## 2 Production of Lipases
### Production of Recombinant Lipases
#### 2.2.1 Prokaryotic Expression Systems  
_E. coli_ is by far the most used prokaryotic microorganism for recombinant production of lipases, followed by the Gram-positive soil bacterium _B. subtilis_. Hence, according to [PERSON] and [PERSON] [42], _E. coli_ represents 50% of the total heterologous production of lipases by eukaryotic and prokaryotic hosts. This dominant position in the production hierarchy has been earned by the more than 40 years of intensive use as production host and the even longer record of serving as one of the most important biological model organisms. As a result, _E. coli_ is likely the best understood organism on the planet with a superior genetic toolbox and profound insights into its genetics and physiology. Together, these advantages have been used to construct highly efficient _E. coli_ based cell factories for heterologous protein production, and there are examples where the new protein constitutes 30% of the total intracellular content of proteins. Moreover, _E. coli_ based processes are typically easy to manipulate and scale-up due to consolidated methodologies of fermentation, cloning and expression with different strains, protocols and studies available. Many strategies are available to optimize a production process. For example, in the context of recombinant lipases, purification can be simplified by fusing the lipase gene to sequences encoding signal peptides (PelB and OmpA) as they will direct the proteins to the periplasmic space. Moreover, yields of soluble protein may be increased by co-expression with chaperones (GroES, GroEL and ClpB). In the first case, the enzyme could be recovered by ultrasonication or other cell lysis processes. The co-expression with chaperones enables the correct folding of the lipase, increases its solubility and decreases protein aggregation. Molecular chaperones are included in a family of unrelated classes of protein that mediate the correct assembly of other polypeptides--i.e., correct folding [66]. The major limitations of using _E. coli_ as an expression host include its inability to secrete large amounts of proteins or to perform post-translational modifications (PTMs)--i.e., protein glycosylation. However, _E. coli_ is a suitable host organism for bacterial lipases, such as from the genera _Pseudomonas, Bacillus, Staphylococcus, Serratia, Burkholeria_, and from metagenomic libraries. Furthermore, _E. coli_ has been explored in studies of the biochemical characterization of lipases produced by previously identified microorganisms [63; 67] or uncultured strains discovered by metagenomics [68; 69]. Table 1 shows different lipase sources, prokaryotic expression systems and lipase properties. Figure 1: Sequential approach used for recombinant lipase production. Commercial lipases are mostly obtained from mesophilic organisms that grow at temperatures between 20 and 45 \({}^{\circ}\)C. However, extremophile microorganisms--psychrophiles (<20 \({}^{\circ}\)C) and thermophiles (>40 \({}^{\circ}\)C)--have drawn attention as natural lipase sources due to the outstanding properties of their enzymes, especially related to thermal stability, pH and tolerance to high salt concentrations [44; 70]. These lipases might be identified and characterized through the construction of metagenomics libraries, and heterologous expression, followed by screening. Activity assays are also conducted in the presence of detergents, organic solvents and enzyme inhibitors to evaluate their industrial potential as biocatalysts. [PERSON] and collaborators [68], for example, have investigated the metagenomics of samples harvested from hot springs (Table 1). After the selection of the best clones, the lipase RK-lip479 was cloned and expressed in the strains _E. coli_ DH5\(\alpha\) and _E. coli_ BL21 (DE3), respectively. The lipase was expressed intracellularly, had maximum activity at 65 \({}^{\circ}\)C and pH 8.0, and maintained 89, 92 and 60% of its activity after 6 h at 55, 65 and 75 \({}^{\circ}\)C [68]. Similarly, the thermostable lipase BaG7 Lip from _B.