对羟基扁桃酸合酶基因的克隆表达及催化特性

Cloning, expression and catalytic characterization of hydroxymandelate synthase

摘要:

【目的】比较两种不同来源基因重组的对羟基扁桃酸合酶(HmaS)，考察其在大肠杆菌中的表达效率。【方法】分别对东方拟无枝酸菌(Amycolatopsis orientalis)和天蓝色链霉菌(Streptomyces coelicolor)来源的hmas进行异源表达，经离子交换层析和凝胶过滤色谱分离纯化获得HmaS，并检测HmaS的酶活和催化特性。【结果】来源于S. coelicolor的HmaSSC2比酶活是来源于A. orientalis的3.6倍；来源于A. orientalis的HmaSAO最适反应温度为28 °C，在弱碱性条件下的酶活稳定性较好；来源于S. coelicolor的HmaSSC2最适反应温度为35 °C，在28?45 °C内保持较高的酶活，具有良好耐热性，在pH 7.0左右酶活最高，更易在偏中性的条件下发挥功能。【结论】HmaSSC2更适用于代谢工程改造大肠杆菌发酵法生产扁桃酸。

Abstract:

[Objective] To investigate the characters of hydroxymandelate synthase (HmaS) gene in Escherichia coli, the activities of HmaS encoded by hmas isolated from Amycolatopsis orientalis and Streptomyces coelicolor were compared. [Methods] The coding sequences of hmas were respectively amplified from the genomic DNAs of A. orientalis and S. coelicolor and were heterologous expressed in E. coli. The expressing proteins were isolated and purified by anion exchange chromatography and gel filtration chromatography, then the enzymatic activity and catalytic properties of HmaS were evaluated. [Results] The activity of HmaSSC2 from S. coelicolor was almost 3.6 times as of A. orientalis. The optimum reaction temperature of HmaSAO is 28 °C with high storage stability in weak alkaline condition. HmaSSC2 has the optimum reaction temperature of 35 °C and maintains high activity between 28 °C and 45 °C. HmaSSC2 functions well under neutral conditions. [Conclusion] The characters of HmaSSC2 from S. coelicolor is more suitable for metabolic engineering of E. coli to produce mandelic acid.