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

doi:10.11944/j.issn.1000-0518.2020.02.190189

摘要/Abstract

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

史红玲, 王文杰, 李祥, 焦铸锦, 刘钺, 唐存多, 阚云超, 姚伦广. 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[J]. 应用化学, 2020, 37(2): 168-174.

SHI Hongling, WANG Wenjie, LI Xiang, JIAO Zhujin, LIU Yue, TANG Cunduo, KAN Yunchao, YAO Lunguang. Enantioselective Biosynthesis of L-Phenylglycine via Cascade Biocatalysis of D-Mandelate Dehydrogenase and L-Leucine Dehydrogenase[J]. Chinese Journal of Applied Chemistry, 2020, 37(2): 168-174.

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