Directed evolution of a D-mandelate dehydrogenase toward D-o-chloromandelic acid and insight into the molecular basis for its catalytic performance

Wang, Hong-Yan; Xie, Yu-Li; Shi, Xin; Shi, Hong-Ling; Xu, Jian-He; Tang, Cun-Duo; Yao, Lun-Guang; Kan, Yun-Chao

Directed evolution of a D-mandelate dehydrogenase toward D-o-chloromandelic acid and insight into the molecular basis for its catalytic performance

2021

Halogenated mandelate and its derivatives are important scaffolds in chemical industry. However, the natural D-mandelate dehydrogenase (DMDH) showed relatively low catalytic activity toward D-o-chloromandelic acid, which largely limits the efficiency of dehydrogenation processes. To improve catalytic activity of Lactobacillus brevis D-mandelate dehydrogenase (LbDMDH) toward halogenated substrate, a D-mandelate dehydrogenase mutant LbDMDHᴺ²⁵³S with higher catalytic activity toward D-o-chloromandelic acid was successfully obtained by directed evolution, which was about 29 times of LbDMDH. In addition, the temperature optimum of LbDMDHᴺ²⁵³S is 50 °C, and closer to the optimal growth temperature for Escherichia coli, indicating that it may be more advantageous in whole cell catalysis using living cells as catalysts. Meanwhile, the Kcₐₜ value toward D-o-chloromandelic acid of LbDMDHᴺ²⁵³S is 2.08 s⁻¹, which is significantly higher than the one (0.09 s⁻¹) of LbDMDH, indicating that LbDMDHᴺ²⁵³S displayed higher catalytic efficiency toward D-o-chloromandelic acid. Then, we preliminarily explained the molecular basis for its catalytic performance by in silico design methods. All of these results established a solid foundation for molecular modification of DMDHs and the other industrial enzymes.

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