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

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.