食品科学 ›› 2021, Vol. 42 ›› Issue (2): 83-89.doi: 10.7506/spkx1002-6630-20191104-030

• 生物工程 • 上一篇    下一篇

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

贾园园,李祥,张振华,张闪,杨露露,唐存多   

  1. (1.南阳师范学院 昆虫生物反应器河南省工程实验室,河南省南水北调中线水源区生态安全重点实验室,河南 南阳 473061;2.车用生物燃料技术国家重点实验室,河南 南阳 473061)
  • 出版日期:2021-01-18 发布日期:2021-01-27
  • 基金资助:
    国家自然科学基金青年科学基金项目(31900916);国家自然科学基金面上项目(31870917); 车用生物燃料技术国家重点实验室开放课题(KFKT2018003);南阳师范学院青年项目(2018QN004); 河南省科研服务平台专项(2016151);河南省南水北调中线水源区水生态安全创新型科技团队专项(17454); 河南省高校科技创新团队项目(20IRTSTHN024);河南省高校省级大学生创新创业训练计划项目(S201910481006)

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

JIA Yuanyuan, LI Xiang, ZHANG Zhenhua, ZHANG Shan, YANG Lulu, TANG Cunduo   

  1. (1. Henan Provincial Engineering Laboratory of Insect Bio-reactor, Henan Key Laboratory of Ecological Security for Water Source Region of Mid-line of South-to-North, Nanyang Normal University, Nanyang 473061, China; 2. State Key Laboratory of Automotive Biofuel Technology, Nanyang 473061, China)
  • Online:2021-01-18 Published:2021-01-27

摘要: 借助pACYCDuet-1和pET28a双质粒共表达系统,构建携带Agrobacterium radiobacter来源扁桃酸消旋酶(Ar mandelate racemase,ArMR)、Lactobacillus harbinensi来源D-扁桃酸脱氢酶(Lh D-mandelate dehydrogenase,LhDMDH)和Exiguobacterium sibiricum DSM 17290来源L-亮氨酸脱氢酶(Es L-leucine dehydrogenase,EsLeuDH)编码基因的重组大肠杆菌,将其命名为E. coli BL21(DE3)/pACYCDuet-1-EsLeuDH-LhDMDH: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LhDMDH、EsLeuDH和ArMR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。

关键词: L-苯甘氨酸;全细胞催化;绿色化学;共表达;级联反应

Abstract: 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 pET28a, which was named as E. coli BL21(DE3)/pACYCDuet-1-EsLeuDH-LhDMDH:pET28a-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.

Key words: L-phenylglycine; whole-cell catalysis; green chemistry; co-expression; cascade reaction

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