碱性果胶酯裂解酶工程菌的构建

Construction of Engineering Strain Producing Pectate Lyase

从本研究室筛选的BacillussubtilisWSHB04-02菌株中扩增出编码碱性果胶酯裂解酶的结构基因PL,将其插入大肠杆菌分泌表达载体pET22b(+)多克隆位点,得到重组载体pET22b(+)PL.序列分析表明,所获PL基因与已报道的B.subtilisSO113的PL基因的同源性为98%.重组载体在大肠杆菌BL21(DE3)中得到表达.SDS-PAGE分析显示,表达产物的分子量(Mr)均为43×103,同核酸序列测定所推导的值相符.该工程菌经IPTG诱导后,胞内酶活为8U/mL.研究发现,碱性果胶酯裂解酶不仅在胞内有酶活,胞外也有酶活,说明所构建的表达体系可使该酶向胞外分泌.

The structure gene PL from Bacillus subtilis WSHB04-02 encoding pectate lyase was amplified by PCR method. Comparison with the structure gene PL from B. subtilis SO113 revealed 98% identity. The pET22b （ ＋ ） vector harboring PL gene was transformed into E. coil BL21DE3. The PL was expressed in E. coil BL21 （DE3）. SDS - PAGE analysis showed that the molecular weight of expressed recombinant PL was about 43×10^3, which was the same as the calculated molecular weight. The intracellular pectate lyase activity of recombinant E. coil reached up to 8 U/mL under the induction of IPTG.