重组表达天冬氨酸-β-脱羧酶及其酶学性质  

Enzymatic Properties of Recombinant Aspartate-β-decarboxylase

作　　者：吴四平 陆阳[1] 张宏娟[2] 刘茜[1] 焦庆才[1] 刘均忠[1] 

机构地区：[1]南京大学医药生物技术国家重点实验室生命科学学院,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》2015年第6期637-641,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：利用p ETDuet-1为载体在宿主细胞E.coli BL21(DE3)中重组表达了粪产碱菌(Alcaligenes faecalis)来源的天冬氨酸-β-脱羧酶(Asd),研究了其酶学性质,考察了p H、p H稳定性、温度、热稳定性、底物浓度对酶活的影响。结果表明,天冬氨酸-β-脱羧酶重组表达成功;最适反应温度和p H分别为45℃和6.0;动力学常数Km和Vmax分别为0.72 mmol/L和10.52 mol/(L·min·g)。0.1 g菌体细胞在10 m L 0.2 mol/L磷酸缓冲溶液中催化2.0 g L-天冬氨酸,40℃,p H=6.0反应15 h,L-天冬氨酸的摩尔转化率达到98.6%。Aspartate-β-decarboxylase is an enzyme that catalyzes the decarboxylation of L-aspartate to produce L-alanine. The gene coding and expressing of this enzyme was cloned from Alcaligenes faecalis.In this study,the vector p ETDuet- 1 was used to recombinantly express the aspartate-β-decarboxylase in Escherichia coli BL21（ DE3）. Several influencing factors of the enzyme reaction,such as p H,p H stability,temperature,thermal stability,and concentration of substrate,were all investigated. The results indicate that the recombinant aspartate-β-decarboxylase was successfully expressed and the reaction was optimal at p H 6. 0 and 45 ℃. The Kmand Vmaxvalues of aspartate-β-decarboxylase were 0. 72 mmol / L and 10. 52 mol /（ L·min·g）. 10 m L of reaction mixture containing 0. 2 mol / L phosphate buffer（ p H= 6. 0）,0. 1 g wet cells,0. 2 mmol / L 5＇-pyridoxal phosphate and 2. 0 g L-aspartate was incubated at40 ℃ for 15 h,the mole conversion rate of L-aspartate was up to 98. 6%.

关 键 词：天冬氨酸-β-脱羧酶 L-天冬氨酸 重组表达 粪产碱菌 L-丙氨酸 生物工程 

分 类 号：Q55[生物学—生物化学]