Rhodococcus erythropolis 手性醇脱氢酶的克隆表达及其性质

【目的】探讨红串红球菌中一种醇脱氢酶的性质及其对酮酯类及酮类底物的催化能力。【方法】从红串红球菌(Rhodococcus erythropolis ATCC 4277)中获取一段长度为1047 bp的醇脱氢酶(adh)基因,插入载体pET-22b(+)后,在大肠杆菌中进行重组表达。15℃的低温下用自诱导培养基诱导24 h,以苯乙酮为底物测定醇脱氢酶酶活。【结果】测得该诱导条件下重组菌体细胞破碎上清中醇脱氢酶酶活力为2.6 U/mg。经温度、pH耐受性等分析,发现该酶最适pH在6.0-6.5之间,耐受温度可以达到60℃,并且在该温度下保持5 h后,酶活也能保留80%。对于β酮酯类底物的催化反应,以对乙酰乙酸乙酯的催化能力最高。用4-氯乙酰乙酸乙酯(COBE)为底物进行全细胞水相催化反应,经手性液相色谱分析,发现在催化产物以R型4-氯-3羟基丁酸乙酯(CHBE)为主。【结论】该酶在酮酯类的底物转化方面有良好的开发潜力及应用前景。

Cloning, expression and characterization of chiral alcohol dehydrogenase from Rhodococcus erythropolis ATCC 4277

[Objective]We characterized alcohol dehydrogenase from Rhodococcus erytropolis to catalyze ketoesters or ketones.[Methods]We cloned alcohol dehydrogenase gene(adh) of 1047 bp from Rhodococcus erythropolis ATCC 4277,inserted the open reading frame of adh into vector pET-22b(+) and expressed in auto-inducing media for 24 h at 15℃.The enzyme activity was determined at 30℃ using acetophenone as substrate.[Results]Under the above conditions,the specific enzyme activity of crude extract was 2.6 U/mg.The optimal pH was between 6.0 and 6.5 and the enzyme can survived up to 60℃.After incubation at 60℃ for 5 h,80% enzyme activity remained.The optimal substrate among β-ketoesters examined was ethyl acetoaetate.Ethyl 4-chloroacetoacetate was catalyzed by whole cell in aqueous phase.After chiral liquid chromatography,the product showed(R)-enantioselective.[Conclusion]The study shows that the enzyme might have potential in β-ketoesters transformation on industrial scale.