重组NADH氧化酶对乳酸脱氢酶乳酸氧化活性的影响

Effects of Recombinant NADH Oxidase on the Lactate Oxidation Activity of Lactate Dehydrogenase

[目的]考察当存在其他利用NADH途径时,发酵型乳酸脱氢酶(lactate dehydrogenase,LDH)催化乳酸氧化能力的改变。[方法]PCR扩增乳酸乳球菌(Lactococcus lactis,L.lactis)中生成H2O的NADH氧化酶基因noxE,将其连接至表达载体并在大肠杆菌中过量表达;对亲和纯化的产物进行SDS-PAGE分析、光谱扫描和活性测定,考察纯化产物是否具有生物学活性;以2,4-二硝基苯肼法测定乳酸脱氢酶的乳酸氧化活性,考察添加NoxE重组蛋白对其活性的影响。[结果]重组NoxE蛋白是种黄素蛋白,具明显的生物学活性,说明noxE表达载体构建成功;添加NoxE后,LDH的乳酸氧化活性提高了3.84倍。[结论]在NADH经呼吸链代谢掉的生理条件下,LDH催化乳酸氧化的能力会明显提高。

[Objective] To compare the lactate oxidation activity of lactate dehydrogenase（LDH） in the presence and absence of another NADH utilization pathway.[Method] The H2O-producing NADH oxidase gene（noxE） was cloned by PCR from Lactococcus lactis genome,ligated into the expression vector and expressed in E.coli.After affinity purification,the recombinant protein was analyzed by SDS-PAGE,UV-vis absorption spectrum and determination of enzyme activity.2,4-Dinitrophenylhydrazine was used to evaluate the effect of NoxE addition on the lactate oxidation activity of LDH.[Result]NoxE was purified as a flavin protein with significant activity.When NoxE was added,the lactate oxidation activity of LDH was increased 3.84-fold.[Conclusion]The lactate oxidation capacity of LDH will be significantly increased under physical conditions where NADH can be consumed via respiration chain.