工业微生物2017，Vol.47Issue(1)：1-9,9.DOI:10.3969/j.issn.1001-6678.2017.01.001
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
Heterologous expression of L-proline-4-hydroxylase and optimization of its catalysis conditions for improved trans-4-hydroxy-L-proline biosynthesis
摘要
Abstract
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.关键词
L-脯氨酸-4-羟化酶/反式-4-羟基-L-脯氨酸/双加氧酶/生物合成/生物催化/优化Key words
L-proline-4-hydroxylase/trans-4-hydroxy-L-proline, dioxygenase, biosynthesis, biocatalysis, optimization引用本文复制引用
周海岩,李会帅,王培,柳志强..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[J].工业微生物,2017,47(1):1-9,9.基金项目
浙江省自然科学基金青年项目(编号:LQ14B060004) （编号:LQ14B060004）
国家自然科学基金青年项目(编号:31500031) （编号:31500031）
浙江省科技计划项目公益技术研究工业项目(编号:2015C31049) （编号:2015C31049）
浙江省教育厅一般科研项目(编号:Y201224739). （编号:Y201224739）
