扩展青霉TS414脂肪酶的克隆与原核表达

提取了扩展青霉Penicillium expansumTS414菌体的总RNA,并以反转录得到的cDNA为模板,成功扩增得到了脂肪酶基因;在此基础上,构建了重组表达载体pGEX-4T-1-PEL,并在大肠杆菌DH5α中实现了扩展青霉脂肪酶的表达;进一步采用低IPTG诱导浓度、低温、长时间的发酵策略等,使扩展青霉脂肪酶得以有活性的表达;同时,通过反复冻融细胞破碎法使其较好地从菌体细胞中分离出来,提取得到的酶液的活力达到0.49 U/mL。以上结果为后续脂肪酶分子进化等研究奠定了坚实的基础,亦可转化为高校或中学生物学实验教学的项目。

Cloning of Penicillium expansum TS414 lipase and its prokaryotic expression

The total RNA of Penicillium expansum TS414 was successfully extracted. Moreover, Peni- cillium expansum TS414 lipase gene was amplified using the corresponding cDNA as the template. Then, the recombinant expression vector pGEX-4T-1-PEL was constructed and transformed into E. coli DH5ot. Further, such strategies as IPTG of a low concentration, a lower fermentation temperature and a relatively long fermentation time were employed in order to express the lipase in an active form. Finally, the method of freeze thawing repeatedly was used to separate the lipase from the cells and the activity of the extracted enzyme solution reached 0. 49 U/mL. The result laid a good foundation for the following research such as modification of the lipase and can further transform into a teaching item of biological experiment for universities or high schools.