柠檬明串珠菌KM20中D-乳酸脱氢酶的特性

Characterization of D-lactate dehydrogenase in Leuconostoc citreum KM20

目的:分析柠檬明串珠菌中D-乳酸脱氢酶(D-LDH)的酶学特性。方法:对柠檬明串珠菌KM20中D-乳酸脱氢酶基因进行克隆表达并构建表达质粒,转化至Escherichia coli BL21(DE3)中实现过表达。结果:经Ni-NTA柱亲和层析纯化后,D-LDH-1与D-LDH-2编码的蛋白分子质量分别为40.0,38.5 kDa;比活力分别为2.18,153.10 U/mg;在丙酮酸还原中两种酶的最适pH值与最适温度均为8.0与40℃;而乳酸氧化时D-LDH-2的最适pH值与最适温度分别为12.0与30℃。D-LDH-1与D-LDH-2对草酰乙酸、苯丙酮酸和2-酮戊二酸具有较强的催化能力,且Ca^(2+)、Cu^(2+)和Na+对其酶活性均具有促进作用,Zn^(2+)与SDS对酶活性有极高的抑制作用。此外,两种酶对丙酮酸的Km值分别为2.98,6.11 mmol/L,对丙酮酸的K_(cat)/K_(m)分别为6.04×10^(2),2.28×10^(4)L/(mol·s),LDH-2对D-乳酸的K_(cat)/K_(m)为65.0 L/(mol·s)。结论:D-LDH-1与D-LDH-2为柠檬酸明串珠菌中催化D-乳酸合成的关键酶。

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.Ca^(2+),Cu^(2+),and Na+promoted the activity of the two enzymes,whereas Zn^(2+)and SDS inhibited.In addition,the K_(cat)/K_(m)of LCK_00027 and LCK_00222 to pyruvate were 6.04×10^(2)L/(mol·s)and 2.28×10^(4)L/(mol·s),respectively.The K_(cat)/K_(m)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.