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

Numerical simulations on refractory lining for steel casting vessels

The manufacturing of several materials adopted in industry, civil construction and our daily life has processes performed at high temperatures, such as melting and heat treatments. Thus, these production processes require products that resist to high temperatures, maintaining their physical and chemical properties in service. Refractory ceramics, due to their properties, have been used for this purpose, having crucial importance in high temperature processes. Refractory linings (composed of refractory ceramics) are used in industrial vessels to produce steel, iron, cement, non-ferrous metals, glass, metallic alloys, in melting process, in petrochemical industry, in incinerators, in mineral processing, in power plants and many other applications. This paper presents the results of a large numerical simulations used to represent the thermomechanical behaviour of steel vessels. Different modelling techniques were used and the results detailed and discussed

A kinetic model of the central carbon metabolism for acrylic acid production in Escherichia coli

Acrylic acid is an economically important chemical compound due to its high market value. Nevertheless, the majority of acrylic acid consumed worldwide its produced from petroleum derivatives by a purely chemical process, which is not only expensive, but it also contributes towards environment deterioration. Hence, justifying the current need for sustainable novel production methods that allow higher profit margins. Ideally, to minimise production cost, the pathway should consist in the direct bio-based production from microbial feedstocks, such as Escherichia coli, but the current yields achieved are still too low to compete with conventional method. In this work, even though the glycerol pathway presented higher yields, we identified the malonyl-CoA route, when using glucose as carbon source, as having the most potential for industrial-scale production, since it is cheaper to implement. Furthermore, we also identified potential optimisation targets for all the tested pathways, that can help the bio-based method to compete with the conventional process.This study was supported by the Portuguese Foundation for Science and Technology(FCT) under the scope of the strategic funding of UIDB/04469/2020 unit. This article is also a result of the project 22231/01/SAICT/2016: “Biodata.pt – Infraestrutura Portuguesa de Dados Biolo´gicos”, by Lisboa Portugal Regional Operational Programme (Lisboa2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). Alexandre Oliveira holds a doctoral fellowship (2020.10205.BD) provided by the FCT. Oscar Dias acknowledge FCT for the Assistant Research contract obtained under CEEC Individual 2018. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.info:eu-repo/semantics/publishedVersio

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)

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

Multilayered Nanostructures Integrated with Emerging Technologies

Surface and interface functionalization are crucial steps to introduce new functionalities in numerous applications, as faster dynamics occur on surfaces rather than bulk. Within this context, the layer-by-layer (LbL) technique is a versatile methodology to controllably form organized nanostructures from the spontaneous adsorption of charged molecules. It enables the assembly of multilayered LbL films on virtually any surface using non-covalent molecular interactions, allowing the nanoengineering of interfaces and creation of multifunctional systems with distinct building blocks (polymers, clays, metal nanoparticles, enzymes, organic macromolecules, etc.). Several applications require thin films on electrodes for sensing/biosensing, and here we explore LbL films deposited on interdigitated electrodes (IDEs) that were 3D-printed using the fusing deposition modeling (FDM) technique. IDEs covered with LbL films can be used to form multisensory systems employed in the analysis of complex liquids transforming raw data into specific patterns easily recognized by computational and statistical methods. We extend the FDM 3D-printing methodology to simplify the manufacturing of electrodes and microchannels, thus integrating an e-tongue system in a microfluidic device. Moreover, the continuous flow within microchannels contributes to faster and more accurate analysis, reducing the amount of sample, waste, and costs

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)

A systematic review of integrated frameworks for resilience and sustainability assessments for critical infrastructures

There is a growing tendency to assess resilience and sustainability of critical infrastructures (CI), given the significant increment in high-impact natural hazard events affecting socio-economic welfare. Historically, these assessments have been conducted separately due to the independent evolution of each concept. However, recent contributions tend to integrate them. This paper provides a state-of-the-art review of integrated assessments for resilience and sustainability in CI, examining concepts, indicators, frameworks, and methodologies. Additionally, a Strengths, Weaknesses, Opportunities, and Threats (SWOT) analysis was performed to gain further insights into the prospects of integrated assessments. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) methodology, eligibility criteria were established, leading to the selection of twelve studies. These works were compared based on five dimensions (economic, environmental, social, technical, and governance) to highlight the differences in the indicators used. While all studies considered the social, environmental, and economic dimensions, some did not further analyze sufficient indicators to evaluate environmental and social effects, with governance often neglected. This study emphasizes the relevance of establishing common metrics for a convergent frame for the resilience and sustainability assessment. The findings presented suggest that integrated assessments lead to a more strategic use of resources toward more resilient CIPortuguese Foundation for Science and Technology (FCT) through grant number PD/ 2020.07208.BD, and by FEDER funds through the Competitivity Factors Oper-ational Programme—COMPETE and by national funds through FCT (Foundation for Science and Technology) within the scope of the project POCI-01-0247- FEDER-039555. It was also partly financed by FCT / MCTES through national funds (PIDDAC) under the R&D Unit Institute for Sustainability and Innovation in Structural Engineering (ISISE), under reference UIDB / 04029/ 2020, and under the Associate Laboratory Advanced Production and Intelligent Systems ARISE under reference LA/P/ 0112/202