Efficient secretory expression, purification, and characterization of lipase in Pseudomonas aeruginosa M18, with multifunctional applications in diagnostics.

Abstract

Lipase (EC 3.1.1.3) is a crucial hydrolase with broad industrial and clinical applications. In this study, the lipA and lipH genes from Pseudomonas aeruginosa were cloned into the pBBR1MCS-2 vector and expressed under the regulation of the highly efficient BSFP_0720 promoter from Burkholderia stabilis FERMP-21014. This system allowed for efficient secretory expression in Pseudomonas aeruginosa without requiring an inducer. The recombinant lipase exhibited both lipase and cholesteryl esterase activities, making it suitable for triglyceride and cholesterol assay kits. Additionally, gene editing was used to knock out the endogenous cholesterol oxidase gene in Pseudomonas aeruginosa, eliminating cross-interference in different assay kits. High-density fermentation using glucose as the carbon source resulted in lipase activity reaching 68 kU/L and cholesteryl esterase activity reaching 214 kU/L after 30 h of fermentation, representing a 356-fold increase compared to natural production. By combining ammonium sulfate precipitation, hydrophobic interaction chromatography, and anion exchange chromatography, a purity of 94.32% was achieved (as determined by CE-SDS). Accelerated stability tests showed that the lyophilized lipase retained over 96% residual activity after storage at 37 °C for 21 days and at 45 °C for 7 days, suggesting its suitability for long-term storage. The enzymatic properties of the lipase demonstrated resistance to common chemicals, high activity in buffers with pH values between 7 and 9, and short-term tolerance to high temperatures (60 °C). These characteristics make the lipase highly adaptable for use in complex clinical samples and various industrial applications. The successful high-efficiency expression and multifunctional utility of this lipase highlight its significant commercial potential in diagnostics and other fields.