Modified Catalytic Performance Of Lactobacillus Fermentum L-lactate Dehydrogenase By Rational Design

L-lactate dehydrogenases can reduce different alpha-keto carboxylic acids asymmetrically and generally have a broad substrate spectrum.The catalytic productα-hydroxy acids have application prospects in many industries such as medicine,food and feed,and daily chemicals.Thus,by rational design,the active pocket of L-lactate dehydrogenase was changed to suit target substrates with substituents of different sizes.The mutated L-lactate dehydrogenase could efficiently reduceα-ketocarboxyl to produce differentα-hydroxy acids,which is of great significance.In this study,the Lf-LDH0845 screened by the previous research group was used to determine the site-directed saturation mutation of the enzyme through rational design.Then19 mutants were obtained by genetic engineering and PCR technology.Substrate specificity exploration and molecular docking of mutants were carried out.Finally,mutant Lf-LDH0845^Y221F was selected to explore the relevant properties.The main research results of the paper are as follows:（1）By molecular docking,four sites for mutation were identified:Arg90,Tyr221,Ile224and Thr230.After experimental verification,Tyr221 of LDH0845 was finally selected for saturation mutation.And subsequent substrate specificity and related properties were finished.（2）19 mutants were obtained through saturation mutagenesis.For pyruvate,the specific activity of LDH0845^Y221K,LDH0845^Y221P and LDH0845^Y221W were increased by 1.9,1.9 and2.3 folds.For 3,4-dihydroxyphenylpyruvate,the specific activity of LDH0845^Y221F,LDH0845^Y221S,LDH0845^Y221I were 42 U?mg^-1,29 U?mg^-1,and 28 U?mg^-1,respectively,were increased by 3.4,2.3,2.3 folds.For phenylpyruvate,the specific activity of LDH0845^Y221F and LDH0845^Y221Q were 191 U?mg^-1 and 208 U?mg^-1,which were increased by 2.8 folds and 3.0folds,respectively.For p-hydroxyphenylpyruvate,the specific activity of LDH0845^Y221F and LDH0845^Y221Pwere 30 U?mg^-1 and 27 U?mg^-1,which were increased by 1.9 folds and 1.7 folds,respectively.For benzoyl formic acid,none of the 19 mutants showed catalytic activity.For glyoxylate,the specific activity of LDH0845^Y221A,LDH0845^Y221Fand LDH0845^Y221G were 65U?mg^-1,40 U?mg^-1 and 45 U?mg^-1,respectively,which were increased by 2.7,1.7 and 1.9 folds.For 4-methyl-2-oxopentanoate,the specific activity of LDH0845^Y221Awas 35 U?mg^-1,which was only 94%of the wild-type enzyme.The specific activity of LDH0845^Y221Fwas 52 U?mg^-1,which was improved.（3）The k_cat values of LDH0845^Y221F for 3,4-dihydroxyphenylpyruvic acid,4-methyl-2-oxopentanoate and glyoxylate are 1.21 s^-1,1.35 s^-1 and 0.72 s^-1,respectively,which are 4.2,1.5and 1.3 folds.The K_m values of LDH0845^Y221F were 4.78 m M,2.28 m M and 2.04 m M,respectively,showing higher affinity.The k_cat/K_m values of LDH0845^Y221F were 0.25,0.59 and0.35 m M^-1·s^-1,and the catalytic efficiency was improved.（4）When the mutant LDH0845^Y221F catalyzed 3,4-dihydroxyphenylpyruvate,the optimum temperature was 25℃and the optimum p H was 6.0.When the temperature was below 20℃and the p H was 4.5 to 6.5,LDH0845^Y221F still retained more than 80%of the enzyme activity.