Study On Production, Purification, Cloning And Expression Of The Lipase From Aureobasidium Pullulans HN2-3

A marine-derived yeast strain HN2-3 isolated from sediment of the salterns was found to secrete a large amount of lipase into the medium. This marine-derived yeast strain was identified to be a strain of Aureobasidium pullulans according to the results of routine yeast identification and molecular methods. The optimal medium for the crude lipase production was 3.0% (w/v) olive oil, 0.4% (w/v) glucose, 0.6% (w/v) ammonium sulfate, 0.1% (w/v) K₂HPO₄, 0.05% (w/v) MgSO₄.7H2O, while the optimal cultivation conditions for the crude lipase production were pH 7.0, 25 oC and 170 rpm. It was found that lipase production was dependent on the time when olive oil was added to the medium. When olive oil was added to 6 h old culture with 0.4% (w/v) glucose, the highest lipase activity was achieved. Under the optimal conditions, over 8.0 U/ml of lipase was produced within 96 h of the fermentation at shake flask level.The lipase in the supernatant of the yeast cell culture was purified to homogeneity with a 3.4-fold increase in specific lipase activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography and anion-exchange chromatography. According to the data on SDS polyacrylamide gel electrophoresis, the molecular mass of the purified enzyme was estimated to be 63.5 kDa. The optimal pH and temperature of the purified enzyme were 8.5 and 35 oC, respectively. The enzyme was greatly inhibited by Hg²⁺, Fe²⁺ and Zn²⁺. The enzyme was strongly inhibited by phenylmethanesulphonyl fluoride, not inhibited by ethylene diamine tetraacetic acid (EDTA), but weakly inhibited by iodoacetic acid. It was found that the purified lipase had the highest hydrolytic activity towards peanut oil.The extracellular lipase structural gene was isolated from cDNA of A. pullulans HN2-3 by using SMART^TM RACE cDNA amplification kit. The gene had an open reading frame of 1245 bp long encoding a lipase. The coding region of the gene was interrupted by only one intron (55 bp). It encodes 414 amino acid residues of a protein with a putative signal peptide of 26 amino acids. The protein sequence deduced from the extracellular lipase structural gene contained the lipase consensus sequence (G-X-S-X-G) and three conserved putative N-glycosylation sites. According to the phylogenetic tree of the lipases, the lipase from A. pullulans was closely related to that from Aspergillus fumigatus (XP₇50543) and Neosartorya fischeri (XP₀01257768) and the identities were 50% and 52%, respectively.The mature peptide encoding cDNA was subcloned into pET-24a (+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21(DE3). The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 47 kDa was found. Enzyme activity assay verified the recombinant protein as a lipase. A maximum activity of 0.96 U/mg was obtained from cellular extract of E. coli BL21(DE3) harboring pET-24a(+)LIP1 . Optimal pH and temperature of the crude recombinant lipase were 8.0 and 35°C, respectively and the crude recombinant lipase had the highest hydrolytic activity towards peanut oil.