Zhang Dong, Zhang Ting, Lei Yuqing, Lin Wenqian, Chen Xingyi, Wu Minchen
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
Haiyan Food and Drug Inspection and Testing Center, Haiyan, China.
Front Bioeng Biotechnol. 2022 Feb 18;10:846489. doi: 10.3389/fbioe.2022.846489. eCollection 2022.
As a valuable versatile building block, L-phenyllactic acid (L-PLA) has numerous applications in the fields of agriculture, pharmaceuticals, and biodegradable plastics. However, both normally chemically synthesized and naturally occurring PLA are racemic, and the production titer of L-PLA is not satisfactory. To improve L-PLA production and reduce the high cost of NADH, an coenzyme regeneration system of NADH was achieved using the glucose dehydrogenase variant GDH and introduced into the L-PLA production process. Here an NADH-dependent L-lactate dehydrogenase-encoding variant gene (L-1) was expressed in GS115. The specific activity of L-LDH1 (Pp) was as high as 447.6 U/mg at the optimum temperature and pH of 40°C and 5.0, which was 38.26-fold higher than that of wild-type L-LDH1 (Pp). The catalytic efficiency ( / ) of L-LDH1 (Pp) was 94.3 mM s, which was 67.4- and 25.5-fold higher than that of L-LDH1(Pp) and L-LDH1 (Ec) expressed in , respectively Optimum reactions of L-PLA production by dual-enzyme catalysis were at 40°C and pH 5.0 with 10.0 U/ml L-LDH1 (Pp) and 4.0 U/ml GDH. Using 0.1 mM NAD, 400 mM (65.66 g/L) phenylpyruvic acid was completely hydrolyzed by fed-batch process within 6 h, affording L-PLA with 90.0% yield and over 99.9% . This work would be a promising technical strategy for the preparation of L-PLA at an industrial scale.
作为一种有价值的多功能结构单元，L-苯乳酸（L-PLA）在农业、制药和生物可降解塑料领域有众多应用。然而，通常化学合成的和天然存在的PLA都是外消旋体，且L-PLA的生产效价并不令人满意。为了提高L-PLA的产量并降低NADH的高成本，利用葡萄糖脱氢酶变体GDH实现了NADH的辅酶再生系统，并将其引入L-PLA生产过程。在此，一个依赖NADH的L-乳酸脱氢酶编码变体基因（L-1）在GS115中表达。L-LDH1（Pp）在40°C和pH 5.0的最佳温度和pH下比活性高达447.6 U/mg，比野生型L-LDH1（Pp）高38.26倍。L-LDH1（Pp）的催化效率（kcat/Km）为94.3 mM-1s-1，分别比在大肠杆菌中表达的L-LDH1（Pp）和L-LDH1（Ec）高67.4倍和25.5倍。双酶催化生产L-PLA的最佳反应条件是40°C和pH 5.0，L-LDH1（Pp）为10.0 U/ml，GDH为4.0 U/ml。使用0.1 mM NAD，通过补料分批工艺在6小时内400 mM（65.66 g/L）苯丙酮酸完全水解，L-PLA产率为90.0%，对映体过量值超过99.9%。这项工作将是在工业规模制备L-PLA的一种有前景的技术策略。
