## 2 Production of Lipases
### Production of Recombinant Lipases
#### 2.2.2 Eukaryotic Expression Systems  
The main studied and used eukaryotic expression systems are the yeasts _K. phaffii, S. cerevisiae, Y. lipolytica_, and the species of filamentous fungus _Aspergillus_ that represent nearly 35, 8, 1 and 3% of all heterologously produced lipases, respectively [42]. These microorganisms are not only efficient producers, but some have the status of being generally recognized as safe (GRAS) (_S. cerevisiae, Y. lipolytica, A. niger_ and _A. oryzae_). Proteins produced in eukaryotic cells can undergo a series of PTMs that can affect their chemical and physical properties [82]. Such modifications can increase protein solubility, stability and secretion rate. In industry, fungal lipases have gained attention because of their stability under different conditions and substrate specificity. Moreover, extraction procedures are simpler in comparison to bacterial lipases due to their extracellular character [34]. Table 2 summarizes different eukaryotic sources of lipases, expression systems and properties. \begin{table}
\begin{tabular}{c c c c c c c c c c c} \hline \hline
**Enzyme** & **Enzyme Source** & \begin{tabular}{c} **Expression Vector** \\ **(Expression Host)** \\ \end{tabular}  & \begin{tabular}{c} **Cloning Vector** \\ **(Recipient Strain)** \\ \end{tabular}  & \begin{tabular}{c} **Expression** \\ **Vector** \\ \end{tabular}  & \begin{tabular}{c} **Molecular** \\ **Mass (GDs)** \\ \end{tabular}  & \begin{tabular}{c} **Optimal** \\ **Temperature** \\ **and pH** \\ \end{tabular}  & \begin{tabular}{c} **Thermal** \\ **Stability** \\ \end{tabular}  & **Additional Remarks** & **Ref. ** \\ \hline \multirow{4}{*}{Lipses \ \begin{tabular}{} \\ MAS1 \\ \end{tabular}  } &
\begin{tabular}{c} Marine \\ _Surg  
\begin{table}
\begin{tabular}{c c c c c c c c c c} \hline \hline
**Eaxyme** & **Enzyme Source** & \begin{tabular}{c} **Expression Vector** \\ **(Expression Host)** \\ \end{tabular}  & \begin{tabular}{c} **Cloning Vector** \\ **(Recipient Serial)** \\ \end{tabular}  & \begin{tabular}{c} **Expression** \\ **Vector** \\ \end{tabular}  & \begin{tabular}{c} **Molecular** \\ **Mass (Q.Da)** \\ \end{tabular}  & \begin{tabular}{c} **Optimal** \\ **Temperature** \\ **and pH** \\ \end{tabular}  & \begin{tabular}{c} **Thermal** \\ **Stability** \\ \end{tabular}  & \begin{tabular}{c} **Additional Remarks** \\ **Ref.** \\ \end{tabular}  \\ \hline FSL & _Fasarium total_ & \(K\), phfil _K33_ & \begin{tabular}{c} pPKCZaA and pPKZaA (_c.c._ col) \\ \end{tabular}  & \ \begin{tabular}{c} pPKCZaA-FSL \\ **and** \\ \end{tabular}  & 30 & 35 \({}^{\circ}\)C, _70_ &
\begin{tabular}{c} Remaining \\ activity of 90\  
_K. phaffii_ is a methylotrophic yeast, which is able to express and produce lipases under the control of methanol-responsive alcohol oxidase promoters. It naturally secretes low amounts of proteins in the extracellular medium, but high levels of secreted recombinant lipase can be achieved using methanol as an inducer. This special characteristic makes the lipase purification step easier and cost-effective. In addition, _K. phaffii_ has a number of advantages in comparison to higher eukaryotes. It is a single-celled microorganism, which is easy to genome engineer and it only requires simple culture conditions. It performs many PTM modifications typical for higher eukaryotes, such as disulfide bond formation, glycosylation, proteolytic processing and appropriate folding. Consequently, it can result in a higher degree of biological active lipases in comparison to bacteria [93]. Compared to _S. cerevisiae_, by using _K. phaffii_ as the production host, the enzyme can be produced in the intracellular space, secreted to the extracellular broth or displayed at cell surface with a reduced hyperglycemia level [42; 43; 50]. Moreover, in comparison to _S. cerevisiae_, _K. phaffii_ can be produced in higher cell densities than _S. cerevisiae_, resulting in a higher protein productivity. _K. phaffii_ also has low nutritional requirements because it is adapted to catabolize raw carbon and nitrogen sources.