Modified catalytic performance of Lactobacillus fermentum l-lactate dehydrogenase by rational design

Abstract

l-Lactate dehydrogenases can reduce alpha-keto carboxylic acids asymmetrically and generally have a broad substrate spectrum. l-Lactate dehydrogenase gene (LF-l-LDH0845) with reducing activity towards 3,4-dihydroxyphenylpyruvate and phenylpyruvate was obtained from Lactobacillus fermentum JN248. To change the substrate specificity of LDH0845 and improve its catalytic activity towards large substrates, site-directed mutation of Tyr221 was performed by analyzing the amino acids in the active center. Kinetic parameters show that the k _cat values of Y221F mutant on 3,4-dihydroxyphenylpyruvate, 4-methyl-2-oxopentanoate, and glyoxylate are 1.21 s⁻¹, 1.35 s⁻¹, and 0.72 s⁻¹, respectively, which are 420%, 150% and 130% of the wild-type LDH0845. This study shows that the mutations of Y221 can significantly change the substrate specificity of LDH0845, making it become a potential tool enzyme for the reduction of alpha-keto carboxylic acids with large functional groups.

Funding

National Key Scientific Instrument and Equipment Development Projects of China,(2013YQ17052504)

Science and Technology Innovative Research Team in Higher Educational Institutions of Hunan Province,(IRT_15R55)

the Natural Science Foundation of Shaanxi province,(2019JQ-725)

Three-Side Innovation Projects for Aquaculture in Jiangsu Province,(KYCX19_1841)