Wang Limin, Cai Yumeng, Zhu Lingfeng, Guo Honglian, Yu Bo
CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China School of Food Engineering and Biological Technology, Tianjin University of Science and Technology, Tianjin, China.
Appl Environ Microbiol. 2014 Dec;80(23):7134-41. doi: 10.1128/AEM.01864-14. Epub 2014 Sep 12.
Bacillus coagulans 2-6 is an excellent producer of optically pure l-lactic acid. However, little is known about the mechanism of synthesis of the highly optically pure l-lactic acid produced by this strain. Three enzymes responsible for lactic acid production-NAD-dependent l-lactate dehydrogenase (l-nLDH; encoded by ldhL), NAD-dependent d-lactate dehydrogenase (d-nLDH; encoded by ldhD), and glycolate oxidase (GOX)-were systematically investigated in order to study the relationship between these enzymes and the optical purity of lactic acid. Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 (a d-lactic acid producer) and Lactobacillus plantarum subsp. plantarum DSM 20174 (a dl-lactic acid producer) were also examined in this study as comparative strains, in addition to B. coagulans. The specific activities of key enzymes for lactic acid production in the three strains were characterized in vivo and in vitro, and the levels of transcription of the ldhL, ldhD, and GOX genes during fermentation were also analyzed. The catalytic activities of l-nLDH and d-nLDH were different in l-, d-, and dl-lactic acid producers. Only l-nLDH activity was detected in B. coagulans 2-6 under native conditions, and the level of transcription of ldhL in B. coagulans 2-6 was much higher than that of ldhD or the GOX gene at all growth phases. However, for the two Lactobacillus strains used in this study, ldhD transcription levels were higher than those of ldhL. The high catalytic efficiency of l-nLDH toward pyruvate and the high transcription ratios of ldhL to ldhD and ldhL to the GOX gene provide the key explanations for the high optical purity of l-lactic acid produced by B. coagulans 2-6.
凝结芽孢杆菌2-6是光学纯L-乳酸的优良生产者。然而，对于该菌株产生高光学纯L-乳酸的合成机制知之甚少。为了研究这些酶与乳酸光学纯度之间的关系，系统地研究了三种负责乳酸产生的酶——NAD依赖的L-乳酸脱氢酶（L-nLDH；由ldhL编码）、NAD依赖的D-乳酸脱氢酶（D-nLDH；由ldhD编码）和乙醇酸氧化酶（GOX）。除了凝结芽孢杆菌外，本研究还将德氏乳杆菌保加利亚亚种DSM 20081（D-乳酸生产者）和植物乳杆菌植物亚种DSM 20174（DL-乳酸生产者）作为比较菌株进行了检测。对这三种菌株中乳酸产生关键酶的比活性进行了体内和体外表征，并分析了发酵过程中ldhL、ldhD和GOX基因的转录水平。L-nLDH和D-nLDH的催化活性在L-、D-和DL-乳酸生产者中有所不同。在天然条件下，凝结芽孢杆菌2-6中仅检测到L-nLDH活性，并且在所有生长阶段，凝结芽孢杆菌2-6中ldhL的转录水平远高于ldhD或GOX基因。然而，对于本研究中使用的两种乳酸杆菌菌株，ldhD的转录水平高于ldhL。L-nLDH对丙酮酸的高催化效率以及ldhL与ldhD和ldhL与GOX基因的高转录比率，为凝结芽孢杆菌2-6产生的L-乳酸的高光学纯度提供了关键解释。
