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

• Published in: Biochemical engineering journal 2021-02, Vol.166, p.107863, Article 107863
• Main Authors: Wang, Hong-Yan, Xie, Yu-Li, Shi, Xin, Shi, Hong-Ling, Xu, Jian-He, Tang, Cun-Duo, Yao, Lun-Guang, Kan, Yun-Chao
• Format: Article
• Language: English
• Subjects: Biosynthesis; D-mandelate dehydrogenase; Directed evolution; Halogenated substrate; In silico design; Molecular basis
• ISSN: 1369-703X; 1873-295X
• DOI: 10.1016/j.bej.2020.107863

[Display omitted] •A D-mandelate dehydrogenase mutant was obtained by directed evolution.•The muatnt has highly specific activity toward D-o-chloromandelic acid.•The muatnt has highly Kcat value toward D-o-chloromandelic acid.•Preliminarily explained the molecular basis for its catalytic performance by in silico design methods. 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.