Editorial: Special issue - Enzyme immobilization

We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)Peer Reviewe

Magnetic micro-macro biocatalysts applied to industrial bioprocesses

The use of magnetic biocatalysts is highly beneficial in bioprocesses technology, as it allows their easy recovering and enhances biocatalyst lifetime. Thus, it simplifies operational processing and increases efficiency, leading to more cost-effective processes. The use of small-size matrices as carriers for enzyme immobilization enables to maximize surface area and catalysts loading, also reducing diffusion limitations. As highly expensive nanoparticles (nm size) usually aggregate, their application at large scale is not recommended. In contrast, the use of magnetic micro-macro (µm-mm size) matrices leads to more homogeneous biocatalysts with null or very low aggregation, which facilitates an easy handling and recovery. The present review aims to highlight recent trends in the application of medium-to-high size magnetic biocatalysts in different areas (biodiesel production, food and pharma industries, protein purification or removal of environmental contaminants). The advantages and disadvantages of these above-mentioned magnetic biocatalysts in bioproces

Hydrogen Peroxide in Biocatalysis. A Dangerous Liaison

Hydrogen peroxide is a substrate or side-product in many enzyme-catalyzed reactions. For example, it is a side-product of oxidases, resulting from the re-oxidation of FAD with molecular oxygen, and it is a substrate for peroxidases and other enzymes. However, hydrogen peroxide is able to chemically modify the peptide core of the enzymes it interacts with, and also to produce the oxidation of some cofactors and prostetic groups (e.g., the hemo group). Thus, the development of strategies that may permit to increase the stability of enzymes in the presence of this deleterious reagent is an interesting target. This enhancement in enzyme stability has been attempted following almost all available strategies: site-directed mutagenesis (eliminating the most reactive moieties), medium engineering (using stabilizers), immobilization and chemical modification (trying to generate hydrophobic environments surrounding the enzyme, to confer higher rigidity to the protein or to generate oxidation-resistant groups), or the use of systems capable of decomposing hydrogen peroxide under very mild conditions. If hydrogen peroxide is just a side-product, its immediate removal has been reported to be the best solution. In some cases, when hydrogen peroxide is the substrate and its decomposition is not a sensible solution, researchers coupled one enzyme generating hydrogen peroxide “in situ” to the target enzyme resulting in a continuous supply of this reagent at low concentrations thus preventing enzyme inactivation. This review will focus on the general role of hydrogen peroxide in biocatalysis, the main mechanisms of enzyme inactivation produced by this reactive and the different strategies used to prevent enzyme inactivation caused by this “dangerous liaison”.This work has been supported by grant CTQ2009-07568 from Spanish Ministerio de Ciencia e Innovación. A. Berenguer-Murcia thanks the Spanish Ministerio de Ciencia e Innovación for a Ramon y Cajal fellowship (RyC-2009-03813). Mr. Hernandez is a holder of a MAEC-AECID fellowship

Ficin: A protease extract with relevance in biotechnology and biocatalysis

Due to the problems raised by the use of animal or microbial recombinant proteases, the use of proteases from vegetable origin is becoming increasingly popular.. Among them, sulfidryl proteases have a special interest. Ficin is an outstanding example of this kind of proteases. This paper aims to be to make a comprehensive review of the recent uses of this enzyme, including for example protein hydrolysis, the production of bioactive peptides and antibodies fragments (researchers point that ficin results are more reproducible than using other proteases), meat tenderization, milk coagulations in cheese making or peptide synthesis. Efforts to get industrial immobilized biocatalysts of the enzyme will be also described. The review shows the huge potential and brilliant prospect that this enzyme can have in the near future.We gratefully recognize the support from the Ministerio de Ciencia e Innovación from Spanish Government (project number CTQ2017-86170-R). The FPU fellowship (Ministerio de Educacion) for Mr. Morellon–Sterling and the fellowship for Mr. Siar from the Algerian Ministry of Higher Education and Scientific Research are also thanked

A novel, highly potent nadph-dependent cytochrome P450 reductase from waste liza klunzingeri liver

The use of marine enzymes as catalysts for biotechnological applications is a topical subject. Marine enzymes usually display better operational properties than their animal, plant or bacterial counterparts, enlarging the range of possible biotechnological applications. Due to the fact that cytochrome P450 enzymes can degrade many different toxic environmental compounds, these enzymes have emerged as valuable tools in bioremediation processes. The present work describes the isolation, purification and biochemical characterization of a liver NADPH-dependent cytochrome P450 reductase (CPR) from the marine fish Liza klunzingeri (LkCPR). Experimental results revealed that LkCPR is a monomer of approximately 75 kDa that is active in a wide range of pH values (6–9) and temperatures (40–60 °C), showing the highest catalytic activity at pH 8 and 50 °C. The activation energy of the enzyme reaction was 16.3 kcal mol−1 K−1. The KM values for cytochrome C and NADPH were 8.83 μM and 7.26 μM, and the kcat values were 206.79 s−1 and 202.93 s−1, respectively. LkCPR displayed a specific activity versus cytochrome C of 402.07 µmol min−1 mg1, the highest activity value described for a CPR up to date (3.2–4.7 times higher than the most active reported CPRs) and showed the highest thermostability described for a CPR. Taking into account all these remarkable catalytic features, LkCPR offers great potential to be used as a suitable biocatalyst

Glutaraldehyde in bio-catalysts design: a useful crosslinker and a versatile tool in enzyme immobilization

Glutaraldehyde is one of the most widely used reagents in the design of biocatalysts. It is a powerful crosslinker, able to react with itself, with the advantages that this may bring forth. In this review, we intend to give a general vision of its potential and the precautions that must be taken when using this effective reagent. First, the chemistry of the glutaraldehyde/amino reaction will be commented upon. This reaction is still not fully clarified, but it seems to be based on the formation of 6-membered heterocycles formed by 5 C and one O. Then, we will discuss the production of intra- and inter-molecular enzyme crosslinks (increasing enzyme rigidity or preventing subunit dissociation in multimeric enzymes). Special emphasis will be placed on the preparation of cross-linked enzyme aggregates (CLEAs), mainly in enzymes that have low density of surface reactive groups and, therefore, may be problematic to obtain a final solid catalyst. Next, we will comment on the uses of glutaraldehyde in enzymes previously immobilized on supports. First, the treatment of enzymes immobilized on supports that cannot react with glutaraldehyde (only inter and intramolecular cross-linkings will be possible) to prevent enzyme leakage and obtain some enzyme stabilization via cross-linking. Second, the cross-linking of enzymes adsorbed on aminated supports, where together with other reactions enzyme/support crosslinking is also possible; the enzyme is incorporated into the support. Finally, we will present the use of aminated supports preactivated with glutaraldehyde. Optimal glutaraldehyde modifications will be discussed in each specific case (one or two glutaraldehyde molecules for amino group in the support and/or the protein). Using preactivated supports, the heterofunctional nature of the supports will be highlighted, with the drawbacks and advantages that the heterofunctionality may have. Particular attention will be paid to the control of the first event that causes the immobilization depending on the experimental conditions to alter the enzyme orientation regarding the support surface. Thus, glutaraldehyde, an apparently old fashioned reactive, remains the most widely used and with broadest application possibilities among the compounds used for the design of biocatalyst

Strategies for the one-step immobilization–purification of enzymes as industrial biocatalysts

In this review, we detail the efforts performed to couple the purification and the immobilization of industrial enzymes in a single step. The use of antibodies, the development of specific domains with affinity for some specific supports will be revised. Moreover, we will discuss the use of domains that increase the affinity for standard matrices (ionic exchangers, silicates). We will show how the control of the immobilization conditions may convert some unspecific supports in largely specific ones. The development of tailor-made heterofunctional supports as a tool to immobilize–stabilize–purify some proteins will be discussed in deep, using low concentration of adsorbent groups and a dense layer of groups able to give an intense multipoint covalent attachment. The final coupling of mutagenesis and tailor made supports will be the last part of the review.This work has been supported by grant CTQ2013-41507-R from Spanish MINECO, grant no.1102-489-25428 from COLCIENCIAS and Universidad Industrial de Santander (VIE-UIS Research Program) (Colombia) and CNPq grant 403505/2013-5 (Brazil). A. Berenguer-Murcia thanks the Spanish MINECO for a Ramon y Cajal fellowship (RyC-2009-03813)

Heterofunctional Supports in Enzyme Immobilization: From Traditional Immobilization Protocols to Opportunities in Tuning Enzyme Properties

A heterofunctional support for enzyme immobilization may be defined as that which possesses several distinct functionalities on its surface able to interact with a protein. We will focus on those supports in which a final covalent attachment between the enzyme and the support is achieved. Heterofunctionality sometimes has been featured in very old immobilization techniques, even though in many instances it has been overlooked, giving rise to some misunderstandings. In this respect, glutaraldehyde-activated supports are the oldest multifunctional supports. Their matrix has primary amino groups, the hydrophobic glutaraldehyde chain, and can covalently react with the primary amino groups of the enzyme. Thus, immobilization may start (first event of the immobilization) via different causes and may involve different positions of the enzyme surface depending on the activation degree and immobilization conditions. Other “classical” heterofunctional supports are epoxy commercial supports consisting of reactive covalent epoxy groups on a hydrophobic matrix. Immobilization is performed at high ionic strength to permit protein adsorption, so that covalent attachment may take place at a later stage. Starting from these old immobilization techniques, tailor-made heterofunctional supports have been designed to permit a stricter control of the enzyme immobilization process. The requirement is to find conditions where the main covalent reactive moieties may have very low reactivity toward the enzyme. In this Review we will discuss the suitable properties of the groups able to give the covalent attachment (intending a multipoint covalent attachment), and the groups able to produce the first enzyme adsorption on the support. Prospects, limitations, and likely pathways for the evolution (e.g., coupling of site-directed mutagenesis and thiol heterofunctional supports of enzyme immobilization on heterofunctional supports) will be discussed in this Review.This work has been supported by Grant CTQ2009-07568 from Spanish Ministerio de Ciencia e Innovacion, Grant No.1102-489-25428 from COLCIENCIAS and Universidad Industrial de Santander (VIE-UIS Research Program) and CNPq and FAPERGS (Brazil). Á.B.-M. thanks the Spanish Ministerio de Ciencia e Innovacion for a Ramon y Cajal fellowship (RyC-2009-03813)

One Pot Use of Combilipases for Full Modification of Oils and Fats: Multifunctional and Heterogeneous Substrates

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)

Evaluation of styrene-divinylbenzene beads as a support to immobilize lipases

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 Reviewe