Characterization of a Highly Solvent-Tolerant SGNH Hydrolase Superfamily Lipolytic Enzyme from <i>Sphaerobacter thermophilus</i>

Thermophilic microbial lipases that retain activity under harsh conditions are a highly desirable tool for catalysis in numerous biosynthetic and biotechnological applications. In this study, a putative SGNH lipase gene, from <i>Sphaerobacter thermophilus</i> (<i>St</i>SGNH1), was overexpressed using a pMCSG7 plasmid in BL21(DE3) cells. The polyhistidine-tagged enzyme was expressed as inclusion bodies that were readily solubilized using Empigen BB detergent, and the protein was purified to homogeneity using immobilized metal affinity chromatography. The classification of <i>St</i>SGNH1 as a thermophilic and alkaliphilic lipase was supported by its ability to optimally catalyze the hydrolysis of medium-length <i>p</i>-nitrophenol esters at elevated temperature (55 °C) and pH (8-11). Evaluation of the <i>St</i>SGNH1 structure generated by AlphaFold indicated that the catalytic domain was composed of a three-layered α/β/α fold, and molecular docking studies yielded insight into which residues proximal to the active site assist in stabilizing the ligand-enzyme interaction and substrate selectivity. Notably, <i>St</i>SGNH1 was able to carry out ester hydrolysis in the presence of elevated concentrations of detergents, chaotropic reagents, and organic solvents, indicating that it would be suitable for employment in industrial reactions. Tryptophan fluorescence measurements in the presence of guanidine hydrochloride were employed to estimate the free energy of folding for <i>St</i>SGNH1 along a reversible folding pathway. The properties of <i>St</i>SGNH1 would be highly desirable for biotechnological applications.