摘要
将肠膜明串珠菌ATCC8293中乳酸脱氢酶(lactate dehydrogenase,LDH)基因ldh克隆至载体pETDuet^TM-1,构建重组表达载体pETldh(6394 bp)并转化到Escherichia coli BL21(DE3)中实现表达。经Ni-NTA柱亲和层析得到较纯的重组LDH,进行酶学性质研究。结果表明,LDH的蛋白分子质量为37 kDa,最适pH值为7.0,温度40℃。在最适酶活力测定条件下,LDH比活力为67.45 U/mg,也证实该酶是转化丙酮酸为D-乳酸的D-LDH。此外,LDH对丙酮酸和还原态烟酰胺腺嘌呤二核苷酸的Km分别为1.27 mmol/L和0.48 mmol/L,对丙酮酸的K(cat)和K(cat)/Km分别为421 s^-1和3.31×10^5 L/(mol·s)。除将丙酮酸作为底物外,对草酰乙酸和苯丙酮酸也具有较强的催化能力。本研究结果为利用肠膜明串珠菌生产乳酸和苯乳酸提供一定理论依据。
In this study,the lactate dehydrogenase gene(ldh)of Leuconostoc mesenteroides ATCC8293 was cloned into pETDuet^TM-1 vector to construct a recombinant plasmid,which was then transformed into Escherichia coli BL21(DE3)for expression.The recombinant enzyme was purified using Ni-NTA column chromatography and its enzymatic properties were characterized.As results,the molecular mass of lactate dehydrogenase(LDH)was measured to be 37 kDa,and the optimal pH and temperature were 7.0 and 40℃,respectively.Under the optimized conditions,the specific activity of LDH was 67.45 U/mg.It was also confirmed that the enzyme was D-LDH,which converts pyruvic acid to D-lactic acid.Furthermore,the Km values of this enzyme for pyruvate and NADH were 1.27 mmol/L and 0.48 mmol/L,respectively,and the Kcat and Kcat/Km values for pyruvate were 421 s^-1 and 3.31×10^5 L/(mol·s),respectively.Besides pyruvate as a substrate,it also had high activities toward oxaloacetate and phenylpyruvate.The results of this study provide a theoretical basis for the production of lactic acid and phenyllactic acid using L.mesenteroides.
作者
李玲
龚金炎
袁海娜
方若思
楚秉泉
肖功年
Namsoo HAN
LI Ling;GONG Jinyan;YUAN Haina;FANG Ruosi;CHU Bingquan;XIAO Gongnian;Namsoo HAN(School of Biological and Chemical Engineering,Zhejiang University of Science and Technology,Hangzhou 310023,China;Division of Animal,Horticultural,and Food Sciences,Chungbuk National University,Cheongju 28644,Korea)
出处
《食品科学》
EI
CAS
CSCD
北大核心
2020年第18期153-158,共6页
Food Science
基金
浙江省重点研发计划项目(2019C02089)
浙江省自然科学基金青年科学基金项目(LQ18C200002)。
关键词
肠膜明串珠菌
乳酸脱氢酶
乳酸
苯乳酸
Leuconostoc mensenteroides
lactate dehydrogenase
lactic acid
phenyllactic acid