## 5 Immobilized Lipase
### Magnetic Nanocarriers  
Materials like magnetite (Fe\({}_{3}\)O\({}_{4}\)) are used as support for immobilization because they allow a rapid separation from the reaction medium when an external magnetic field is applied [116]. The development of magnetic nanoparticles (MNPs) as a support for enzymes immobilization has been recently reviewed [117]. Typical magnetic nanomaterials include iron oxide (Fe\({}_{3}\)O\({}_{4}\) and \(\gamma\)-Fe\({}_{2}\)O\({}_{3}\)), alloy-based (CoPt\({}_{3}\) and FePt), pure metal (Fe and Co), and spinel-type ferromagnet (MgFe\({}_{2}\)O\({}_{4}\), MnFe\({}_{2}\)O\({}_{4}\), and CoFe\({}_{2}\)O\({}_{4}\)) [117]. Examples of lipase immobilized in various types of magnetic nanoparticles are shown in Table 4.  
Magnetic nanoparticles (MNPs) have good biocompatibility and non-toxicity but tend to aggregate and oxidize, so they need to be functionalized on the surface and use a cross-linking agent to bind the enzyme. One way is by using silica coating where a silica shell is formed on the surface by using amino-functional reagents like 3-aminopropyl triethoxysilane (APTES). Fe\({}_{3}\)O\({}_{4}\) particles were encapsulated with mesoporous silicon and modified with APTES or 3-mercaptoethanol trimethoxysilane (MPTMS) followed by binding of the lipase with glutaraldehyde. APTES-modified Fe\({}_{3}\)O\({}_{4}\) particles were reported to give better yield of biodiesel (90%) than MPTMS particles [75]. _Burkholderria_ sp. lipase on Fe\({}_{3}\)O\({}_{4}\) MNPs also achieved 90% conversion [123].  
Another way to protect MNPs is with organic polymers, including synthetic polymers and biopolymers. The polymer can be either incorporated into the precursor solution to form Fe\({}_{3}\)O\({}_{4}\) MNPs  
\begin{table}
\begin{tabular}{l l l l l l} \hline \hline
**Lipase Source** & **Carrier** & **Immobilization** & **Yield \({}^{\circ}\) (\%)** & **No. Cycles** & **References** \\ \hline _B. capsaicin_ & Silica coated Hydroxyapatite and glutaraldehydehyde & Encapsulation & 98 & 4 & [118] \\ _A. terreus_ & Iron oxide polydopamine & Covalent bonding & 92 & 7 & [64] \\ _T. Lungiensis \&_ & Core-shell structured iron oxide & Covalent bonding & 99 & 11 & [119] \\ _C. Antarctica_ & Co\({}^{2+}\) chelated, with & & & & \\ _P. fluorescens_ & 3-glycidoxymethyltrimethoxysilane & Adsorption & 95 & 10 & [70] \\ _R. micheli and_ & Silica core shell iron oxide with & & & & \\ _T. Lunginsma_ & & tryethylamine & Covalent bonding & 93.1 & 5 & [60] \\ _C. Antarctica_ & Poly(urea-urethane) encapsulated & & Encapsulation & 95 & 8 & [120] \\ _A. terreus_ & Magnetic iron oxide with & & & & \\ _C. Antarctica_ & 1-Butyl-3-methylimidazolium & & & & \\ _C. Antarctica_ & & tetrafluoroplate \&_ & & & \\ _S.aminopropyl_firethoxysilane & & & & & \\ _B. capsaicin_ & Polysiloxane–polyvinyl alcohol & & & & \\ hybrid magnetic-polymer composite & & Covalent bonding & 96.5 & - & [122] \\ \hline \hline \end{tabular}  
* Biodiesel yield (wt/wt.%) was determined as the methyl esters amount produced by the lipases in the reaction process divided by the initial amount of esters.  
\end{table}
Table 4: Biodiesel production with various immobilized lipases using magnetic nanoparticles as carriers.  
or externally to create the core shell [117]. The polymer surface provides numerous functional groups that facilitate the enzyme binding. Magnetic chitosan binds the lipase by covalent attachment [124].  
Separation of nanobiocatalysts is difficult in an oily system [119] but the magnetic properties of MNPs can facilitate the separation of enzyme from reaction media. In this way, the reaction may be immediately finished as well as using the enzyme for further uses [117].