Co-expression, purification, and characterization of an acidophilic and n-hexane-tolerant lipase with its foldase from Burkholderia gladioli Bsp-1

Lipases are versatile biocatalysts widely applied in hydrolysis and synthesis reactions, yet bacterial acidophilic and solvent-tolerant lipases remain poorly characterized. In this study, an acid- and organic solvent-resistant lipase (LipC) and its cognate foldase (LifB) from Burkholderia gladioli Bsp-1 were cloned and heterologously expressed in Escherichia coli. Soluble and catalytically active LipC was obtained only upon co-expression with LifB, demonstrating a strict foldase-dependent folding requirement. Phylogenetic analysis classified LipC as a LipA-type bacterial lipase. Biochemical characterization revealed that LipC exhibited maximal activity at pH 3.5 and showed remarkable stability under acidic conditions, retaining more than 70% of its activity after prolonged incubation at pH 4.0. The enzyme displayed optimal activity at 55 ℃ and maintained moderate thermal stability. Notably, LipC retained high activity in nonpolar organic solvents, with significant activation observed in n-hexane and cyclohexane. Substrate specificity and kinetic analysis indicated a preference for medium-chain fatty acid esters, with the highest catalytic efficiency toward p-nitrophenyl caprate (C10). Collectively, these results identify LipC as a foldase-dependent bacterial lipase combining acidophilic behavior and solvent tolerance, expanding the current understanding of bacterial lipases and highlighting their potential relevance for biocatalysis under acidic and nonaqueous conditions. KEY POINTS: • LipC is a foldase-dependent bacterial lipase requiring LifB for functional expression. • LipC exhibits strong acidophilicity with optimal activity at pH 3.5. • LipC is highly activated by nonpolar solvents such as n-hexane and cyclohexane.