Enhancing cryo-enzymatic efficiency in cold-adapted lipase from Psychrobacter sp. C18 via site-directed mutagenesis

Abstract

As industrial demands for cold-active enzymes have been increased, psychrophilic lipases present a promising solution with potential for innovation and growth in food, pharmaceutical, and detergent industries. Cold-adapted enzymes achieve high catalytic efficiency at low temperatures through their structural flexibility and conformational adaptability. Therefore, in this study, the lipase gene from Psychrobacter sp. C18 was cloned and subjected to site-directed mutagenesis based on computer aided predictions to enhance the enzyme's cold-adapted properties and flexibility. Mutations were strategically selected in loops of the active site to improve the enzyme's accessibility to the substrate under cold conditions. The P163G, L186G, and Q239W mutations were selected for further analysis. Enzyme activity, along with its stability and structural flexibility, was assessed using techniques including UV–Vis spectroscopy, fluorescence, and circular dichroism (CD) spectroscopy. The obtained data revealed that the optimal temperature for the wild-type lipase was 30 °C, which shifted to lower temperatures in the mutants: 15 °C for P163G and L186G, and 20 °C for Q239W. Additionally, the optimal pH of the mutant lipases shifted to more alkaline conditions compared to the wild-type enzyme. While the thermal and pH stability of the mutant enzymes slightly decreased, these findings can be attributed to their enhanced flexibility. Far-UV CD spectroscopy revealed a reduction in α-helical content of the mutant enzymes. Molecular dynamics simulations corroborated these findings, confirming increased structural flexibility in all three mutants compared to the wild-type enzyme. This research underlines the importance of applying engineered cold-adapted enzymes for industrial application.