来自詹氏乳杆菌的耐热D-乳酸脱氢酶的酶学性质研究

【目的】D-乳酸脱氢酶是催化丙酮酸合成D-乳酸的关键酶。由于其不耐热，从而限制了D-乳酸高温发酵菌株的构建。本文从詹氏乳杆菌中克隆新型D-乳酸脱氢酶研究其酶学性质，为构建D-乳酸高温发酵菌株，进一步降低D-乳酸生产成本奠定基础。【方法】通过克隆詹氏乳杆菌的D-乳酸脱氢酶，将其进行体外表达，并与来自植物乳杆菌中的D-乳酸脱氢酶的最适温度、最适pH、动力学参数及热稳定性和热失活性相比较，研究詹氏乳杆菌D-乳酸脱氢酶的耐热性。【结果】詹氏乳杆菌的D-乳酸脱氢酶最适温度(45 °C)比植物乳杆菌中的D-乳酸脱氢酶的最适温度(30 °C)高很多，热失活的时间和温度均要比植物乳杆菌中D-乳酸脱氢酶高很多。同时其催化效率(kcat/Km)是植物乳杆菌D-乳酸脱氢酶的3倍左右。【结论】詹氏乳杆菌的D-乳酸脱氢酶具有更好的耐热性和更高的催化活力。

Biochemical characterization of a novel thermostable D-lactate dehydrogenase from Lactobacillus jensenii

Objective] NAD-dependent D-lactate dehydrogenase (D-LDH) catalyzed the conversion of pyruvate to D-lactic acid. However, the weakness of thermostability of D-LDHs reported to date hindered the recombinant strain construction for high-temperature fermentation process. Finding a novel thermostable D-LDH would lay the foundation for constructing the efficient D-lactate producer under high-temperature fermentation conditions and thus will reduce the operation cost of producing D-lactic acid. Methods] D-LDH was cloned from Lactobacillus jensenii strain and then expressed in Escherichia coli to determine its optimal reaction temperature, optimal reaction pH, kinetic parameter, thermostability and thermal inactivation, which is compared with the mesophilic D-LDH from Lactobacillus plantarum ssp. plantarum. Results] D-LDH from Lactobacillus jensenii strain had higher optimal reaction temperature (45 °C), better thermostability and 3 times higher catalytic activity (kcat/Km) than those of D-LDH from Lactobacillus plantarum ssp. plantarum (optimal reaction temperature was just 30 °C). Conclusion] D-LDH from Lactobacillus jensenii strain had better thermostability and higher catalytic activity, which is useful component for industrial applications.