丙酮酸甲酸裂解酶基因在大肠杆菌中的克隆、表达及酶学性质的研究

丙酮酸甲酸裂解酶（pyruvate format-lyas，PFL）是厌养或兼性厌养微生物中，代谢途径的关键酶之一，为了进一步研究其功能，我们以大肠杆菌JM109菌株基因组DNA为模板，进行PCR扩增大肠杆菌中的pfl基因，为测序方便将所得DNA片段连接到pMD18-T载体上，将测序正确后的pfl基因连接到表达载体pET-22b(＋)中，重组表达载体在大肠杆菌BL21(DE3)中诱导表达, 通过SDS-PAGE电泳分析，在分子量为85kDa处出现新生的蛋白条带。利用金属亲和层析对添加了6×组氨酸标签的PFL进行纯化，对PFL的酶学性质进行了研究。结果表明：此酶的最适温度为35 ℃，最适pH为7.5，米氏常数Km＝2.3mmol，Tm＝49.9℃。

Cloning, Expression and Characterization of Pyruvate Formate-lyase in Escherichia coli

Pyruvate formate-lyase(PFL) is one of the key enzymes in the metabolic pathway of anaerobic or facultative anaerobic microorganism. In order to further study the function of PFL, the pfl gene was amplified by PCR using the E. coli JM109 genomic DNA, and the amplified fraction was ligated into the vector pMD18-T for DNA sequencing. The identified pfl gene was cloned into the expression vector pET-22b and the recombinant expression vector was induced and expressed in E. coli BL21(DE3). A new protein band appeared by analysis of SDS-PAGE compared to the control, whose molecular weight was 85kD. The PFL with 6×His-Tag was purified by metal affinity chromatography. The characterization of purified PFL was carried out. The result showed that the temperature optimum and pH optimum were 35℃ and 7.5 , respectively. The Km value of the enzyme was 2.3mmol, and the value of melting temperature was 49.9℃.