d-(−)-Pantolactone (DPL) is a key intermediate for the production of d-(+)-pantothenate (vitamin B5). Deracemization of d,l-pantolactone (D,L-PL) through oxidizing l-(+)-pantolactone (LPL) to ketopantoyl lactone (KPL) and subsequently reducing KPL to DPL is a promising route for synthesizing DPL. Herein, a newly mined l-pantolactone dehydrogenase from Rhodococcus hoagie (RhoLPLDH) was used for the oxidative dehydrogenation of LPL. To alleviate inclusion bodies formed by membrane-bound RhoLPLDH intracellular expression in E. coli, strategies involving chaperone assistance and decreasing induction temperature were used to achieve RhoLPLDH soluble expression. To enhance its activity, directed evolution and hydrophilicity-based engineering yielded increased catalytic activity and thermostability. 1 M LPL was efficiently converted to KPL by engineering strain CM5 co-expressing RhoLPLDH^{L254I/V241I/I156L/F224Q/N164K} and chaperone. A “two stages in one-pot” method was employed in deracemization of 1 M D,L-PL with 91.2% yield. These results demonstrated that CM5 catalyst exhibits great potential in enzyme cascade deracemization for the production of DPL.

d -(−)-泛内酯 (DPL) 是生产d -(+)-泛酸（维生素 B5）的关键中间体。通过将l -(+)-泛内酯 (LPL) 氧化为酮泛甲酰内酯 (KPL) 并随后将 KPL 还原为 DPL，将 d , l -泛内酯 (D, L -PL) 脱消旋化是合成 DPL 的一条很有前途的途径。在此，一种新从红球菌( Rho LPLDH) 中提取的l-泛内酯脱氢酶被用于 LPL 的氧化脱氢。减轻大肠杆菌中膜结合Rho LPLDH 细胞内表达形成的包涵体, 涉及伴侣辅助和降低诱导温度的策略被用来实现Rho LPLDH 可溶性表达。为了增强其活性，定向进化和基于亲水性的工程提高了催化活性和热稳定性。通过共表达Rho LPLDH ^{L254I/V241I/I156L/F224Q/N164K}和分子伴侣的工程菌株 CM5 将 1 M LPL 有效转化为 KPL。采用“一锅法两步法”脱外消旋1 MD, L -PL，产率为91.2%。这些结果表明，CM5 催化剂在用于生产 DPL 的酶级联脱消旋化中表现出巨大潜力。