Cloning Of Aspergillus Tamarii FS132 Lipase Gene And Its Expression In Pichia Pastoris

A lipase-producing strain FS132 was isolated from the volcanic vent soil in SinKiang Province. The strain was identified by 18S rRNA molecular marker. The lipase gene (ATL) was further cloned and then expressed in Pichia pastoris. The results not only make a foundation for further investigating the mechanism of lipase synthesis in Aspergillus tamari, but also provide a new kind of lipase gene resource for improvement the existing lipase. The main results of this study were as follows:(1) A lipase-producing strain FS132 was isolated from the volcanic vent soil in SinKiang Province. It was a mildew based on its colony morphology. The 18S rRNA gene of FS132 was cloned and sequenced. According to the phylogenetic analysis, the 18S rRNA marker of FS 132 was showed highly homology to that ofAspergillus tamari.(2) Cloning and sequence analysis of the ATL-DNA and ATL-cDNA: the genomic DNA and total RNA of A. tamari FS 132 were extracted respectively. Specific primers were designed based on the homology lipase gene in the literature. The ATL-DNA and ATL-cDNA was amplified using the techniques of PCR and RT-PCR. The total size of ATL-cDNA was made of 921 bp, and the open read frame of ATL gene was 1024 bp, which containing 2 introns with sizes of 51 bp and 52 bp respectively.(3) To identify the function of cloned ATL-cDNA, the recombinant expression vector was constructed, and the Pichia pastoris GS115 was used as a host for expressing ATL gene. The ATL gene of A. tamarii FS132 was expressed in GS 115 and secreted into culture aider the addition of 0.5% methanol to the medium. Result of SDS-PAGE showed that a 36.7 kDa recombinant protein was secreted from the host yeast. And clear zone was developed on the tributyrin plate. The lipase activity in the supernatant of the culture was 20U/ml assayed by NaOH titrate method.(4) The optimal culture conditions for lipase producing of FS132 was obtained as following: the optimal culture temperature of 36℃, initiation pH of 7.0 for the culture medium, oxygen-consumption of 25 mL medium for each 250mL flask, and culture time of 45 hr.