Abstract：
The malic enzyme (ME) catalyzes the synthesis of L-malic acid (L-MA) from pyruvic acid and CO2 with NADH as the reverse reaction of L-MA decarboxylation. Carboxylation requires excess pyruvic acid, limiting its application. In this study, it was determined that CO2 was the carboxyl donor by parsing the effects of HCO3- and CO2, which provided a basis for improving the L-MA yield. Moreover, the concentration ratio of pyruvic acid to NADH was reduced from 70:1 to 5:1 using CO2 to inhibit decarboxylation and to introduce the ME mutant A464S with a 2-fold lower Km than that of the wild type. Finally, carboxylation was coupled with NADH regeneration, resulting in a maximum L-MA yield of 77 % based on the initial concentration of pyruvic acid. Strategic modifications, including optimal reactant ratios and efficient mutant ME, significantly enhanced L-MA synthesis from CO2, providing a promising approach to the biotransformation process.
摘要：
苹果酶（ME）催化由丙酮酸和CO2合成L-苹果酸（L-MA），NADH是L-MA脱羧的逆反应。羧基化需要过量的丙酮酸,限制其应用。在这项研究中,通过分析HCO3-和CO2的作用，确定CO2为羧基供体，为提高L-MA收率提供了依据。此外，使用CO2抑制脱羧并引入Km比野生型低2倍的ME突变体A464S，丙酮酸与NADH的浓度比从70：1降低到5：1。最后,羧化与NADH再生偶联，导致基于丙酮酸初始浓度的77%的最大L-MA产率。战略修改，包括最佳反应物比例和有效的突变ME，显著增强了从CO2合成L-MA，为生物转化过程提供了一种有前途的方法。
