How site-directed mutagenesis of the lid domain affects the properties of lipases

Lipases (EC 3.1.1.3 – triacylglycerol hydrolases) are versatile enzymes widely used in biocatalysis due to their ability to catalyse both hydrolysis and synthesis reactions. However, only a few lipases meet the stringent requirements of industrial processes, which demand high catalytic activity, substrate specificity and selectivity, as well as structural stability. To meet these requirements, protein engineering techniques such as site-directed mutagenesis have been used to optimize lipases. One of the main targets for these modifications is the lid domain of lipases, an α-helix structure that regulates access to the active site and plays a role in interfacial activation. Alterations in this domain can significantly affect properties such as activity, specificity, enantioselectivity, and thermal or solvent stability. This review discusses recent advances in the application of site-directed mutagenesis to the lid domain of microbial lipases. We categorize the main mutation strategies based on their effects, ranging from adjustments in lid hydrophobicity and flexibility to the incorporation of stabilizing elements such as disulphide bonds. By analysing these studies alongside homology-based approaches, we aim to provide insights that may support the rational design of recombinant lipases with enhanced properties for industrial applications.