## 3 Lipase Characteristics and Engineering
### Lipase Engineering  
Protein engineering is the process of customizing new enzymes with improved features by altering their primary amino acid sequences. Given the considerable diversity of possible alterations, this procedure has already produced remarkable results in the design of optimized lipases used in important industrial areas. Protein engineering strategies, such as rational design and directed evolution have led to interesting results in the improvement of different lipases. Engineering a protein by rational design uses prior knowledge of protein structure and in-depth computational modeling approaches to deliberately design new biocatalystss [109]. In part, the number of lipase structures deposited in the Protein Data Bank (PDB, [[https://www.rcsb.org/](https://www.rcsb.org/)]([https://www.rcsb.org/](https://www.rcsb.org/))) and lipase sequences information in various databases has substantially facilitated the rational design  
Figure 2: Overview of the three-dimensional structure of two microbial lipases. (**A**) Crystallographic structure of _Candida rugosa_ lipase with its \(\alpha\)/\(\beta\)-hydrolase fold in gray exemplifies the lid in its open conformation (PDB access number 1 CRL) and its \(\alpha\)/\(\beta\)-hydrolase in a wheat tint exemplifies the lid in its closed conformation (PDB access number 1 TRH). (**B**) Crystallographic structure of _Rhizomucor mehei_ lipase with its \(\alpha\)/\(\beta\)-hydrolase fold in gray exemplifies the lid in its open conformation (PDB access number 4 TGL) and its \(\alpha\)/\(\beta\)-hydrolase fold in cyan exemplifies the lid in its closed conformation (PDB access number 3 TGL). The lids in both structures are in red. All figures were prepared using molecular visualization software, PYMOL. of these proteins. Furthermore, many different types of modeling software have been created, which makes this methodology easier to use and enhances the success rate of modeling predictions. Usually, the information derived from computer modeling identifies certain amino acids (hot-spots) that should be altered to lead to a change in the lipases' properties. By using computational predictions, [PERSON] et al. [110] constructed four mutants of _S. marcescens_ lipase. The MutG2P and MutG59P mutants showed higher catalytic efficiency and thermal stability than the wild-type (WT) enzyme. Furthermore, other computational approaches have been developed to predict the effects of mutations on lipases. [PERSON] et al. [37] used a strategy denoted as RIF that consists of three computational methods--i.e., Rosetta ddg monomer, I Mutant 3.0, and FoldX--to rationally design and optimize the thermal stability of _R. miehei_ lipase. The most stable mutant, harboring the mutations T18K/T22I/E230I/S56C-N63C/V189C-D238C, exhibited a 14.3 \({}^{\circ}\)C higher melting temperature and a 12.5 fold increase in half-life at 70 \({}^{\circ}\)C. [PERSON] et al. [111] selected different mutants of CALB based on the B-factor value of amino acid residues and computational modeling by RosettaDesign. The melting temperature of the mutant R249L was increased by 2.3 \({}^{\circ}\)C compared to that of WT CALB. Moreover, the rational strategy was applied to block the primary acyl-binding tunnel of CALA lipase to create variants with high specificity for medium-chain-length fatty acids [112]. Likewise, rational design was used to engineer CALA, aiming to develop a highly selective variant for _trans_-fatty acids [113; 114]. The dynamic behavior of lipases in different conditions has also been investigated using computational approaches, such as molecular dynamics (MD) simulations. The MD simulations have made progress in this direction since they provide atomistic information about the dynamic molecular interactions, which determine the protein stability and function [115]. [PERSON] and [PERSON] [116] carried out MD simulations of a mutant porcine pancreatic lipase in open and closed conformations using different solvents to explain the dynamics of lid opening.