重组融合蛋白大肠杆菌不对称合成(R)-2-羟基-3-苯基丙酸

Construction of fusion protein for asymmetric synthesis of (R)-2-hydroxy-3-phenylpropionic acid

将来自于Lactobacillus plantarum的突变D-乳酸脱氢酶(D-LDH^(Y52V))和Candida boidinii的甲酸脱氢酶(FDH)进行融合,重组成双功能融合蛋白,为生物法合成(R)-2-羟基-3-苯基丙酸(D-PLA)提供新的方法。利用重叠延伸PCR技术,将对底物苯丙酮酸(PPA)亲合力更高的突变蛋白D-LDH^(Y52V)与FDH通过连接肽(Gly4Ser)3融合,将融合基因克隆至表达载体p ET-28a(+),并转化Escherichia coli BL21(DE3),经IPTG诱导得到高效表达。SDS-PAGE电泳分析结果表明,融合蛋白分子质量为79.7 k Da,在重组菌中获得了正确表达;酶活性测定结果表明,融合蛋白具有D-LDH和FDH的双重生物学活性。在不对称转化PPA合成(R)-2-羟基-3-苯基丙酸(DPLA)的应用中,融合蛋白体系比D-LDH^(Y52V)单独表达体系的D-PLA产量提高了40.71%。通过5 h底物分批补加发酵,D-PLA产量为17.34 g/L,底物转化率为77.64%,生产强度3.47 g/(L·h)。双功能融合蛋白增强了辅酶再生的效率,有效促进了(R)-2-羟基-3-苯基丙酸的不对称合成。

The bifunctional fusion protein system consisting of a mutant D-lactic dehydrogenase （D-LDHr52v） and formate dehydrogenase （ FDH ） was constructed for asymmetric synthesis of （R） -2-hydroxy-3-phenylpropionic acid and cofactor regeneration. The d-ldhr52V-fdh gene was constructed with the flexible 15 amino acids linker （Gly4Ser） 3 by o- verlap extension PCR, and the final full length product was cloned into the pET-28a（ ＋ ） vector for protein expression in Escherichia coli BL21 （DE3） , wherein protein expression was induced by IPTG. The expression of fusion protein was analyzed through SDS-PAGE. The result showed a specific band of about 79.7 kDa after expression of E. coli BL21 （DE3） containing plasmid pET28a-d-ldhr52V-fdh, its size was identical with the expected molecular weight of the fusion protein. The specific activity of crude recombinant protein extracts was then found to exhibit both D-LDH and FDH activity. In asymmetric reductions of phenylpyruvic acid （PPA） , cells expressing fusion protein generated 40.71% more of the D-PLA in the batch fermentation compared with cells expressing D-LDHrSzV. Fed-batch fermen- tation was conducted by intermittent feed PPA, after 5 h transformation, the final D-PLA concentration reached 17.34 g/L with the conversion ratio of 77.64%. Results showed that the fusion protein D-LDHrSZV-FDH was successfully constructed, and it could provide foundation for further study of asymmetric synthesis of （R）-（ ＋ ）-2-hydroxy-3-phe- nylpropionic acid with simultaneous cofaetor regeneration.