Thermomyces lanuginosus脂肪酶(简称TLL)是具有重要商业应用价值的脂肪酶之一。运用RT-PCR技术,从Thermomyces lanuginosus ZJB09222基因组中克隆得到885 bp脂肪酶基因cDNA序列,其结构基因编码蛋白包含292个氨基酸。将脂肪酶基因cDNA序列开放阅读框克隆到大肠杆菌表达载体pET-28b中,转化大肠杆菌BL21(DE3),构建了基因工程菌E.coli BL21/pET28b-TLL。诱导表达后SDS-PAGE电泳显示该脂肪酶分子量约为32 ku。筛选获得廉价重组菌培养基,表达条件优化结果表明,当OD600约为0.6～0.8时,加入IPTG至终浓度为0.1 mmol/L,在28℃诱导培养7 h,酶活达到41 U/mL。

Thermomyces lanuginosus lipase is one of the most important industrial enzymes.Based on published DNA sequence,the full length cDNA of the lipase was cloned using RT-PCR method.It revealed that the cDNA of the T.lanuginosus lipase gene encoded a protein lipase of 292 amino acid residues.The open reading frame of the cDNA was cloned into the plasmid pET-28b,which was then transformed into E.coil BL21(DE3) strain.The Mr of the expression product was 32 ku by SDS-PAGE.The recombinant E.coli BL21/pET28b-TLL exhibited highest activity of 41 U/mL at 28℃ for 7 h induction with 0.1 mmol/L IPTG after screening of the best medium.