Production and characterization of a thermostable lipase from Bacillus subtilis

In this study, 27 bacterial strains isolated from the oil factory soil samples and identified as Bacillus sp. Eight of these strains (29,62%) showed lipolytic activity. Strain TKL-25 which showed the best lipase activity of these strains selected as the lipase producer. Strain TKL-25 showed the best lipase activity on a tributyrin plate (the largest hydrolysis zone) was selected as a producer of lipase and identified as Bacillus subtilis using VITEK-2 identification system. The best enzyme production (optimum conditions) took place at pH 10.0, 40°C.The enzyme was active at pH 4.0-9.0, 20-110°C, optimum activity was determined at pH 5.0, 80°C. After a treatment of 30 min. with five different buffer systems in the range of pH=3.0-10.0, residual activity of the enzyme was detected as 56% at pH 8.0, 43% at pH 3.6, 41% at pH 7.6. At the other pH buffers enzyme activity was conserved less than 30%. After a treatment of 30 min. at 50-120°C, activity of the enzyme was conserved 176% at 50°C, 209% at 160°C, 176% at 70°C, 192% at 80°C, 161% at 90°C, 171% at 100°C, 135% at 110°C, 124% at 120°C.When the enzyme incubated with the inhibitors and metal ions at two concentrations (1-5 mM) for 30 min., activity was conserved in different levels. Those are PMSF (5 mM) 54%, ß-merkaptoetanol (%0.5) 27%, MgCl2 (5 mM) 23%, CaCl2 (5 mM) 21%, MnCl2 (5 mM) 21%, SDS 21% respectively. The activity was totally inhibited with FeCl2.The enzyme was treated with organic solvents at two concentrations (20-40%) for 30 min. Lipolytic activity was induced 9% at the presence of 20% chloroform and it was retained 68% at 20% chloroform, 30% at 20% ethanol, 24% at 20% methanol, 20% at 20% acetone, 23% at 40% glycerol, it was completely inhibited at the presence of 40% methanol, 2-propanol and 2-butanol. A single protein band with the molecular mass of 17.5 kDa was determined by SDS-PAGE analysis.Bacillus subtilis TKL-25 strain is a mesophilic bacteria, the enzyme from this has an acido-thermophilic-thermostable feature. This will provide a great advantage in the industrial productions along the fermentation process for the energy requirement.