肺炎克雷伯氏菌D-乳酸脱氢酶基因的克隆及其在大肠杆菌中的表达

以肺炎克雷伯氏菌（Klebsiella pneumoniae）基因组DNA为模板，应用PCR技术扩增并克隆出D-乳酸脱氢酶的基因（ldhD），将其连接到表达载体pET-22b(+)质粒上，构建pET-22b(+)-ldhD重组质粒，测序结果100%正确。将重组质粒pET-22b(+)-ldhD转化到大肠杆菌表达菌株BL21(DE3)中，通过氨苄霉素抗性平板筛选，构建大肠杆菌BL21(DE3)-pET-22b(+)-ldhD基因工程菌。重组菌株经IPTG诱导表达，SDS-PAGE蛋白电泳分析，目标条带出现在分子量约37000处，表明D-乳酸脱氢酶基因ldhD在大肠杆菌BL21(DE3)中成功表达。采用紫外分光光度法测定其酶活，底物丙酮酸终浓度为10mmol/L时，在丙酮酸还原为D-乳酸反应方向D-乳酸脱氢酶表现出119.04U/mL的酶活力；底物D-乳酸终浓度为50mmol/L时，在D-乳酸转化为丙酮酸的逆反应方向中表现出0.89U/mL的酶活力。比酶活则分别为9.16U/mg和 0.07U/mg。通过Lineweaver-Burk双倒数作图法，计算出酶反应的米氏常数K_M为10.54mmol/L。经摇瓶发酵后，通过高效液相色谱测定产物，D-乳酸的产量达到3.09g/L。

Cloning of D-lactate dehydrogenase gene from Klebsiella pneumoniae and expression in Escherichia coli

The gene ldhD encoding D-lactate dehydrogenase(D-LDH) was cloned and amplified from genomic DNA of Klebsiella pneumoniae by PCR.The PCR product was inserted into the expression vector pET-22b(+) under the control of T7 promoter.It was then transformed into E.coli Top10 and sequenced in order to verify that it was free of mutant.The verified recombinant vector was transformed into expression strain E.coli BL21(DE3) which is deficient in D-lactate dehydrogenase.The D-lactate dehydrogenase activity of the recombinant strain was determined by spectral assay after IPTG induction.SDS-PAGE results showed that the cloned ldhD was expressed in the E.coli.The target strip of the expression product had a molecular weight of about 37000.The measured enzyme activity was 119.04U/mL for reduction and 0.89U/ mL for oxidation.The resulting protein mass concentration was 13.0 mg/mL and the calculated specific enzyme activity was 9.16U/mg.According to a Lineweaver-Burk plot,the KM value of the reaction in which pyruvate was converted to D-lactate was assayed as 10.54 mmol/L.After fermentation in a shake flask for 24h,a yield of 3.09g/L of D-lactate acid was obtained.