## 4 Relevant Immobilization Methods for Recombinant Lipases  
The use of enzymes in various industrial areas has significantly increased due to several associated benefits. However, some factors, such as low enzymatic stability, may limit the use of lipases and other industrial enzymes [120; 121]. One of the most important and widely used techniques to improve enzyme properties is their immobilization, in which the biocatalysts are attached to an inert support. The immobilization of enzymes provides several benefits, such as (1) the production of efficient and stable biocatalysts; (2) possibility of recovery and reusability of enzymes [122; 123]; (3) easy purification of the products when compared to the free enzymes; (4) increasing the efficiency; (5) permitting their applications in continuous fixed-bed operation [122]. The immobilization methods can be separated into two classes--namely, the physical and chemical methods. Physical methods include support and enzyme interactions of different types, including hydrogen bonds and hydrophobic interactions. In the latter method, the formation of covalent bonds can be achieved through amide, ether, thio-ether or carbamate bonds between the support and the enzyme [124; 125; 126]. The physical methods specifically include entrapment and adsorption, while chemical methods include cross-linking and covalent binding. Different enzyme immobilization methods are grouped and listed in Figure 3.  
The immobilization of an enzyme by adsorption is a simple technique with high commercial value due to its simplicity, regular use in large-scale processes, low cost, retention of high enzyme activity, and relatively chemical-free enzyme binding [127]. Supports with different degrees of hydrophobicity were previously used such as butyl Sepabeads and octadecyl Sepabeads [126], decaoctil-Sepabeads [128], macroporous resin HPD826 [129], polypropylene powder [124; 130; 131] and pore-expanded mesoporous silica (SBA-15) [132]. The use of hydrophobic supports is of particular interest, because these supports mimic the enzymes' natural media and can often promote hyperactivation, highly selective adsorption, purification, increased enantioselectivity, and strong but reversible immobilization, making support reuse possible after the enzyme has been deactivated [133; 134]. Figure 3: Different methods of enzyme immobilization. Hydrophobic core-shell polymeric supports, including Accurel, PMMA/PMMA (poly(methyl methacrylate) on core and shell) and PMMA-co-DVB/PMMA-coDVB (methyl methacrylate copolymerized with divinylbenzene on core and shell) obtained by a combined suspension and emulsion polymerization process have demonstrated great potential in the immobilization of a recombinant _C. antarctica_ lipase [135]. This technique involves the lipase interfacial activation versus the hydrophobic surface of the support, which is the main cause of the lipase improved activity and stabilization [135; 136]. One example of commercial lipase immobilized on a hydrophobic support is Novozym 435 (N435), supplied by Novozymes. The enzyme is expressed in _A. niger_ and produced by interfacial activation of lipase B from _C. antarctica_ on the resin, Lewatit VP OC 1600. This resin is a macroporous support formed by poly(methyl methacrylate) crosslinked with divinylbenzene. N435 is considered one of the most widely used commercial biocatalysts in both academy and industry [137]. In physical adsorption, enzyme leaching is a critical problem that limits the use of the immobilized enzyme in different reaction conditions [138]. To solve this problem, entrapment is employed to restrict the enzyme in the polymer frameworks [139]. In the entrapment method, the enzyme is locked inside the lattice of a polymer matrix or membrane so that it is retained while allowing the penetration of the substrate. That can be achieved with a variety of materials, including polymers, sol-gels, polymer/sol-gel composites and other inorganic materials.