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  

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

作　　者：史红玲[1] 王文杰 李祥 焦铸锦[1] 刘钺 唐存多[1,2] 阚云超[1] 姚伦广[1] SHI Hongling;WANG Wenjie;LI Xiang;JIAO Zhujin;LIU Yue;TANG Cunduo;KAN Yunchao;YAO Lunguang(Henan 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;State Key Laboratory of Automotive Biofuel Technology,Nanyang,Henan 473061,China)

机构地区：[1]南阳师范学院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,河南南阳473061 [2]南阳师范学院车用生物燃料技术国家重点实验室,河南南阳473061

出　　处：《应用化学》2020年第2期168-174,共7页Chinese Journal of Applied Chemistry

基　　金：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);南阳师范学院青年项目(2018QN004)资助~~

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、NH+4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12h,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。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,NH+4 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.

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 

分 类 号：O622[理学—有机化学]