应用化学 ›› 2020, Vol. 37 ›› Issue (2): 168-174.DOI: 10.11944/j.issn.1000-0518.2020.02.190189

• 研究论文 • 上一篇    下一篇

D-扁桃酸脱氢酶和L-亮氨酸脱氢酶级联催化的L-苯甘氨酸对映选择性生物合成

史红玲a,王文杰a,李祥a,焦铸锦a,刘钺b,唐存多ab*(),阚云超a,姚伦广a   

  1. a昆虫生物反应器河南省工程实验室和河南省南水北调中线水源区生态安全重点实验室 南阳师范学院 河南 南阳 473061
    b车用生物燃料技术国家重点实验室 南阳师范学院 河南 南阳 473061
  • 收稿日期:2019-07-08 接受日期:2019-11-20 出版日期:2020-02-01 发布日期:2020-02-06
  • 通讯作者: 唐存多
  • 基金资助:
    国家自然科学基金(3190110303, 31870917)车用生物燃料技术国家重点实验室开放课题 (KFKT2018003)和南阳师范学院青年项目(2018QN004)资助

Enantioselective Biosynthesis of L-Phenylglycine via Cascade Biocatalysis of D-Mandelate Dehydrogenase and L-Leucine Dehydrogenase

SHI Honglinga,WANG Wenjiea,LI Xianga,JIAO Zhujina,LIU Yueb,TANG Cunduoa*(),KAN Yunchaoa,YAO Lunguanga   

  1. aHenan Provincial Engineering Laboratory of Insect Bio-reactor and Henan Key Laboratory of Ecological Security for Water Source Region of Mid-line of South-to-North,Nanyang,Henan 473061,China
    bState Key Laboratory of Automotive Biofuel Technology,Nanyang,Henan 473061,China
  • Received:2019-07-08 Accepted:2019-11-20 Published:2020-02-01 Online:2020-02-06
  • Contact: TANG Cunduo
  • Supported by:
    Supported by the National Natural Science Foundation of China(No.3190110303, No.31870917), the State Key Laboratory of Motor Vehicle Biofuel Technology(No.KFKT2018003), and the Special Funded Projects of Nanyang Normal University(No.2018QN004)

摘要:

L-苯甘氨酸是重要的手性非天然α-氨基酸,可广泛用于合成多种食品添加剂及药物中间体,探索其绿色合成工艺具有重要的意义。 本研究将新型高活性的D-扁桃酸脱氢酶(LhDMDH)和L-亮氨酸脱氢酶(EsLeuDH)偶联,在辅酶内循环的前提下,仅需较低浓度的辅酶即可实现生物催化D-扁桃酸合成L-苯甘氨酸。 通过对加酶量、氧化型烟酰胺腺嘌呤二核苷酸(NAD+)浓度、NH4+浓度和底物浓度等因素进行优化,获得了一个最经济的反应条件:200 mmol/L的D-扁桃酸、6.5 kU/L的加酶量、0.1 mmol/L NAD+、0.5 mol/L NH4+的条件下、30 ℃,反应12 h,在此条件下,产物的得率和对映体过量(e.e.)值分别可达98%和99%以上,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, D-扁桃酸脱氢酶, L-亮氨酸脱氢酶, 生物催化, 级联反应

Abstract:

L-Phenylglycine is an important class of chiral non-natural amino acids, and can be used to synthesize a variety of important pharmaceutical intermediate. Exploiting the green synthesis process of phenylacetone acid has significant economic value. In this study, novel and highly active D-mandelate dehydrogenase (LhDMDH) and L-leucine dehydrogenase (EsLeuDH) are coupled to achieve bio-transformation of D-mandelic acid into L-phenylglycine on the premise of coenzyme circulation, and this reaction only requires a lower concentration of coenzyme. By optimizing the transformation conditions including added amount of enzyme, β-nicotinamide adenine dinucleotide (NAD+) concentration, NH4+ concentration and substrate concentration, we obtain the most economical condition: 200 mmol/L D-mandelic acid, 6.5 kU/L enzyme, 0.1 mmol/L NAD +, 0.5 mol/L NH4+ and 30 ℃ for 12 h. The product yield and enantionmeric excess (e.e.) value can reach more than 98% and 99% under the most economical condition, respectively. This transformation has large industrialization potential, and lays a solid foundation for achieving large-scale biosynthesis of L-phenylglycine.

Key words: L-Phenylglycine, D-mandelate dehydrogenase, L-leucine dehydrogenase, biocatalysis, cascade reaction