利用人工氧还酶体系催化L-苹果酸氧化脱羧反应

利用含人工氧还酶体系的粗酶液代替纯酶催化反应,以省去酶分离纯化过程.由苹果酸酶突变体ME-t(MEL310R/Q401C)和非天然辅酶烟酰胺5-氟胞嘧啶二核苷酸(NFCD+)组成的人工氧还酶体系可以催化氧化L-苹果酸生成丙酮酸,并得到非天然辅酶的还原态(NFCDH).利用含人工氧还酶体系的粗酶液催化反应,只得到单一产物丙酮酸,其选择性与纯酶催化的相同.来自粪肠球菌Enterococcus faecalis的NADH氧化酶(NOX)可再生NFCD+.与含NAD+,ME粗酶液和NOX粗酶液的偶联反应体系相比,含NFCD+,ME-t粗酶液和NOX粗酶液的体系获得的丙酮酸产率高9%,而副产物乳酸明显减少.可见人工氧还酶体系使用更方便,且产物选择性更高,有望代替纯酶催化反应.这为降低生物催化剂的成本,扩大生物催化反应的应用提供了一种新的策略.

Oxidative Decarboxylation of L-Malate by Using a Synthetic Bioredox System

A synthetic bioredox system for the oxidative decarboxylation of L-malate to pyruvate was presented, with which the enzyme purification steps can be omitted. The bioredox system consisted of ME-t (mutant malic enzyme ME L310R/Q401C) and non-natural coenzyme nicotinamide flucytosine dinucleotide (NFCD+) catalyzes the oxidation of L-malate to pyruvate and concurrently generates the reduced coenzyme, NFCDH. Escherichia coli BL21(DE3) pET24b-ME-t cell lysates catalyze the oxidation of L-malate and produce pyruvate only, indicating that the cell lysates have the same substrate selectivity as the purified enzyme. NADH oxidase (NOX) from Enterococcus faecalis can oxidize NFCDH to regenerate NFCD+. Compared with the reaction system containing NAD+, ME lysates, and NOX lysates, the system containing NFCD+, ME-t lysates, and NOX lysates converts L-malate to pyruvate in 9% higher yield and with significantly less lactate formation. These results suggest that the synthetic bioredox system composed of ME-t and NFCD+ is easy to use and has higher product selectivity than the corresponding natural system dependent on NAD+. The synthetic bioredox system has the potential to substitute the purified enzyme. The results provide new opportunities for design and application of redox biocatalysts.