基于表达和反应耦联的羟基化酶催化合成反式-4-羟基-L-脯氨酸

Synthesis of trans-4-hydroxy-L-proline by hydroxylase via coupling enzyme expression and catalytic reaction

反式-4-羟基-L-脯氨酸(trans-4-Hpro)是药物合成中一种重要的中间体,可以用来合成碳青霉烯类抗生素类高附加值药物。目前,已有许多通过生物催化转化trans-4-Hpro的研究,但如何利用有限的酶资源实现其高效转化是难点。本文中,笔者基于已知脯氨酸4羟基化酶(GenBank No.BAA20094.1)的基因序列,利用基因挖掘工具获得Kutzneria albida来源的新型羟基化酶Ka PH1,以L-脯氨酸为底物,构建羟基化酶表达和反应过程耦联催化合成trans-4-Hpro的耦联反应体系。结果表明,Escherichia coli pET-28a-Ka PH1(E.coli Ka PH1)菌株具有高效催化合成反式-4-羟基-L-脯氨酸的能力,通过对E.coli Ka PH1催化体系的优化,以20 mmol/L L-脯氨酸作为底物,外源添加等摩尔浓度的2酮戊二酸,产量最高达到1246 mg/L,比优化前(1051 mg/L)提高了18.5%。进一步对表达和反应的耦联过程进行调控,反应体系中不添加2酮戊二酸,在底物浓度为20 mmol/L时,产量最高达到2223 mg/L,转化率为84.8%;在底物浓度为100 mmol/L时,产量最高达到7860 mg/L,转化率为59.9%。

Trans-4-hydroxy-L-proline(trans-4-Hpro)is an important intermediate in pharmaceutical synthesis,which can be used to synthesize high value-added drugs,such as carbapenem antibiotics.At present,there have been many studies on biotransformation of trans-4-Hpro,but how to use the limited enzyme resources to achieve its efficient transformation is difficult.Based on the gene sequence of L-proline 4-hydroxylase(GenBank No.BAA20094.1),a novel hydroxylase Ka PH1 from Kutzneria albida was obtained by gene mining.Ka PH1 was highly efficient for the synthesis of trans-4-hydroxy-L-proline in Escherichia coli pET-28a-Ka PH1.Then the efficient coupling system was established by optimizing the enzyme expression of Ka PH1 and the synthesis of trans-4-hydroxy-L-proline with L-proline as the substrate.The yield of trans-4-hydroxy-L-proline reached 1246 mg/L with 20 mmol/L L-proline through the optimization of whole cell transformation system,18.5%higher than the initial level(1051 mg/L).Furthermore,after regulation of the coupling process involving enzyme expression and catalytic reaction,the optimized yield of trans-4-hydroxy-L-proline was achieved as 2223 mg/L with the conversion rate of 84.8%for 20 mmol/L L-proline;when the substrate concentration was 100 mmol/L,the optimized yield of trans-4-hydroxy-L-proline reached the highest level(7860 mg/L)with the conversion rate of 59.9%.