Engineering of Nocardioides acrostichi lipase: utilizing natural variation and mutational free energy to alter expression and functional properties

The rapidly growing biocatalysis market demands cost-effective, sustainable technologies. Lipases are vital, versatile enzymes used in food, pharmaceuticals, and biofuels. Nocardioides acrostichi lipase shows promising potential for biotechnological applications, but its low extracellular production limits its industrial utility. Here, we employed a rational design integrating natural variation identified from sequence analysis and predicted mutational Gibbs free energy (ΔΔG) changes. We designed and experimentally tested three multi-site mutants. Engineered variants showed significantly improved extracellular expression and diverse enzymatic profiles. A mutant NaL-2 showed tolerance to both high temperature (> 70% activity at 70 °C) and extreme alkaline condition (stable at pH 11–13), which is a rare trait for lipase. The results provide novel lipase variants and strategies for enhancing enzyme stability and functionality, with implications for biotechnological applications.