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Lipases are the most widely used enzymes in biocatalysis, and the most utilized method for enzyme immobilization is using hydrophobic supports at low ionic strength. This method allows the one step immobilization, purification, stabilization, and hyperactivation of lipases, and that is the main cause of their popularity. This review focuses on these lipase immobilization supports. First, the advantages of these supports for lipase immobilization will be presented and the likeliest immobilization mechanism (interfacial activation on the support surface) will be revised. Then, its main shortcoming will be discussed: enzyme desorption under certain conditions (such as high temperature, presence of cosolvents or detergent molecules). Methods to overcome this problem include physical or chemical crosslinking of the immobilized enzyme molecules or using heterofunctional supports. Thus, supports containing hydrophobic acyl chain plus epoxy, glutaraldehyde, ionic, vinylsulfone or glyoxyl groups have been designed. This prevents enzyme desorption and improved enzyme stability, but it may have some limitations, that will be discussed and some additional solutions will be proposed (e.g., chemical amination of the enzyme to have a full covalent enzyme-support reaction). These immobilized lipases may be subject to unfolding and refolding strategies to reactivate inactivated enzymes. Finally, these biocatalysts have been used in new strategies for enzyme coimmobilization, where the most stable enzyme could be reutilized after desorption of the least stable one after its inactivation. ; We gratefully recognize the financial support from MINECO-Spanish Government (project number CTQ2017-86170-R), Colciencias (Colombia, project number FP 44842-076-2016), Colciencias, Ministerio de Educación Nacional, Ministerio de Industria, Comercio y Turismo e ICETEX, Convocatoria Ecosistema Científico – Colombia Científica. Fondo Francisco José de Caldas, Contract RC-FP44842-212-2018, Generalitat Valenciana (PROMETEO/2018/076), FAPERGS (project number 17/2551-0000939-8), FUNCAP (project number BP3-0139-00005.01.00/18) and CONACYT (Mexico, project number CB-2016-01, 286992).

Eversa is an enzyme recently launched by Novozymes to be used in a free form as biocatalyst in biodiesel production. This paper shows for first time the immobilization of Eversa (a commercial lipase) on octyl and aminated agarose beads and the comparison of the enzyme properties to those of the most used lipase, the isoform B from Candida antarctica (CALB) immobilized on octyl agarose beads. Immobilization on octyl and aminated supports of Eversa has not had a significant effect on enzyme activity versus p-nitrophenyl butyrate (pNPB) under standard conditions (pH 7), but immobilization on octyl agarose beads greatly enhanced the stability of the enzyme under all studied conditions, much more than immobilization on aminated support. Octyl-Eversa was much more stable than octyl-CALB at pH 9, but it was less stable at pH 5. In the presence of 90% acetonitrile or dioxane, octyl-Eversa maintained the activity (even increased the activity) after 45 days of incubation in a similar way to octyl-CALB, but in 90% of methanol, results are much worse, and octyl-CALB became much more stable than Eversa. Coating with PEI has not a clear effect on octyl-Eversa stability, although it affected enzyme specificity and activity response to the changes in the pH. Eversa immobilized octyl supports was more active than CALB versus triacetin or pNPB, but much less active versus methyl mandelate esters. On the other hand, Eversa specificity and response to changes in the medium were greatly modulated by the immobilization protocol or by the coating of the immobilized enzyme with PEI. Thus, Eversa may be a promising biocatalyst for many processes different to the biodiesel production and its properties may be greatly improved following a suitable immobilization protocol, and in some cases is more stable and active than CALB. ; We gratefully recognize the support from the MINECO from Spanish Government, (project number CTQ2017-86170-R) and Colciencias (Colombia) (project number FP 44842-076-2016). ; Peer reviewed

The effect of an additive on enzyme stability used to be considered an intrinsic feature of a lipase. However, in this paper we have found that the effect of additive on enzyme stability depends on the immobilization protocol. After assaying the effects of diverse chloride salts with different cations on different lipases activity, no relevant effect was detected. Free enzymes or the covalently immobilized enzymes are not stabilized by these cations for any of the studied lipases. However, Mn2+ and Ca2+ (at a concentration of 5 mM) are able to greatly stabilize the lipases from Rhizomucor miehei (RML) and Candida rugosa (CRL) when they are present during the inactivation, but only if the enzymes are immobilized on octyl-agarose (stabilization factor ranging from 20 to 50). The effect was only detected when using more than 2.5 mM of the cations, and reached the maximum value at 5 mM, suggesting a saturation mechanism of action. The stabilization seemed to be based on a specific mechanism, and required the recognition sites to be saturated by the cations. Mg2+ has no effect on enzyme stability for both enzymes, but it is able to suppress the stabilization promoted by the other two cations using CRL; while it has no effect on the cation stabilization when using RML. This is the first report of a cation induced enzyme stabilization effect that depends on the lipase immobilization protocol. ; We gratefully recognize the support from the MINECO from Spanish Government, (project number CTQ2013-41507-R). The predoctoral fellowships for Ms Rueda (Colciencias, Colombian Government and Becas Iberoamérica "Jóvenes Investigadores", Banco Santander) and for Ms Rodríguez (CONICET and SPU, Argentine Government) and Dr dos Santos (CNPq, Brazil) are also recognized. ; Peer Reviewed

A commercial and very hydrophobic styrene-divinylbenzene matrix, MCI GEL® CHP20P, has been compared to octyl-Sepharose® beads as support to immobilize three different enzymes: lipases from Thermomyces lanuginosus (TLL) and from Rhizomucor miehie (RML) and Lecitase ® Ultra, a commercial artificial phospholipase. The immobilization mechanism on both supports was similar: interfacial activation of the enzymes versus the hydrophobic surface of the supports. Immobilization rate and loading capacity is much higher using MCI GEL® CHP20P compared to octyl-Sepharose® (87.2 mg protein/g of support using TLL, 310 mg/g using RML and 180 mg/g using Lecitase® Ultra). The thermal stability of all new preparations is much lower than that of the standard octyl-Sepharose® immobilized preparations, while the opposite occurs when the inactivations were performed in the presence of organic co-solvents. Regarding the hydrolytic activities, the results were strongly dependent on the substrate and pH of measurement. Octyl-Sepharose ® immobilized enzymes were more active versus p-NPB than the enzymes immobilized on MCI GEL® CHP20P, while RML became 700-fold less active versus methyl phenylacetate. Thus, the immobilization of a lipase on this matrix needs to be empirically evaluated, since it may present very positive effects in some cases while in other cases it may have very negative ones. © 2014 by the authors. ; We gratefully recognize the support from the Spanish Government, grant CTQ2009-07568 and CTQ2013-41507-R and CNPq (Brazil). The predoctoral fellowships for García-Galán (Spanish Government) and dos Santos (CNPq, Brazil) are also recognized. The authors wish to thank Ramiro Martínez (Novozymes, Spain) for kindly supplying the enzymes used in this research. The help and comments from Ángel Berenguer (Instituto de Materiales, Universidad de Alicante) are kindly acknowledged. We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI) ; Peer Reviewed

Two different heterofunctional octyl-amino supports have been prepared using ethylenediamine and hexylendiamine (OCEDA and OCHDA) and utilized to immobilize five lipases (lipases A (CALA) and B (CALB) from Candida antarctica , lipases from Thermomyces lanuginosus (TLL), from Rhizomucor miehei (RML) and from Candida rugosa (CRL) and the phospholipase Lecitase Ultra (LU). Using pH 5 and 50 mM sodium acetate, the immobilizations proceeded via interfacial activation on the octyl layer, after some ionic bridges were established. These supports did not release enzyme when incubated at Triton X-100 concentrations that released all enzyme molecules from the octyl support. The octyl support produced significant enzyme hyperactivation, except for CALB. However, the activities of the immobilized enzymes were usually slightly higher using the new supports than the octyl ones. Thermal and solvent stabilities of LU and TLL were significantly improved compared to the OC counterparts, while in the other enzymes the stability decreased in most cases (depending on the pH value). As a general rule, OCEDA had lower negative effects on the stability of the immobilized enzymes than OCHDA and while in solvent inactivation the enzyme molecules remained attached to the support using the new supports and were released using monofunctional octyl supports, in thermal inactivations this only occurred in certain cases. ; We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI). ; We gratefully recognize the support from the MINECO from Spanish Government, (project number CTQ2013-41507-R). The predoctoral fellowships for Rueda (Colciencias, Colombian Government and Becas Iberoamérica "Jóvenes Investigadores", Banco Santander) and dos Santos and Albuquerque (CNPq, Brazil) are also recognized. The authors wish to thank Ramiro Martínez (Novozymes, Spain) for kindly supplying some of the enzymes used in this research.

Novozym 435 (N435) is a commercially available immobilized lipase produced by Novozymes. It is based on immobilization via interfacial activation of lipase B from Candida antarctica on a resin, Lewatit VP OC 1600. This resin is a macroporous support formed by poly(methyl methacrylate) crosslinked with divinylbenzene. N435 is perhaps the most widely used commercial biocatalyst in both academy and industry. Here, we review some of the success stories of N435 (in chemistry, energy and lipid manipulation), but we focus on some of the problems that the use of this biocatalyst may generate. Some of these problems are just based on the mechanism of immobilization (interfacial activation) that may facilitate enzyme desorption under certain conditions. Other problems are specific to the support: mechanical fragility, moderate hydrophilicity that permits the accumulation of hydrophilic compounds (e.g., water or glycerin) and the most critical one, support dissolution in some organic media. Finally, some solutions (N435 coating with silicone, enzyme physical or chemical crosslinking, and use of alternative supports) are proposed. However, the N435 history, even with these problems, may continue in the coming future due to its very good properties if some simpler alternative biocatalysts are not developed. ; We gratefully recognize the financial support from MINECO from the Spanish Government (project number CTQ2017-86170-R, Colciencias, Ministerio de Educación Nacional, Ministerio de Industria, Comercio y Turismo e ICETEX, Convocatoria Ecosistema Científico – Colombia Científica. Fondo Francisco José de Caldas, Contrato RC-FP44842-212-2018 and Colciencias (Colombia) (project number FP44842-076-2016), Generalitat Valenciana (PROMETEO/2018/076), FAPERGS (project number 17/2551-0000939-8), CONICET (R. Argentina), FUNCAP (project number BP3-0139-00005.01.00/18) and ANPCyT (PICT 2015-0932 and PICT CABBIO 4687).

Supports based on poly-styrene-divinylbenzene (PSD) are commercially available since a long time ago. However, they are not commonly used as enzyme immobilization matrices. The main reason for this lies in the negative effect of the very hydrophobic surface on enzyme stability that produces the instantaneous enzyme inactivation in many instances. However, they have recently regained some impact in enzyme immobilization. They are easy to modify, and have been prepared with different modifiers. We will pay special attention to the coating of these supports with ionic liquids, which permits to have the ionic liquid phase anchored to the solid and modulate the enzyme properties without risk of losing these expensive and potentially toxic compounds. Thus, this review will present the covalent or physical immobilization of enzymes on PSD supports, submitted to different modifications. Moreover, lipases immobilized via interfacial activation on some naked PSD supports have shown some unexpected improvement in their catalytic properties, with uses in reactions like hydrolysis, esterification or transesterification. ; We gratefully recognize the support from the Spanish Government, CTQ2013-41507-R and CNPq (Brazil). The predoctoral fellowships for Ms. García-Galán (Spanish Government), Mr K. Hernandez (I3P-CSIC) and Mr dos Santos (CNPq, Brazil) are also recognized. ). Á. Berenguer-Murcia thanks the Spanish Ministerio de Ciencia e Innovacion for a Ramon y Cajal fellowship (RyC-2009-03813).

A new heterofunctional support, octyl-glyoxyl agarose, is proposed in this study. The supports were prepared by simple periodate oxidation of the commercial octyl-agarose, introducing 25 μmol of glyoxyl groups per wet gram of support. This support was assayed with three different lipases (those from Candida antarctica (form B), Thermomyces lanuginosus (TLL) or Rhizomucor miehei) and the artificial phospholipase Lecitase Ultra. Used at pH 7, the new support maintained as first immobilization step the lipase interfacial activation. Thus, it was possible to have the purification and immobilization of the enzyme in one step. Moreover, stabilization of the open form of the lipase was achieved. The covalent enzyme/support bonds cannot be obtained if the immobilized enzyme was not incubated at alkaline pH value. This incubation at pH 10 of the previously immobilized enzymes produced a smaller decrease in enzyme activity when compared to the direct immobilization of the enzymes on glyoxyl-agarose at pH 10, because the immobilization via interfacial activation promoted a stabilization of the lipases. Except in the case of TLL (covalent attachment involved 70% of the enzyme molecules), covalent immobilization yield was over 80%. The non-covalent attached enzyme molecules were discarded by washings with detergent solutions and the new biocatalysts were compared to the octyl-agarose immobilized enzymes. While the stability in thermal and organic solvents inactivations was increased for Lecitase Ultra, CALB and RML, TLL improved its stability in organic media but its thermal stability decreased after covalent attachment of the interfacially activated enzyme. This stabilization resulted in octyl-glyoxyl-lipase preparations which presented higher activity in the presence of organic solvents. Finally, while octyl-agarose released enzyme molecules after incubation at high temperatures or in the presence of organic solvents and detergents, the covalently immobilized enzyme remained attached to the support even after boiling the enzyme in SDS, eliminating the risks of product contamination. ; We gratefully recognize the support from the MINECO of Spanish Government, CTQ2013-41507-R. The predoctoral fellowships for Ms. Rueda (Colciencia, Colombian Goberment) and Mr dos Santos (CNPq, Brazil) are also recognized. ; Peer Reviewed