## 3 Lipase Characteristics and Engineering
### Lipase Engineering
At higher temperatures, it was possible to observe the lid opening by the mutants Asp250 Val and Glu254 Leu, suggesting the important role of these residues in holding the lid in closed conformation, which can affect the activity of this enzyme. A computational design scheme based on MD simulations was also applied to improve the thermal stability of the lipase LIP2 from _Y. lipolytica_[117]. Based on the four structural parameters RMSD, Rg, SASA and the number of internal hydrogen bonds, it was found that the V213P mutant would have higher thermal stability than its WT parent. V213P also has an optimal temperature of 42 \({}^{\circ}\)C, which is 5.0 \({}^{\circ}\)C higher than that of the wild type. Directed evolution (also called molecular evolution) does not require that the amino acid sequence and three-dimensional structure should be previously available, as random mutagenesis generates libraries with random mutations in the gene of interest. Those random mutations can be induced by error-prone PCR, chemicals, irradiation, DNA shuffling or staggered extension process (StEP). Owing to the high number of possible clones and enzyme variants, it is necessary to conduct high-throughput and time-consuming screening for the selection of the best enzyme variants created. Besides rational design and directed evolution, there is a third approach for engineering an enzyme to acquire better properties. Semi rational design is the combination of the former two approaches. It can be summarized as the generation of libraries of mutant genes via the saturation mutagenesis method. In this technique, one or a few amino acid residues are replaced by the other 19 amino acid possibilities through degenerate codons. Semi rational design has been proven to be a rapid laboratory method for engineering proteins, once a relatively smaller size of libraries are generated, facilitating the further screening and selection step [50; 118; 119]. Curiously, [PERSON] and collaborators [76] scrutinized how many changes are necessary to transform a non-attractive lipase into an enzyme with better properties and potential for use in industrial applications. Lip3-encoding gene from _Drosophila melanogaster_ was cloned into the vector pETMCSIII and expressed in the _E. coli_ BL21 (DE3) system. Concomitantly, error-prone PCR was used to generate libraries of mutant variants harboring random mutations along the lipase gene. After seven rounds of directed evolution, screening and selection, only five mutant lipases stood out from tens of hundreds of lipase variants. The best variant, R7_59A, had eight substitutions in its sequence compared to the WT lipase. The lipase R7_59A showed an activity 4, 5, 6 and 12 fold higher than WT towards coconut oil, tributyrin, glyceryl trioctanoate, and glyceryl trioleate substrates, respectively. Using crude lysate, there was an improvement of 228 times in the 57_59A activity against pNP-C8 compared to the WT. Furthermore, the 57_59A mutant had a 6.3, 1.5, 7.8 and 2.6 fold increment in the activity against pNP-C3, pNP-C8, pNP-C16 and pNP-C18 substrates compared to WT. The induced mutations in the best five variants were present in amino acid residues related to the cap domain stability, residues near to the catalytic triad and in residues of the hinge points of the cap domain. According to the study, those lipase variants could be targeted in further evolutions aiming to use them in detergent formulation [76].