编码1,3-丙二醇氧化还原酶同工酶基因yqhD的克隆与高效表达

Cloning and Over Expression of Gene yqhD Encoding 1,3-Propenediol Oxidor Eductase Isoenzyme in Escherichia coli

利用PCR技术从大肠杆菌(Escherichia coli)中扩增出1.16 kb的编码1,3-丙二醇氧化还原酶同工酶的基因yqhD,将其连接到温控表达载体pHsh,得到重组载体pHsh-yqhD,重组载体在大肠杆菌JM109中得到高效表达。SDS-PAGE分析显示:融合表达产物的相对分子质量均为43 000,同核酸序列测定所推导的值相符。对含有yqhD的基因工程菌进行表达研究表明:42℃诱导4 h,1,3-丙二醇氧化还原酶同工酶的酶活力达到100 IU/mg,而对照菌株的酶活力仅为0.5IU/mg。

The structure gene yqhD from E. coli encoding 1, 3-propanediol oxidoreductase isoenzyme was amplified using PCR method. The temperature control expression vector pHsh harboring yqhD gene was transformed into E. coli JM109 to yield the recombinant expression vector pHsh-yqhD. The yqhD was over-expressed in E. coli JM109. SDS-PAGE analysis showed that the relative molecular weights of the expressed recombinant products were about 43 000, which was corresponding to the prediction by gene sequence. Compared with E. coli JM109 （pHsh）, the 1,3-propanediol oxidoreductase isoenzyme activity with recombinant E. coli reached up to 100 U/rag protein when inducted at 42 ℃ for 4 h,which was much higher than that with E. coli JM109 0. 5 U/mg protein.