产脂肪酶微生物的筛选鉴定及脂肪酶lipA基因克隆

Seperation,identification and cloing of lipA from lipase-prducing microorganism

以橄榄油为唯一碳源对富含油污土壤中产脂肪酶微生物进行富集培养,以溴甲酚紫为指示剂进行初筛,结合橄榄油乳化液滴定法测定酶活力,得到酶活力较高的产脂肪酶菌株Y19,其粗酶活力为16.88 U/mL,经16S rDNA初步鉴定为枯草芽孢杆菌。通过特定引物PCR扩增菌株Y19脂肪酶lipA基因,构建pET32a-lipA重组表达载体并在大肠杆菌中诱导表达。以表达菌的细胞裂解物进行生物柴油转酯试验,GC/MS结果显示,重组脂肪酶lipA粗酶液催化的转酯产物中脂肪酸甲酯成分与生物柴油十分相近。

The research added olive oil as the single carbon resources to enrichment culture,and bromocresol purple to screening culture as indicator to obtain lipase-producing microorganism from oil-riched soil.Emulsified olive oil method was used to obtain a lipase-producing strain Y19,which was identified to be Bacillus subtilis by 16S rDNA method,with crude lipase acitivity of 16.88 U/mL.The lipA gene was amplified by PCR.The pET32a-lipA recombinant carrier was established and expressed in E.coli.The bio-diesel trasesterification reaction produced by the recombinant lipA crude lipase was similar to biodiesel molecule through GC/MS test.