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The use of enzymes in industrial processes requires the improvement of their features in many instances. Enzyme immobilization, a requirement to facilitate the recovery and reuse of these water-soluble catalysts, is one of the tools that researchers may utilize to improve many of their properties. This review is focused on how enzyme immobilization may improve enzyme stability. Starting from the stabilization effects that an enzyme may experience by the mere fact of being inside a solid particle, we detail other possibilities to stabilize enzymes: generation of favorable enzyme environments, prevention of enzyme subunit dissociation in multimeric enzymes, generation of more stable enzyme conformations, or enzyme rigidification via multipoint covalent attachment. In this last point, we will discuss the features of an "ideal" immobilization protocol to maximize the intensity of the enzyme-support interactions. The most interesting active groups in the support (glutaraldehyde, epoxide, glyoxyl and vinyl sulfone) will be also presented, discussing their main properties and uses. Some instances in which the number of enzyme-support bonds is not directly related to a higher stabilization will be also presented. Finally, the possibility of coupling site-directed mutagenesis or chemical modification to get a more intense multipoint covalent immobilization will be discussed. ; We gratefully recognize the financial support from Ministerio de Ciencia e Innovación-Spanish Government (project number CTQ2017-86170-R) and CSIC for the project AEP045. Diego Carballares thanks a FPI fellowship to Ministerio de Ciencia e Innovación, Roberto Morellon-Sterling recognizes a FPU to Ministerio de educación.

Lipases are among the most utilized enzymes in biocatalysis. In many instances, the main reason for their use is their high specificity or selectivity. However, when full modification of a multifunctional and heterogeneous substrate is pursued, enzyme selectivity and specificity become a problem. This is the case of hydrolysis of oils and fats to produce free fatty acids or their alcoholysis to produce biodiesel, which can be considered cascade reactions. In these cases, to the original heterogeneity of the substrate, the presence of intermediate products, such as diglycerides or monoglycerides, can be an additional drawback. Using these heterogeneous substrates, enzyme specificity can promote that some substrates (initial substrates or intermediate products) may not be recognized as such (in the worst case scenario they may be acting as inhibitors) by the enzyme, causing yields and reaction rates to drop. To solve this situation, a mixture of lipases with different specificity, selectivity and differently affected by the reaction conditions can offer much better results than the use of a single lipase exhibiting a very high initial activity or even the best global reaction course. This mixture of lipases from different sources has been called "combilipases" and is becoming increasingly popular. They include the use of liquid lipase formulations or immobilized lipases. In some instances, the lipases have been coimmobilized. Some discussion is offered regarding the problems that this coimmobilization may give rise to, and some strategies to solve some of these problems are proposed. The use of combilipases in the future may be extended to other processes and enzymes. ; This research was funded by Ministerio de Ciencia e Innovación-Spanish Government (project number CTQ2017-86170-R) and Generalitat Valenciana (PROMETEO/2018/076).

Immobilization and purification of enzymes are usual requirements for their industrial use. Both purification and immobilization have a common factor: they use a solid activated support. Using a support for enzyme purification means having mild conditions for enzyme release and a selective enzyme–support interaction is interesting. When using a support for immobilization, however, enzyme desorption is a problem. The improvement of enzyme features through immobilization is a usual objective (e.g., stability, selectivity). Thus, a support designed for enzyme purification and a support designed for enzyme immobilization may differ significantly. In this review, we will focus our attention on the requirements of a support surface to produce the desired objectives. The ideal physical properties of the matrix, the properties of the introduced reactive groups, the best surface activation degree to reach the desired objective, and the properties of the reactive groups will be discussed. ; We gratefully recognize the support from the Spanish Government, CTQ2013-41507-R, Colciencia (Colombia) and CNPq (Brazil). The predoctoral fellowships for Mr dos Santos (CNPq, Brazil) are also recognized. Á. Berenguer-Murcia thanks the Spanish Ministerio de Ciencia e Innovaciûn for a Ramón y Cajal fellowship (RyC-2009-03813).

This paper describes the optimization of the immobilization of the peroxidase from Roystonea regia (RPTP) using the technique of the crosslinking enzyme aggregates. The immobilization was optimized using response surface methodology and after evaluating three different precipitants, ethanol was finally selected. Three variables were analyzed, glutaraldehyde concentration, precipitant concentration and time before collecting the RPTP-CLEAs. The immobilization yield was around 75%. The activity of the RPTP-CLEA was very high, this was even more patent at pH 3, where the free RPTP was fully inactive and the RPTP-CLEA maintained 40% of the maximum activity. In stress inactivations, the RPTP-CLEA maintained the very high thermostability that presented the free enzyme at 90 °C and pH 7 (half-live of 50 min). At pH 3, the free enzyme suffered subunit dissociation as a first step of the inactivation, this is not possible using RPTP-CLEAs and provided a very high thermostabilization (that depends on the enzyme concentration). The stability of the enzyme in the presence of hydrogen peroxide is good at low concentrations of this reagent (e.g., 10 mM), but if the concentration is higher (e.g., 300 mM), the enzyme stability drops. The immobilization provides an improved stability in the presence of this oxidant, but the values reached may not be high enough for some applications. The RPTP-CLEAs may be used for the decoloration of methyl orange solutions using 5 mM of hydrogen peroxide for 4 cycles (4 h each cycle) without apparent decrease in activity (but only degrading around 50% of the substrate). Using 225 mM of this oxidative reagent, the activity slowly decreased after each cycle (but enabling the full destruction of the colorant). This immobilized enzyme may be used even at pH 3 and 225 mM hydrogen peroxide, conditions where the free enzyme is completely inactive. ; We gratefully recognize the support from the MINECO of Spanish Government, CTQ2013-41507-R and project 1391 from the Department of research and extension of the Health facultyUIS. The predoctoral fellowship for Ms Rueda (Colciencia, Colombian Government). ; 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

This review mainly focuses on the use of glucose oxidase in the production of D-gluconic acid, which is a reactant of undoubtable interest in different industrial areas. The enzyme has been used in numerous instances as a model reaction to study the problems of oxygen supply in bioreactors. One of the main topics in this review is the problem of the generated side product, hydrogen peroxide, as it is an enzyme-inactivating reagent. Different ways to remove hydrogen peroxide have been used, such as metal catalysts and use of whole cells; however, the preferred method is the coupling glucose oxidase with catalase. The different possibilities of combining these enzymes have been discussed (use of free enzymes, independently immobilized enzymes or co-immobilized enzymes). Curiously, some studies propose the addition of hydrogen peroxide to this co-immobilized enzyme system to produce oxygen in situ. Other cascade reactions directed toward the production of gluconic acid from polymeric substrates will be presented; these will mainly involve the transformation of polysaccharides (amylases, cellulases, etc.) but will not be limited to those (e.g., gluconolactonase). In fact, glucose oxidase is perhaps one of most successful enzymes, and it is involved in a wide range of cascade reactions. Finally, other applications of the enzyme have been reviewed, always based on the production of D-gluconic acid, which produces a decrease in the pH, a decrease in the oxygen availability or the production of hydrogen peroxide; in many instances, cascade reactions are also utilized. Thus, this review presents many different cascade reactions and discusses the advantages/drawbacks of the use of co-immobilized enzymes. ; We gratefully recognize the financial support from Ministerio de Ciencia e Innovación-Spanish Government and FEDER funds (project number CTQ2017-86170-R, RTI2018-095291-BI00, MAT2017-87579-R) and Generalitat Valenciana (PROMETEO/2018/076). DC thank to Ministerio de Ciencia e Innovacion-Spanish Government by a FPI. PWT thanks to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001.

Lecitase Ultra is a chimera produced by the fusion of the genes of the lipase from Thermomyces lanuginosus and the phospholipase A1 from Fusarium oxysporum. The enzyme was first designed for the enzymatic degumming of oils, as that problem was not fully resolved before. It is commercialized only as an enzyme solution by Novo Nordisk A/S. This review shows the main uses of this promising enzyme. Starting from the original degumming use, the enzyme has found applications in many other food modification applications, like production of structured phospholipids (e.g., derivatives of phosphatidylcholine), tuning the properties of flour, etc. Moreover, the enzyme has been used in fine chemistry (resolution of racemic mixtures), in the production of aromas and fragrances, polymers modification, etc. Some papers show the use of the enzyme in biodiesel production. Moreover, we present the different technologies applied to obtain a suitable immobilized biocatalyst, remarking the immobilization via interfacial activation and how heterofunctional acyl supports may solve some of the limitations. Immobilized enzyme physical and chemical modifications have also been presented. Finally, Lecitase Ultra has been one of the model enzymes in a new strategy to coimmobilize lipases and other less stable enzymes. ; We gratefully recognize the financial support from MINECO from 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, Colciencias (Colombia, project number FP 44842-076-2016), 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).

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).

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).

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).

[EN] Magnetic-chitosan particles were prepared following three different protocols enabling the preparation of particles with different sizes – nano (Nano-CMag, Micro (Micro-CMag) and Macro (Macro-CMag) – and used for pectinase immobilization and clarification of grape, apple and orange juices. The particle size had a great effect in the kinetic parameters, Nano-CMag biocatalyst presented the highest V value (78.95 mg. min), followed by Micro-CMag and Macro-CMag, with V of 57.20 mg.min and 46.03 mg.min, respectively. However, the highest thermal stability was achieved using Macro-CMag, that was 8 and 3-times more stable than Nano-CMag and Micro-CMag biocatalysts, respectively. Pectinase immobilized on Macro-CMag kept 85% of its initial activity after 25 batch cycles in orange juice clarification. These results suggested that the chitosan magnetic biocatalysts presented great potential application as clarifying catalysts for the fruit juice industry and the great importance of the chitosan particles preparation on the final biocatalyst properties. ; This work was supported by grants and scholarships (L Dal Magro) from Capes, CNPq (process 403505/2013-5) and FAPERGS (process 17/2551-0000939-8). We gratefully recognize the support from the MINECO from Spanish Government, (project number CTQ2017-86170-R) The authors wish to thank Mr. Ramiro Martínez (Novozymes, Spain), Amazon group and LNF Latinoamericana for kindly supplying the enzymes used in this research, as well as Vitivinicola Jolimont (Canela, Brazil) for providing the grape juice.