Screening, Optimization Of Medium, Property And Gene Cloning Of Lipase From Bacteria Associated With Marine Sponge

Lipase is used widely in industry. However, the challenges still lie in exploiting novel lipase for research and applied industry. Marine microorganisms have been proved to be an important source for novel enzymes. In this study, the screening of lipase, medium optimization and lipase properties, gene cloning was investigated for bacteria associated with the South China Sea sponge. To our knowledge, there is no data about lipase from bacterial associated with marine sponge.Forty-four bacteria isolated from sponge Stelletta tenuis, Halichondria regosa and Craniella australiensis were screened for lipase production, twenty-nine of them were found to be with lipase activity. Three bacteria with stronger lipase activity were identified as Bacillus pumilus B106, Bacillus sp. B22 and Pseudomonas sp.A40 though 16S rDNA sequencing, among which Bacillus pumilus B106 showed the highest lipase production ability. The maximum cell dry weight and the maximum lipase activity reached 6.38g/L at 35h, 25.28U/ml at 45h, respectively. The enzymic activity was 2.81-fold and 2.28-fold higher than that of Bacillus sp. B22 (8.98U/m) and Pseudomonas sp.A40(11.11U/ml), respectively. Thus, Bacillus pumilus B106 was selected for medium optimization, lipase property and gene cloning.On the basis of one-factor test, Plackett–Burman experimental design was used to evaluate the seven medium components and identify the optimal values of the significant medium components. As a result, the optimal medium was composed of tween80 (5.0 ml L^-1), Mg²⁺ (17.15 gL^-1), PO₄^3- (2.0 gL^-1), yeast extract (5.0 gL^-1), maize oil (12.5 ml L^-1) beef extract (5 gL^-1) and CaCl₂ (2.282 gL^-1) with artificial sea water (ASW) and an initial pH7.0. The fermentation condition was 5% inoculum, 180rpm at 28℃for 70h. The maximum lipase activity was 86.37U/ml at 60h and the maximum cell dry weight was 7.44g/L with this optimum medium, which were 3.54-fold and 1.31-fold higher than that of the basal medium, respectively.Characteristics such as pH, temperature, salinity and organic solvents were also studied. The optimal temperature was 50℃and the optimal pH was 8.0. Meanwhile, for the test of salinity, relative activity above 78% was observed in the range of 0～150‰. These results fully represented the advantage of marine lipase. The lipase from Bacillus pumilus B106 showed a different stability in organic solvents including methanol, ethanol, DMSO. For example, an inhibition effect on the lipase was observed for 2-propanol. Eighty-two percent and 29% of relative activity was measured with 10% and 40% (v/v) 2-propanol. Methanol showed inhibition or stimulation under different concentrations. With 10% and 20% (v/v) methanol, 139% and 127% of relative activity was measured respectively, which indicated the lipase activity was enhanced; however, with 30% and 40% (v/v) methanol, 65% and 33% of relative activity was measured, respectively, which indicated the lipase activity was inhibited. Under low concentration of ethanol, 89% and 86% of relative activity was measured with 10% and 20% ethanol, respectively, which showed weak inhibition, while under high concentration of ethanol, 29% of relative activity was measured with 40% ethanol which showed strong inhibition.The sequence of lipase complete genes was cloned using PCR methods from Bacillus pumilus B106. Lipase gene contains an open reading frame (ORF) of 648bp and encods a 215aa lipase protein. The amino acid sequence is 98% identical to the lipase gene from Bacillus pumilus (AAR84668). The amino acid sequence of lipase was composed of 53.02% polar amino acids and 46.98% hydrophobic amino acids. In the secondary structure of the lipase, there were 25.58% a-helixes and 23.72%β-sheets. Besides, there were four potential patterns through PROSITE motif analysis. Based on protein domain prediction, the lipase belongs to alpha/beta hydrolases family. Phylogenetic analysis demonstrats the lipase from Bacillus pumilus B106 showed high identity above 90% with bacillus sp. including Bacillus pumilus and Bacillus licheniformis.