Purification and characterization of lipase from Geobacillus thermodenitrificans HBB268

This study was conducted to optimize the lipase production capacity of Geobacillus thermodenitrificans HBB268 strain, as well as to purify and characterize the intracellular lipase. The optimization of medium components for lipase production was performed using PBD and CCD statistical methods, determining the optimal concentrations of 18.27 g/L peptone, 18.41 g/L meat extract, and 1.26 g/L glucose. Under these conditions, a maximum lipase activity of 352 U/mL was achieved after 18 hours of incubation. In the purification steps, lipase was initially 3.05-fold purified using ammonium sulfate precipitation at 60% saturation. Subsequently, the enzyme was purified further using Phenyl-Sepharose® CL-4B hydrophobic interaction chromatography (4-fold), Q Sepharose® ion exchange chromatography (55-fold), and finally Butyl-Sepharose chromatography (372-fold), resulting in a specific activity of 5104.76 U/mL. SDS-PAGE analysis determined the molecular weight of the lipase to be approximately 55 kDa. The maximum lipase activity was observed at pH 7.00 and 55 °C, while its stability was maintained in basic environments ranging from pH 8.00 to 10.00. Kinetic analyses revealed a Vmax of 75.56 U/mL and a Km of 26.07 μM, indicating that the enzyme has a high substrate affinity for p-nitrophenyl laurate. Lipase activity was largely preserved in the presence of +1-valent metal ions. In commercial detergents, Perwoll® at 1% concentration significantly preserved lipase stability. Additionally, lipase, when used with detergent, slightly increased its efficiency in removing oil stains from cotton fabrics. It was also observed that thiol group reductants enhanced lipase activity, whereas thiol group oxidants reduced it. The primary amino acids responsible for catalysis in the active site of the lipase were determined to be cysteine, tryptophan, and serine. Among polyhydric alcohols, sorbitol was found to enhance the thermostability of lipase, while apolar solvents increased its stability.