Genetic characterization and mutagenesis of fat-degrading enzymes from Bacillus cereus for enhanced lipid degradation and esterification

Lipases are key biocatalysts for industrial lipid processing. Thirty bacterial isolates were screened for lipolytic activity using qualitative and quantitative assays, identifying Bacillus cereus B18 as the most potent producer (clear zone 30 ± 0. 15ₘm; A 410 0. 2435 ± 0. 15). Phylogenetic analysis confirmed its close relationship with reference B. cereus strains. Controlled 5L bioreactor fermentation, with and without ozone supplementation, significantly enhanced lipase production. Sequential purification steps (ammonium sulfate, DEAE-cellulose, Sephadex G-100) increased specific activity up to 8827Uₘg -1 while maintaining 77 to 92% activity. Optimization of culture parameters, including carbon and nitrogen sources, inoculum size, pH, temperature, and incubation period, further improved enzyme yield. GC-MS analysis of degraded fat revealed 35 fatty acids, comprising 60% saturated and 40% unsaturated. Molecular analysis detected point mutations in conserved α/β hydrolase folds and GxSxG motifs, contributing to enzyme heterogeneity. These findings establish B. cereus B18 as a robust lipase producer with high potential for industrial applications in fat biodegradation and lipid-based bioproduct synthesis. • Genetic characterization of lipase and other fat-degrading enzymes from Bacillus cereus was performed to identify key catalytic genes. • Site-directed mutagenesis of selected lipase genes enhanced enzymatic efficiency and substrate specificity. • Engineered enzymes demonstrated improved lipid hydrolysis and esterification activity under optimized conditions. • Functional validation confirmed that mutant variants outperform wild-type enzymes in both degradation of triglycerides and synthesis of esters. • Findings provide a biotechnological framework for industrial lipid processing, including biodiesel production and value-added lipid bioconversions.