Ni_2025_Enzyme.Microb.Technol_194_110798

Optimizing lipases for the transesterification of long-chain fatty acid esters plays a vital role in enhancing biodiesel yields, especially from recycled cooking oils. In this study, we applied error-prone PCR to create a diverse mutagenesis library based on Bacillus subtilis lipase A(BSLA). The I157V mutant, selected through 96-well high-throughput screening, exhibited over sixfold greater catalytic efficiency (k(cat)/K(m)) than the wild-type enzyme. Insights from molecular docking and dynamic simulations pointed to the mutation's effect on aligning substrates more effectively within the enzyme's catalytic center. This alignment minimized non-productive binding modes and reinforced the enzyme-substrate interactions, which in turn boosted catalytic output. In biodiesel production using waste cooking oil as feedstock, the I157V mutant achieved a methyl ester yield of 87 % (w/w) after 10 h, representing an -45 % increase compared to 60 % (w/w) for the wild type. These findings not only advance enzyme engineering for industrial biocatalysis but also underline the potential of targeted mutations in supporting sustainable energy solutions.