芽孢杆菌P38中乳酸脱氢酶对其产L-乳酸光学纯度的影响

Effect of lactate dehydrogenases on the optical purity of L-lactic acid produced in Bacillus sp.P38

【目的】研究芽孢杆菌(Bacillus sp.)P38中乳酸脱氢酶对其产高光学纯L-乳酸(光学纯度>99%)的影响。【方法】全基因组测序显示在该菌中存在3个乳酸代谢关键酶,分别为L-乳酸脱氢酶(L-LDH)、D-乳酸脱氢酶(D-LDH)和苹果酸或L-乳酸脱氢酶(M/L-LDH)。通过将这3个酶进行异源表达、纯化与酶学特性分析,结合Native-PAGE、实时荧光定量PCR等方法,初步确定该菌高产光学纯L-乳酸的机理。【结果】Bacillus sp.P38中L-LDH对丙酮酸的催化活性(Kcat/Km值)最高,分别是D-LDH的2.9倍和M/L-LDH的4.3倍。其中M/L-LDH主要起L-LDH的功能。Native-PAGE实验中未检测到D-LDH活性。Bacillus sp.P38所有发酵阶段ldh L的转录水平均高于ldh D和ldh M/L。【结论】L-LDH是Bacillus sp.P38产高光学纯L-乳酸的主要关键酶。

[Objective] This study is to investigate the effect of lactate dehydrogenases on the optical purity of L-lactic acid produced in Bacillus sp. P38. [Methods] Genome annotation result shows that there are three enzymes responsible for lactic acid production： L-lactate dehydrogenase（L-LDH）（encoded by ldh L）, D-LDH（encoded by ldh D）, and one possible malate/lactate-LDH（M/L-LDH）（encoded by ldh M/L）. These enzymes were investigated both in vivo and in vitro to study the relationship between enzymatic activities, gene transcriptions and the optical purity of lactic acid. [Results] M/L-LDH was found mainly to act as L-LDH. The L-LDH catalytic efficiency toward pyruvate was 2.9-fold higher than that of D-LDH and 4.3-fold higher than that of M/L-LDH. The D-LDH activity was not detectable in Bacillus sp. P38 under active staining. The relative transcription levels of ldh L in Bacillus sp. strain P38 were much higher than those of ldh D and ldh M/L at different growth phases, and the transcription ratio of ldh L to ldh D increased from the logarithmic phase to decline phase. [Conclusion] L-LDH is the key enzyme for high optical purity of L-lactic acid produced by Bacillus sp. P38.