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

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.