In this work we aim to understand the roles of evolutionarily conserved catalytic residues of the family VIII esterase LipBL by a combination of site-directed mutagenesis, biochemical methods and homology modelling. We performed site-directed mutagenesis to identify active-site residues and studied the role of the conserved G-X-S-X-G motif in enzyme function for what is believed to be the first time in this family of esterases. We describe the biochemical characterization of recombinant LipBL and mutated variants to elucidate the roles of conserved amino acids in its lipolytic activity and study the biophysical properties of LipBL.