Objective:This study focused on investigating the enzymatic characteristics of D-lactate dehydrogenase(D-LDH)in Leuconostoc citreum KM20.Methods:The D-lactate dehydrogenase gene(LDH)from L.citreum KM20 was cloned and expressed to construct expression plasmid,and then transformed into Escherichia coli BL21(DE3)for overexpression.Results:The enzymes encoded by LCK 00027 and LCK_00222 were purified by Ni-NTA column affinity chromatography with molecular mass of 40.0 kDa and 38.5 kDa,respectively.The specific activities were 2.18 U/mg and 153.10 U/mg,respectively.The optimal pH and temperature for pyruvate reduction were 8.0 and 40 ℃,respectively,while for the LCK_00222 encoding enzyme lactic acid oxidation the values were 12.0 and 30 ℃,respectively.The two enzymes had high activities toward oxaloacetic acid,sodium phenylpyruvate,and 2-oxoglutaric acid.Ca2+,Cu2+,and Na+promoted the activity of the two enzymes,whereas Zn2+and SDS inhibited.In addition,the Kcat/Km of LCK_00027 and LCK_00222 to pyruvate were 6.04× 102 L/(mol·s)and 2.28× 104 L/(mol·s),respectively.The Kcat/Km of LCK_00222 encoding enzyme to D-lactic acid was 65.0 L/(mol·s).Conclusion:D-LDH-1 and D-LDH-2 are key enzymes catalyzing the synthesis of D-lactic acid in Leuconostoc citrate.
维普
万方数据