## 1 Introduction  
Microbial lipases have been extensively applied in several biotechnological processes including biofuel production and food biotechnology [1; 2; 3; 4; 5]. As with most enzymes, price is still a disadvantage for its industrial implementation when compared with conventional--but less green--catalysts [6; 7]. Among other complementary strategies, lipase immobilization on mesoporous supports has been probed as an efficient tool to solve such limitations by allowing enzyme re-use and stabilization [6; 8; 9]. Applications of immobilized lipases include production of cocoa butter substitute; production of bioactive compounds, flavors, and fragrances; and enantioselective resolutions. The most used immobilized lipases are Lipozyme RM IM(r), Lipozyme TL IM(r), and Novozyme(r) 435 [10; 11; 12; 13]. The latter, based on CAL B (_Candida antarctica_ lipase B) immobilized on the Lewati(r) VP OC 1600 support, is a paradigmatic example of how to obtain a biotechnological solution for the industrial production of biodiesel from vegetable oil [7; 14; 15]. Immodilization on Glyoxyl-agarose support (Gx) is recognized as one of the best strategies to stabilize enzymes, surpassing not only other immobilization approaches but also tools derived from molecular biology [16; 17]. In some cases, optimal Gx enzyme derivatives can be up to 60,000-fold more stable than the free enzyme under very harsh conditions such as in high temperatures (>60 \({}^{\circ}\)C)or in concentrated inactivating organic solvents (\(>\)50% \(v/v\)) and, eventually, reactivation can be achieved [18; 19]. Thus, immobilization on Gx provides an enormous application potential for white biotechnology especially under non-conventional reaction conditions [6; 20]. However, immobilization on Gx cannot be performed at neutral pH when most enzymes are stable, constituting one of its crucial disadvantages [21]. Such a limitation relies essentially on: (i) the reversible nature of the imine bond formed between the enzyme and the support; (ii) the fact that equilibrium is shifted towards reactants (imine bond hydrolysis) since the immobilization media are essentially water; and (iii) the low reactivity at pH 7.0 of nucleophilic (primary amine) enzyme groups against the aldehyde groups of the Gx support [17; 20]. Hence, the probability of immobilization on Gx increases in pH conditions where more enzyme nucleophilic groups are able to establish bonds with the support [17; 22]. Since the most abundant enzyme nucleophilic is epsilon-NH\({}_{2}\) (pK\({}_{4}\) 10.5) [16; 17; 20; 23], immobilizations on Gx are usually performed at pH 10 or above. This implies that the applicability of Gx, with few exceptions (e.g., some multimeric proteins [24; 25; 26]), is limited to enzymes that must be not only rich in lysyl residues but also alkaline-tolerant [17; 20; 23]. Different approaches have been proposed to partially overcome such a limitation by increasing the reactivity of the enzymes with Gx at pHs lower than 10: (i) The chemical animation of enzymes with ethylenediamine in addition to increasing the number of available protein primary amine groups. This allows a decrease in the immobilization pH on Gx to 9.0, as the introduced chemical groups have a lower pKa than epsilon-NH\({}_{2}\) (9.2 and 10.5, respectively) [27; 28]. However, it cannot be applied to proteins which are unstable during the amination process or at pH \(\geq\) 9.0, such as those applied during the immobilization of the resulting aminated enzymes [22; 29]. (ii) The addition of reducing agents such as cyanoborohydride or aminoboranes in some cases decrease the immobilization pH on Gx below 9.0 by reducing the initial reversible enzyme-Gx imine bonds to irreversible secondary amino bonds.