Cloning And Expressing Of Lipase Gene From Geotrichum Candidum Y162

Lipase-producing strains isolation, lipase gene cloning and expression have enriched lipase gene resources and created a platform for theory and application of lipases. A fungus producing high-activity lipase was isolated from oil-contaminated soil samples from Luzhou, Sichuan Province. 376bp internal transcribed spacer (ITS) sequence (Accession number: EF159152) was amplified using universal primers ITS1 and ITS4. Senquence blast showed 97% identical to Geotrichum sp. TUGM12 (Accession number: AY787702). Combined with morphological characteristics, the strain was identified as Geotrichum candidum, named G. candidum Y162.The lipase from G. candidum Y162 exhibited maximum activity at 40℃and pH 9.0, and was fairly stable in pH from 6.0 to 8.5 and temperature below 35℃. The enzyme activity was strongly inhibited by Hg2+, Ag+, EDTA , SDS , and slightly inhibited by Mn2+,,Cu2+, Fe3+, while significantly stimulated by Ba2+and enhanced by K+, Mg2+, Ca2+, Zn2+.In order to improve lipase production, the fermentation conditions of lipase production by G. candidum Y162 were optimized with response surface methodology, and the data were analysed by using Statistical Analysis System (SAS) 9.0 software. Initially, screening design methodology Plackett-Burman was used to evaluate the effects of eleven factors related to lipase production and three statistically significant factors: soybean flour, corn steep liquor and culture time were selected. Subsequently, the path of steepest ascent was used to approach the optimal region of lipase production. Then, the optimal combined conditions for maximum enzyme activity were further optimized by response surface methodology and determined as follows: soybean flour 2.51%, corn steep liquor 2.12% and culture time 101.97h.Under the optimal conditions, the enzyme activity was improved from 9.60 U/mL to 21.75 U/mL, enhanced 2.27-fold, which indicates that response surface methodology is an effective means for optimizing lipase production conditions by G. candidum Y162.By means of bioinformatics, alignment of nucleotide sequence of lipase gene reported from Geotrichum link was performed. Primers were designed based on the conservative nucleotide sequence and the lipase gene of G. candidum Y162 was cloned using genomic DNA as template for the first time in China (Accession number: DQ841279). Nucleotide sequencing revealed that the open reading frame has 1692 nucleotides, encoding 563 amino acid residues including a signal sequence of 19 amino acid residues. The mature lipase gene comprises of 544 amino acid residues, which is 86% identical to complete cds of G. fermentans mRNA for lipase I precursor(Accession number: AB000260). Subsequently, the lipase gene was cloned into expression vector pPIC9K, and then transformed into Pichia pastoris GS115. Cultures of recombined P. pastoris accumulated active enzyme in the supernatant to levels of 30.4U/mL after induction for 96 hours on flask scale. This yield approaches the data previously reported in foreign literatures. The molecular mass of the recombinant GCL determined by SDS-PAGE was 60KDa.