Improvement of biological indicator by increasing the catalytic efficiency of geobacillus stearothermophilus triacylglycerol lipase (GstLipC)

Geobacillus stearothermophilus spores are highly resistant to extreme temperatures, making them valuable bioindicators in sterilization processes. Spore germination is a highly regulated process initiated by the sequential activation of specific enzymes, with various proteins known to play critical roles in this mechanism. In this study, a GDSL family lipase from Geobacillus stearothermophilus, implicated in spore germination, was functionally characterized. Four site-directed mutations were introduced into the enzyme based on bioinformatics analysis, and the mutant variants were compared with the wild-type enzyme. The mutant lipase genes were cloned into the pNW33N plasmid and transformed into Geobacillus stearothermophilus. Sporulation and germination assays were performed on both wild-type and recombinant strains. Germination kinetics were quantitatively assessed using spectrophotometric measurements of OD600. The obtained results showed that both the wild-type and mutant GDSL lipases were successfully characterized, and the mutations led to measurable changes in the Km values of the enzyme. In addition, observable differences were detected between the germination processes of wild-type and mutant spores