重组大肠杆菌全细胞催化D,L-扁桃酸对映选择性制备L-苯甘氨酸

Enantioselective L-Phenylglycine Production from D,L-Mandelic Acid Using Engineered Escherichia coli Whole Cells

借助pACYCDuet-1和pET28a双质粒共表达系统,构建携带Agrobacteriumradiobacter来源扁桃酸消旋酶(Ar mandelate racemase,Ar MR)、Lactobacillus harbinensi来源D-扁桃酸脱氢酶(Lh D-mandelate dehydrogenase,Lh DMDH)和ExiguobacteriumsibiricumDSM17290来源L-亮氨酸脱氢酶(EsL-leucine dehydrogenase,Es LeuDH)编码基因的重组大肠杆菌,将其命名为E. coli BL21(DE3)/pACYCDuet-1-EsLeuDHLhDMDH:pET28a-ArMR。在低温、低浓度诱导剂的诱导下,该重组菌成功表达了具有各自催化活性的3种重组酶,其发酵液中Lh DMDH、Es LeuDH和Ar MR的活性分别为195.8、56.2 U/mL和174.5 U/mL。以诱导后的全细胞为催化剂、D,L-扁桃酸为底物,在D,L-扁桃酸初始浓度50 mmol/L、pH 9.5的500 mmol/L NH4Cl-NH3·H2O缓冲液体系下,180 r/min、30℃反应48 h后,L-苯甘氨酸得率可达77.48%,其对映体过量值大于99%。本研究具有较大的产业化潜力,为实现L-苯甘氨酸规模化的生物合成奠定了坚实的基础。

In this study, a recombinant Escherichia coli strain carrying D-mandelate dehydrogenase, L-leucine dehydrogenase and mandelate racemase encoding genes was established by using a dual plasmid co-expression system of pACYCDuet-1 and pET28 a, which was named as E. coli BL21(DE3)/pACYCDuet-1-EsLeuDH-LhDMDH:pET28 a-ArMR. Under low temperature and in the presence of low concentrations of the inducer, the recombinant strain successfully expressed three recombinant enzymes with catalytic activities, and the activities of D-mandelate dehydrogenase, L-leucine dehydrogenase and mandelate racemase in its fermentation broth were 195.8, 56.2 and 174.5 U/mL respectively. Using the induced whole cells as the catalyst and D,L-mandelic acid as the substrate, the yield of L-phenylglycine was 77.48% after reaction at 30 ℃ for 48 h in 500 mmol/L NH4Cl-NH3·H2O buffer at pH 9.5 with an agitation rate of 180 r/min with an enantiomeric excess(e.e.) value of greater than 99%. This study has potential for industrial application, laying a solid foundation for large-scale biosynthesis of L-phenylglycine.