白地霉ch-3脂肪酶Ⅰ基因在大肠杆菌中的表达

Expression of lipase I gene from Geotrichum candidum ch-3 in Escherichia coli

构建了白地霉脂肪酶Ⅰ的基因工程菌,为进一步进行蛋白质工程改造和脂肪酶应用奠定了基础。从新疆昌吉市油脂化工厂含油冻土中分离得到1株低温脂肪酶产生菌-白地霉ch-3。该菌发酵上清液中的脂肪酶最适作用温度为35℃,在0℃仍可保持66%的相对酶活力。应用PCR技术从白地霉ch-3基因组DNA中克隆得到脂肪酶Ⅰ基因lip1,将该基因与原核表达质粒载体pET-22b(+)连接,构建重组质粒pETl-ip1,转化E.coliBL21(DE3),酶切鉴定,筛选得到重组菌。十二烷基磺酸钠-聚丙希酰胺(SDS-PAGE)显示重组脂肪酶Ⅰ的相对分子质量约为5.8×104,酶活为2.73 U/mL,表明lip1基因的表达产物具有正常的生物学活性。白地霉ch-3脂肪酶Ⅰ基因lip1能够在大肠杆菌中有效地表达。

A cold-adapted lipase-producing strain Geotrichum candidum ch-3 was isolated from frozen soil containing oil and fat. The optimal temperature of the lipase in the culture supernatant was 35 ℃. It retained 66 % activity at 0 ℃. Lipase Ⅰ gene lipl from G. candidum ch-3 was amplified by PCR and inserted into pET-22b （ ＋ ） to generate recombinant pET-lipl, pET-lipl was transformed into E. coli BL21 （DE3）. The Mr of the expression product was 5.8 ×10^4 by SDS-PAGE. The lipase activity was 2. 73 U/mL, which indicated the cloned DNA fragment encoded a lipase. Lipase gene lipl of G. candidum ch-3 was efficiently expressed in E. coli.