Abstract
Lipases from Pseudomonas species are valuable biocatalysts, but their heterologous expression is often complicated by the need for different protocols to ensure proper folding and activity. In this study, we successfully produced a soluble and active form of LipGoM, a lipase derived from Pseudomonas sp., that employs a refolding strategy after solubilization in urea. Our findings indicate that refolding LipGoM in the presence of its native foldase, LifGoM, is essential for restoring its enzymatic activity. Furthermore, we identified that optimizing the refolding conditions, specifically pH, ionic strength, protein concentration, and the addition of glycerol, reducing agents, and ions, was critical for success. The lipase-lipid GoM mixture, Lip-Lif GoM, exhibited maximal hydrolytic activity toward p-nitrophenyl-octanoate at 55 °C and pH 8.0, demonstrating notable stability in the presence of detergents and organic solvents. These results demonstrate the indispensable role of its cognate foldase, LifGoM, in activating LipGoM, as well as the significance of buffer composition, particularly glycerol, for the effective refolding of the enzyme and its subsequent activity.