## 4 Relevant Immobilization Methods for Recombinant Lipases
It has been observed that the entrapment of lipases in silica sol-gels increases thermal stability and enhances reaction rates by several orders of magnitude [140]. The entrapment of CALB was successfully achieved with poly 1-vinyl-3-ethylimidazolium bromide (VEImBr) [139], and the immobilization of the whole cells or cell lysates of _E. coli_, producing a thermostable lipase from _T. languinos_ (r_E.coli_/lip), was also reported using silica xerogel and nanocarbon in silica composites [141]. For chemical methods, the enzyme is firmly immobilized on the chemically modified support through covalent binding and cross-linking, effectively preventing enzyme leakage. Cross-linking is an improvement on the covalent attachment because the enzyme is cross-linked to the support with the help of a cross-linker. The most commonly used cross-linking reagent is glutaraldehyde, as it is economical and easily obtainable in large quantities [142]. CALB was immobilized by covalent binding on strongly hydrophobic polystyrene microspheres (poly glycidyl methacrylate) activated by epoxy groups, and this immobilization has been proven to play a key role in improving the properties of the lipase [143]. The carrier hollow silica microspheres (MAT540 TM) activated by six bisepoxides enclosing different spacers was applied for the immobilization of CALB. The immobilized preparation was used in the kinetic resolution of racemic 1-phenylethanol and five racemic amines using shaken flasks and continuous-flow packed-bed microreactors with good yields and high enantiomeric excess (ee \(>\) 99%, for all) [144]. Furthermore, the recombinant CALB expressed in _K. phaffii_ was immobilized by cross-linked enzyme aggregate (CLEA) technology and tested for the synthesis of olvanil [145]. A recombinant lipase from _Thermus thermophilus_ WL expressed in _E. coli_ was immobilized onto the surface, being rich in the amine functional groups (-NH\({}_{2}\)), of the 3-APTES-modified Fe\({}_{3}\)O\({}_{4}\)@SiO2 nanoparticles [125]. The covalent bond on the aldehyde activated-agarose in the presence of DTT (glyoxyl-DTT-agarose) at pH 7.0 and on glyoxyl-agarose at pH 10.2 was applied for the immobilization of the lipase from Archaeon _Pyrococcus furiosus_ (Pf2001) produced in _E. coli_ B21 [126]. In the last decade, applications of new materials for the immobilization of lipases have drawn special interest. These new material groups include magnetized nano-sized materials [146; 147], which provide large surface areas for the attachment of the enzyme and increase the probability of its efficient activity. By using this technique, the improvement of the downstream processing is clear as it facilitates the immobilized enzyme separation from the reaction mixture. [PERSON] et al. [147] immobilized a recombinant _Pseudomonas aeruginosa_ lipase through surface conjugation with gold nanoparticles and observed higher lipolytic activity in theoconjugate compared to the free enzyme. [PERSON] et al. [148] and [PERSON] et al. [149] immobilized a _Candida rugosa_ lipase onto the silica nanoparticles surface and glutaraldehyde-activated aminopropyl glass beads, resulting in easy recovery, and enzyme reuse for ester synthesis. In this context, when _Candida antarctica_ lipase was immobilized on chitosan-coated magnetic nanoparticles of iron activated with glutaraldehyde for the production of the biolubricant ester, the biocatalyst gave half of the initial conversion after seven cycles of esterification [150]. Another special area of interest has been the applications of hybrid and composite materials for lipase immobilization. The hybrid origin materials are synthesized from combinations of precursors of inorganic, organic and mixed inorganic and organic origin and offer high stability, good affinity for enzymes and the presence of many chemical functional groups compatible with the chemical moieties present in the protein structure [133].