Improving catalytic activity of Lactobacillus harbinensis d-mandelate dehydrogenase toward d-o-chloromandelic acid by laboratory evolution

Abstract

In the pharmaceutical and chemical industry, l-o-chlorophenylglycine and its derivatives are very important building blocks. Nevertheless, the catalytic activity of natural d-mandelate dehydrogenase (DMDH) toward d-o-chloromandelic acid (D-o-CMA) was very low, which greatly limits its industrial application. Taking Lactobacillus harbinensisd-mandelate dehydrogenase (LhDMDH) as parent, laboratory evolution was performed to improve its catalytic performance toward d-o-chloromandelic acid. An A59G/N252G mutant of LhDMDH with a higher catalytic performance toward d-o-chloromandelic acid was obtained, its catalytic activity was approximately 8.4 times than that of LhDMDH. Furthermore, the optimal reaction temperature of A59G/N252G is 50 °C. Meanwhile, the Kcat value toward d-o-CMA of A59G/N252G is 18.33 s − 1, which is about 10 times of LhDMDH (1.81 s − 1). Lastly, using the in silico design approach, the molecular basis about its catalytic performance improvement was preliminarily explored. Therefore, these researchs established solid practice foundation for the molecular modification of d-mandelate dehydrogenase for catalytic performance.