吲哚摩尔转化率达91.8%。To achieve the purpose of efficient,low cost catalytic synthesis of L-tryptophan,tryptophanase from Enterobacter aerogenes was recombinant expressed in Escherichia coli BL21（ DE3） using plasmid p ET30 a as vectors. The enzymatic properties of the recombinant tryptophanase were studied,and several influencing factors of the enzyme reaction,such as temperature,initial p H,mass concentration of pyruvic acid,n（ indole） / n（ pyruvic acid） were investigated with pyruvic acid,indole and ammonium as substrates. Then,pyruvate fermentation broth,indole and ammonium were transformed to Ltryptophan by the recombinant tryptophanase. The results indicated that the recombinant tryptophanase was successfully expressed,and the optimal conditions for the enzymatic conversion were 35 ℃,initial p H = 9. 0,c（ pyruvic acid） = 0. 17 mol / L,and n（ indole） ∶ n（ pyruvic acid） = 0. 6 ∶ 1. In 100 m L reaction mixture which contained 0. 57 mol / L of pyruvic acid,after 28 h,the concentration of Ltryptophan synthesized by tryptophanase whole cell was 0. 25 mol / L,by adding 6. 5 m L 4. 27 mol / L of indole alcohol solution. And,the mole conversion rate of indole was up to 91. 8%.

关 键 词：产气肠杆菌 色氨酸酶 吲哚 丙酮酸发酵液 L-色氨酸 生物工程 

分 类 号：TQ464.7[化学工程—制药化工]