Lactate are proved to be attractive electron donor for the production of n-caproic acid (CA) that is a high value-added fuel precursor and chemical feedstock, but little is known about molecular mechanism of lactate transformation. In the present study, the gene for L-lactate dehydrogenase (LDH, EC.1.1.1.27) from a Ruminococcaceae strain CPB6 was cloned and expressed in Escherichia coli BL21 (DE3) with plasmid pET28a. The recombinant LDH exhibited molecular weight of 36-38 kDa in SDS-PAGE. The purified LDH was found to have the maximal oxidation activity of 29.6 U/mg from lactate to pyruvate at pH 6.5, and the maximal reduction activity of 10.4 U/mg from pyruvate to lactate at pH 8.5, respectively. Strikingly, its oxidative activity predominates over reductive activity, leading to a 17-fold increase for the utilization of lactate in E. coli/pET28a-LDH than E. coli/pET28a. The CPB6 LDH gene encodes a 315 amino acid protein sharing 42.19% similarity with Clostridium beijerinckii LDH, and lower similarity with LDHs of other organisms. Significant difference were observed between the CPB6 LDH and C. beijerinckii and C. acetobutylicum LDH in the predicted tertiary structure and active center. Further, X-ray crystal structure analysis need to be performed to verify the specific active center of the CPB6 LDH and its role in the conversion of lactate into CA.

中文翻译：

---

瘤胃球菌科细菌CPB6 L-乳酸脱氢酶基因的克隆与鉴定

乳酸被证明是生产正己酸（CA）的有吸引力的电子供体，正己酸是一种高附加值的燃料前体和化学原料，但对乳酸转化的分子机制知之甚少。在本研究中，来自瘤胃球菌科 CPB6 菌株的 L-乳酸脱氢酶基因 (LDH, EC.1.1.1.27) 被克隆并在带有质粒 pET28a 的大肠杆菌 BL21 (DE3) 中表达。重组LDH在SDS-PAGE中显示出36-38kDa的分子量。发现纯化的 LDH 在 pH 6.5 从乳酸到丙酮酸的最大氧化活性为 29.6 U/mg，在 pH 8.5 从丙酮酸到乳酸的最大还原活性为 10.4 U/mg。引人注目的是，它的氧化活性优于还原活性，导致大肠杆菌中乳酸的利用增加了 17 倍。大肠杆菌/pET28a-LDH 比大肠杆菌/pET28a。CPB6 LDH 基因编码一个 315 个氨基酸的蛋白质，与拜氏梭菌 LDH 具有 42.19% 的相似性，与其他生物的 LDH 相似性较低。在预测的三级结构和活性中心中，在 CPB6 LDH 与 C. beijerinckii 和 C. acetobutylicum LDH 之间观察到显着差异。此外，需要进行 X 射线晶体结构分析来验证 CPB6 LDH 的特定活性中心及其在乳酸转化为 CA 中的作用。乙酰丁酸 LDH 在预测的三级结构和活性中心。此外，需要进行 X 射线晶体结构分析来验证 CPB6 LDH 的特定活性中心及其在乳酸转化为 CA 中的作用。乙酰丁酸 LDH 在预测的三级结构和活性中心。此外，需要进行 X 射线晶体结构分析来验证 CPB6 LDH 的特定活性中心及其在乳酸转化为 CA 中的作用。