L-脯氨酸-4-羟化酶的异源表达和合成反式-4-羟基-L-脯氨酸的条件优化

摘要

L-脯氨酸-4-羟化酶(L-Proline-4-hydroxylase,P4H)是依赖α-酮戊二酸(α-KG)和Fe2+的双加氧酶成员之一,在反式4-羟基-L-脯氨酸(trans-4-hydroxy-L-proline,t-4Hyp)等重要手性化合物的生物合成中发挥关键作用.本研究构建了来源于Bradyrhizobium japonicum USDA 6的P4H重组大肠杆菌Escherichia coli BL21(DE3)/pET-28b-p4hBJ,SDS-PAGE和酶活检测结果表明,该菌株具有表达可溶性P4H和催化合成t-4Hyp的能力.通过优化,确定了该重组菌全细胞催化合成t-4Hyp较优的反应体系和条件:10 mLpH 6.5 80 mmol/LMES缓冲液、9 mmol/L L-Pro,6 mmol/L L-抗坏血酸,6 mmol/L α-KG,0.8 mmol/L FeS04·7H2O,反应温度为35℃;在20 g/L湿细胞的催化反应中,t-4Hyp的合成量达到34.86 mg/L,比优化前(17.53 mg/L)提高了98.86％.该工作为进一步利用P4H生物催化法合成t-Hyp奠定了一定的技术基础.%L-Proline-4-hydroxylase (P4H) is a member of Fe (Ⅱ)/c-ketoglutarate (cα-KG)-dependent dioxygenase superfamily,playing an important role in biosynthesis of trans-4-hydroxy-L-proline (t-4Hyp) and other important chiral building-block chemicals.In this work,recombinant Escherichia coli BL21 (DE3)/pET-28b-p4hBJ harboring P4H gene p4HBJ,originated from Bradyrhizobium japonicum USDA 6 was constructed.The results of SDS-PAGE and enzyme activity solubly analysis showed that P4H was expressed solubly and actively in E.coli BL21 (DE3)/pET-28b-p4hBJ.Furthermore,the whole cell catalysis conditions with E.coli BL21 (DE3)/pET-28b-p4hBJ was investigated and optimized for t-4Hyp biosynthesis.The optimum reaction system and conditions were as follows:10 mL pH 6.5 MES buffer,9 mmol/L L-Pro,6 mmol/L L-ascorbic acid,6 mmol/L α-KG,0.8 mmol/L FeSO4 · 7H2O;the reaction temperature was 35 ℃.Under the optimized conditions,a t-4Hyp yield of 34.86 mg/L was achieved via whole cell catalysis reaction with 20 g/L wet cells,which was 98.86％ higher than that of pre-optimization.The work provided an important theoretical foundation for the further research of t-4Hyp biosynthesis with P4H as biocatalyst.