Immobilization of Enzymes

A special issue of Catalysts (ISSN 2073-4344). This special issue belongs to the section "Biocatalysis".

Deadline for manuscript submissions: closed (30 September 2019) | Viewed by 93966

Special Issue Editors

Department of Food, Environmental and Nutritional Sciences (DeFENS), University of Milano, Milano, Italy
Interests: biocatalysis; heterologous protein expression; enzyme immobilization; enzyme engineering; multi-enzymatic processes; acylases; ketoreductases; nucleoside phosphorylases; fermentations
Department of Drug Sciences, University of Pavia, 27100 Pavia, Italy
Interests: biocatalysis; enzyme immobilization; circular bioeconomy
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Special Issue Information

Dear Colleagues,

Enzymes are extremely versatile and powerful catalysts used in several fields (fine chemicals, pharmaceuticals, food, plastics, cosmetics, biofuels); moreover, they often represent an environmentally friendly alternative to conventional chemical catalysts. However, the competitiveness of biocatalyzed processes with respect to the “conventional” chemical ones is often hampered by the poor long-term operational stability of the biocatalysts and their difficult recovery and re-use. These drawbacks can generally be overcome by enzyme immobilization that, by definition, produces “an enzyme confined in a defined region of space with retention of catalytic activity, increased stability and which can be used repeatedly and continuously”. Thus, it is not surprising that enzyme immobilization is frequently considered as a pre-requisite to the development of an enzyme as a biocatalyst for synthetic applications.

Many efforts have been devoted over the years to the development of various immobilization techniques and materials. Nevertheless, the optimal immobilization technique often needs to be tailored for each enzyme to fit both the peculiar enzyme features and the final process. Combining information derived from protein sequence, 3-D structure, reaction mechanism and process features with data on physical/chemical properties of the carrier is pivotal to develop ad hoc immobilization strategies.

For this Special Issue, we welcome contributions from all aspects of enzyme immobilization, may they be related to fundamental science or practical applications, that can be outlined by the following keywords:

  • Enzyme immobilization techniques
  • Enzyme co-immobilization for cascade reactions
  • Rational design of immobilization
  • Improved enzyme properties via immobilization
  • Novel supports for enzyme immobilization
  • Enzyme modification to improve immobilization on solid support
  • Biotransformations catalyzed by immobilized enzymes

Dr. Immacolata Serra
Prof. Daniela Ubiali
Guest Editors

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Keywords

  • Enzyme immobilization techniques
  • Enzyme co-immobilization for cascade reactions
  • Rational design of immobilization
  • Improved enzyme properties via immobilization
  • Novel supports for enzyme immobilization
  • Enzyme modification to improve immobilization on solid support
  • Biotransformations catalyzed by immobilized enzymes

Published Papers (23 papers)

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23 pages, 6881 KiB  
Article
Comparative Study on Enzyme Immobilization Using Natural Hydrogel Matrices—Experimental Studies Supported by Molecular Models Analysis
by Karolina Labus, Kamila Wolanin and Łukasz Radosiński
Catalysts 2020, 10(5), 489; https://doi.org/10.3390/catal10050489 - 01 May 2020
Cited by 30 | Viewed by 4058
Abstract
Currently, great attention is focused on conducting manufacture processes using clean and eco-friendly technologies. This research trend also relates to the production of immobilized biocatalysts of industrial importance using matrices and methods that fulfill specified operational and environmental requirements. For that reason, hydrogels [...] Read more.
Currently, great attention is focused on conducting manufacture processes using clean and eco-friendly technologies. This research trend also relates to the production of immobilized biocatalysts of industrial importance using matrices and methods that fulfill specified operational and environmental requirements. For that reason, hydrogels of natural origin and the entrapment method become increasingly popular in terms of enzyme immobilization. The presented work is the comparative research on invertase immobilization using two natural hydrogel matrices—alginate and gelatin. During the study, we provided the molecular insight into the structural characteristics of both materials regarding their applicability as effective enzyme carriers. In order to confirm our predictions of using these hydrogels for invertase immobilization, we performed the typical experimental studies. In this case, the appropriate conditions of enzyme entrapment were selected for both types of carrier. Next, the characterization of received invertase preparations was made. As a final experimental result, the gelatin-based hydrogel was selected as an effective carrier for invertase immobilization. Hereby, using mild conditions and a pro-ecological, biodegradable matrix, it was possible to obtain very stable and reactive biocatalyst. The choice of gelatin-immobilized invertase preparation was compatible with our predictions based on the molecular models of hydrogel matrices and enzyme used. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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12 pages, 1169 KiB  
Communication
Immobilization of Old Yellow Enzymes via Covalent or Coordination Bonds
by Francesca Tentori, Teodora Bavaro, Elisabetta Brenna, Danilo Colombo, Daniela Monti, Riccardo Semproli and Daniela Ubiali
Catalysts 2020, 10(2), 260; https://doi.org/10.3390/catal10020260 - 20 Feb 2020
Cited by 13 | Viewed by 3925
Abstract
Ene-reductases (ERs) belonging to the old yellow enzyme (OYE) family have been thoroughly investigated for the stereospecific reduction of activated prochiral C=C double bonds. In this work, OYE3 was immobilized both by covalent binding on glyoxyl-agarose (OYE3-GA), and by affinity-based adsorption on EziG [...] Read more.
Ene-reductases (ERs) belonging to the old yellow enzyme (OYE) family have been thoroughly investigated for the stereospecific reduction of activated prochiral C=C double bonds. In this work, OYE3 was immobilized both by covalent binding on glyoxyl-agarose (OYE3-GA), and by affinity-based adsorption on EziGTM particles (OYE3-EziG). The immobilized OYE3-GA was demonstrated to be active (activity recovery = 52%) and to retain almost 100% of its activity under the enzymatic assay conditions (50 mM phosphate buffer pH 7, 28 °C) for six days, whereas the activity of the non-immobilized enzyme dropped to 50% after two days. In the case of EziGTM, the highest activity recovery (54%) was achieved by using the most hydrophilic carrier (EziGTM Opal) that was selected for the full characterization of this type of enzyme preparation (stability, recycling, re-use, enzyme leakage). OYE3-EziG was slightly less stable than OYE3-GA under the same experimental conditions. OYE3-GA could be recycled and re-used for up to 12 reaction cycles in the bioreduction of α-methyl-trans-cinnamaldehyde; after 12 runs, the highest conversion achieved was 40%. In the case of the co-immobilized OYE3/GDH-EziG, the conversion dropped to 56% after two reaction cycles. No enzyme leakage was detected over 48 h for both OYE3-GA and OYE3/GDH-EziG (50 mM phosphate buffer pH 7, 28 °C). These seed results pave the way for a true optimization of the immobilization of OYE3, as well as for the use of immobilized OYE3 for preparative applications both in batch and continuous flow conditions. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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17 pages, 2648 KiB  
Article
Increased Stability of Oligopeptidases Immobilized on Gold Nanoparticles
by Marcelo Yudi Icimoto, Adrianne Marlise Mendes Brito, Marcos Paulo Cyrillo Ramos, Vitor Oliveira and Iseli Lourenço Nantes-Cardoso
Catalysts 2020, 10(1), 78; https://doi.org/10.3390/catal10010078 - 04 Jan 2020
Cited by 7 | Viewed by 2674
Abstract
The metallopeptidases thimet oligopeptidase (THOP, EC 3.4.24.25) and neurolysin (NEL, EC 3.4.24.26) are enzymes that belong to the zinc endopeptidase M13 family. Numerous studies suggest that these peptidases participate in the processing of bioactive peptides such as angiotensins and bradykinin. Efforts have been [...] Read more.
The metallopeptidases thimet oligopeptidase (THOP, EC 3.4.24.25) and neurolysin (NEL, EC 3.4.24.26) are enzymes that belong to the zinc endopeptidase M13 family. Numerous studies suggest that these peptidases participate in the processing of bioactive peptides such as angiotensins and bradykinin. Efforts have been conducted to develop biotechnological tools to make possible the use of both proteases to regulate blood pressure in mice, mainly limited by the low plasmatic stability of the enzymes. In the present study, it was investigated the use of nanotechnology as an efficient strategy for to circumvent the low stability of the proteases. Recombinant THOP and NEL were immobilized in gold nanoparticles (GNPs) synthesized in situ using HEPES and the enzymes as reducing and stabilizing agents. The formation of rTHOP-GNP and rNEL-GNP was characterized by the surface plasmon resonance band, zeta potential and atomic force microscopy. The gain of structural stability and activity of rTHOP and rNEL immobilized on GNPs was demonstrated by assays using fluorogenic substrates. The enzymes were also efficiently immobilized on GNPs fabricated with sodium borohydride. The efficient immobilization of the oligopeptidases in gold nanoparticles with gain of stability may facilitate the use of the enzymes in therapies related to pressure regulation and stroke, and as a tool for studying the physiological and pathological roles of both proteases. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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12 pages, 931 KiB  
Communication
A Multi-Enzymatic Cascade Reaction for the Synthesis of Vidarabine 5′-Monophosphate
by Marina Simona Robescu, Immacolata Serra, Marco Terreni, Daniela Ubiali and Teodora Bavaro
Catalysts 2020, 10(1), 60; https://doi.org/10.3390/catal10010060 - 01 Jan 2020
Cited by 18 | Viewed by 3767
Abstract
We here described a three-step multi-enzymatic reaction for the one-pot synthesis of vidarabine 5′-monophosphate (araA-MP), an antiviral drug, using arabinosyluracil (araU), adenine (Ade), and adenosine triphosphate (ATP) as precursors. To this aim, three enzymes involved in the biosynthesis of nucleosides and nucleotides were [...] Read more.
We here described a three-step multi-enzymatic reaction for the one-pot synthesis of vidarabine 5′-monophosphate (araA-MP), an antiviral drug, using arabinosyluracil (araU), adenine (Ade), and adenosine triphosphate (ATP) as precursors. To this aim, three enzymes involved in the biosynthesis of nucleosides and nucleotides were used in a cascade mode after immobilization: uridine phosphorylase from Clostridium perfringens (CpUP), a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP), and deoxyadenosine kinase from Dictyostelium discoideum (DddAK). Specifically, CpUP catalyzes the phosphorolysis of araU thus generating uracil and α-d-arabinose-1-phosphate. AhPNP catalyzes the coupling between this latter compound and Ade to form araA (vidarabine). This nucleoside becomes the substrate of DddAK, which produces the 5′-mononucleotide counterpart (araA-MP) using ATP as the phosphate donor. Reaction conditions (i.e., medium, temperature, immobilization carriers) and biocatalyst stability have been balanced to achieve the highest conversion of vidarabine 5′-monophosphate (≥95.5%). The combination of the nucleoside phosphorylases twosome with deoxyadenosine kinase in a one-pot cascade allowed (i) a complete shift in the equilibrium-controlled synthesis of the nucleoside towards the product formation; and (ii) to overcome the solubility constraints of araA in aqueous medium, thus providing a new route to the highly productive synthesis of araA-MP. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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16 pages, 4246 KiB  
Article
Biochemical and Structural Characterization of Cross-Linked Enzyme Aggregates (CLEAs) of Organic Solvent Tolerant Protease
by Muhammad Syafiq Mohd Razib, Raja Noor Zaliha Raja Abd Rahman, Fairolniza Mohd Shariff and Mohd Shukuri Mohamad Ali
Catalysts 2020, 10(1), 55; https://doi.org/10.3390/catal10010055 - 01 Jan 2020
Cited by 13 | Viewed by 3210
Abstract
Cross-linked enzyme aggregates (CLEAs) is an immobilization technique that can be used to customize enzymes under an optimized condition. Structural analysis on any enzyme treated with a CLEA remains elusive and has been less explored. In the present work, a method for preparing [...] Read more.
Cross-linked enzyme aggregates (CLEAs) is an immobilization technique that can be used to customize enzymes under an optimized condition. Structural analysis on any enzyme treated with a CLEA remains elusive and has been less explored. In the present work, a method for preparing an organic solvent tolerant protease using a CLEA is disclosed and optimized for better biochemical properties, followed by an analysis of the structure of this CLEA-treated protease. The said organic solvent tolerant protease is a metalloprotease known as elastase strain K in which activity of the metalloprotease is measured by a biochemical interaction with azocasein. Results showed that when a glutaraldehyde of 0.02% (v/v) was used under a 2 h treatment, the amount of recovered activity in CLEA-elastase was highest. The recovered activity of CLEA-elastase and CLEA-elastase-SB (which was a CLEA co-aggregated with starch and bovine serum albumin (BSA)) were at an approximate 60% and 80%, respectively. The CLEA immobilization of elastase strain K allowed the stability of the enzyme to be enhanced at high temperature and at a broader pH. Both CLEA-elastase and CLEA-elastase-SB end-products were able to maintain up to 67% enzyme activity at 60 °C and exhibiting an enhanced stability within pH 5–9 with up to 90% recovering activity. By implementing a CLEA on the organic solvent tolerant protease, the characteristics of the organic solvent tolerant were preserved and enhanced with the presence of 25% (v/v) acetonitrile, ethanol, and benzene at 165%, 173%, and 153% relative activity. Structural analysis through SEM and dynamic light scattering (DLS) showed that CLEA-elastase had a random aggregate morphology with an average diameter of 1497 nm. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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11 pages, 1547 KiB  
Article
Tris-(Nitrilotriacetic Acid)-Decorated Polymer Conjugates as Tools for Immobilization and Visualization of His-Tagged Proteins
by Jana Beranová, Tomáš Knedlík, Adéla Šimková, Vladimír Šubr, Libor Kostka, Tomáš Etrych, Pavel Šácha and Jan Konvalinka
Catalysts 2019, 9(12), 1011; https://doi.org/10.3390/catal9121011 - 01 Dec 2019
Cited by 6 | Viewed by 3766
Abstract
Recombinant proteins are commonly expressed with artificial affinity tags for purification, immobilization and characterization. The most frequently used tag, His-tag, is a sequence of consecutive histidine residues fused to the protein of interest. Specialized small molecules that bind His-tag are primarily used for [...] Read more.
Recombinant proteins are commonly expressed with artificial affinity tags for purification, immobilization and characterization. The most frequently used tag, His-tag, is a sequence of consecutive histidine residues fused to the protein of interest. Specialized small molecules that bind His-tag are primarily used for purification, while antibodies are used for protein analysis. However, various issues may be encountered with the use of antibodies. Low inherent stability, the difficulty of introducing chemical modifications, and often-unreliable batch-to-batch consistency are among the limiting factors that call for better alternatives. Recently described polymer conjugates of N-(2-hydroxypropyl) methacrylamide and low-molecular-weight functional ligands, so-called iBodies, are antibody mimetics capable of replacing antibodies in biochemical applications. We tailored this system for methods utilizing His-tag by accessorizing the polymer carrier with tris-nitrilotriacetic acid targeting ligands. These anti-polyHis iBodies are additionally accessorized with fluorophores, enabling detection, and biotin ligands, enabling immobilization. Here, we characterized anti-polyHis iBodies and explored their use as antibody mimetics. We tested their stability, as well as the influence of different metal mediators and His-tag lengths on binding. With high affinity and stability, iBodies represent a new alternative for immobilization and visualization of His-tagged proteins. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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14 pages, 2708 KiB  
Article
Human Deoxycytidine Kinase Is a Valuable Biocatalyst for the Synthesis of Nucleotide Analogues
by Katja F. Hellendahl, Sarah Kamel, Albane Wetterwald, Peter Neubauer and Anke Wagner
Catalysts 2019, 9(12), 997; https://doi.org/10.3390/catal9120997 - 27 Nov 2019
Cited by 8 | Viewed by 2995
Abstract
Natural ribonucleoside-5’-monophosphates are building blocks for nucleic acids which are used for a number of purposes, including food additives. Their analogues, additionally, are used in pharmaceutical applications. Fludarabine-5´-monophosphate, for example, is effective in treating hematological malignancies. To date, ribonucleoside-5’-monophosphates are mainly produced by [...] Read more.
Natural ribonucleoside-5’-monophosphates are building blocks for nucleic acids which are used for a number of purposes, including food additives. Their analogues, additionally, are used in pharmaceutical applications. Fludarabine-5´-monophosphate, for example, is effective in treating hematological malignancies. To date, ribonucleoside-5’-monophosphates are mainly produced by chemical synthesis, but the inherent drawbacks of this approach have led to the development of enzymatic synthesis routes. In this study, we evaluated the potential of human deoxycytidine kinase (HsdCK) as suitable biocatalyst for the synthesis of natural and modified ribonucleoside-5’-monophosphates from their corresponding nucleosides. Human dCK was heterologously expressed in E. coli and immobilized onto Nickel-nitrilotriacetic acid (Ni-NTA) superflow. A screening of the substrate spectrum of soluble and immobilized biocatalyst revealed that HsdCK accepts a wide range of natural and modified nucleosides, except for thymidine and uridine derivatives. Upon optimization of the reaction conditions, HsdCK was used for the synthesis of fludarabine-5´-monophosphate using increasing substrate concentrations. While the soluble biocatalyst revealed highest product formation with the lowest substrate concentration of 0.3 mM, the product yield increased with increasing substrate concentrations in the presence of the immobilized HsdCK. Hence, the application of immobilized HsdCK is advantageous upon using high substrate concentration which is relevant in industrial applications. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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10 pages, 1547 KiB  
Communication
Enhanced Performance of Immobilized Xylanase/Filter Paper-ase on a Magnetic Chitosan Support
by Aldo Amaro-Reyes, Azariel Díaz-Hernández, Jorge Gracida, Blanca E. García-Almendárez, Monserrat Escamilla-García, Teresita Arredondo-Ochoa and Carlos Regalado
Catalysts 2019, 9(11), 966; https://doi.org/10.3390/catal9110966 - 16 Nov 2019
Cited by 15 | Viewed by 2904
Abstract
Enzyme immobilization on different supports has emerged as an efficient and cost-effective tool to improve their stability and reuse capacity. This work aimed to produce a stable immobilized multienzymatic system of xylanase and filter paper-ase (FPase) onto magnetic chitosan using genipin as a [...] Read more.
Enzyme immobilization on different supports has emerged as an efficient and cost-effective tool to improve their stability and reuse capacity. This work aimed to produce a stable immobilized multienzymatic system of xylanase and filter paper-ase (FPase) onto magnetic chitosan using genipin as a cross-linking agent and to evaluate its biochemical properties and reuse capacity. A mixture of chitosan magnetic nanoparticles, xylanase, and FPase was covalently bonded using genipin. Immobilization yield and efficiency were quantified. The activity of free and immobilized enzymes was quantified at different values of pH, temperature, substrate concentration (Km and Vmax), and reuse cycles. The immobilization yield, immobilization efficiency, and activity recovery were 145.3% ± 3.06%, 14.8% ± 0.81%, and 21.5% ± 0.72%, respectively, measured as the total hydrolytic activity. Immobilization confers resistance to acidic/basic conditions and thermal stability compared to the free form. Immobilization improved 3.5-fold and 78-fold the catalytic efficiency (Kcat/Km) of the xylanase and filter paper-ase activities, while immobilized xylanase and FPase could be reused for 34 min and 43 min, respectively. Cross-linking significantly improved the biochemical properties of immobilized enzymes, combined with their simplicity of reuse due to the paramagnetic property of the support. Multienzyme immobilization technology is an important issue for industrial applications. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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16 pages, 1676 KiB  
Article
Developing a Novel Enzyme Immobilization Process by Activation of Epoxy Carriers with Glucosamine for Pharmaceutical and Food Applications
by Immacolata Serra, Ilaria Benucci, Marina Simona Robescu, Claudio Lombardelli, Marco Esti, Cinzia Calvio, Massimo Pregnolato, Marco Terreni and Teodora Bavaro
Catalysts 2019, 9(10), 843; https://doi.org/10.3390/catal9100843 - 12 Oct 2019
Cited by 7 | Viewed by 3971
Abstract
In this paper, we describe the development of an efficient enzyme immobilization procedure based on the activation of epoxy carriers with glucosamine. This approach aims at both creating a hydrophilic microenvironment surrounding the biocatalyst and introducing a spacer bearing an aldehyde group for [...] Read more.
In this paper, we describe the development of an efficient enzyme immobilization procedure based on the activation of epoxy carriers with glucosamine. This approach aims at both creating a hydrophilic microenvironment surrounding the biocatalyst and introducing a spacer bearing an aldehyde group for covalent attachment. First, the immobilization study was carried out using penicillin G acylase (PGA) from Escherichia coli as a model enzyme. PGA immobilized on glucosamine activated supports has been compared with enzyme derivatives obtained by direct immobilization on the same non-modified carriers, in the synthesis of different 3′-functionalized cephalosporins. The derivatives prepared by immobilization of PGA on the glucosamine-carriers performed better than those prepared using the unmodified carriers (i.e., 90% versus 79% cefazolin conversion). The same immobilization method has been then applied to the immobilization of two other hydrolases (neutral protease from Bacillus subtilis, PN, and bromelain from pineapple stem, BR) and one transferase (γ-glutamyl transpeptidase from Bacillus subtilis, GGT). Immobilized PN and BR have been exploited in the synthesis of modified nucleosides and in a bench-scale packed-bed reactor for the protein stabilization of a Sauvignon blanc wine, respectively. In addition, in these cases, the new enzyme derivatives provided improved results compared to those previously described. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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11 pages, 2884 KiB  
Article
Proteases Immobilization for In Situ Time-Limited Proteolysis on MALDI Chips
by Michal Rosulek, Petra Darebna, Petr Pompach, Lukas Slavata and Petr Novak
Catalysts 2019, 9(10), 833; https://doi.org/10.3390/catal9100833 - 03 Oct 2019
Cited by 2 | Viewed by 3128
Abstract
A large number of different enzyme immobilization techniques are used in the field of life sciences, clinical diagnostics, or biotechnology. Most of them are based on a chemically mediated formation of covalent bond between an enzyme and support material. The covalent bond formation [...] Read more.
A large number of different enzyme immobilization techniques are used in the field of life sciences, clinical diagnostics, or biotechnology. Most of them are based on a chemically mediated formation of covalent bond between an enzyme and support material. The covalent bond formation is usually associated with changes of the enzymes’ three-dimensional structure that can lead to reduction of enzyme activity. The present work demonstrates a potential of an ambient ion-landing technique to effectively immobilize enzymes on conductive supports for direct matrix-assisted laser desorption/ionization (MALDI) mass spectrometry analyses of reaction products. Ambient ion landing is an electrospray-based technique allowing strong and stable noncovalent and nondestructive enzyme deposition onto conductive supports. Three serine proteolytic enzymes including trypsin, α-chymotrypsin, and subtilisin A were immobilized onto conductive indium tin oxide glass slides compatible with MALDI mass spectrometry. The functionalized MALDI chips were used for in situ time-limited proteolysis of proteins and protein–ligand complexes to monitor their structural changes under different conditions. The data from limited proteolysis using MALDI chips fits to known or predicted protein structures. The results show that functionalized MALDI chips are sensitive, robust, and fast and might be automated for general use in the field of structural biology. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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14 pages, 2517 KiB  
Article
Enzyme Immobilization for Solid-Phase Catalysis
by Yi Fang, Aihua Zhang, Shaohua Li, Michael Sproviero and Ming-Qun Xu
Catalysts 2019, 9(9), 732; https://doi.org/10.3390/catal9090732 - 29 Aug 2019
Cited by 4 | Viewed by 5050
Abstract
The covalent immobilization of an enzyme to a solid support can broaden its applicability in various workflows. Immobilized enzymes facilitate catalyst re-use, adaptability to automation or high-throughput applications and removal of the enzyme without heat inactivation or reaction purification. In this report, we [...] Read more.
The covalent immobilization of an enzyme to a solid support can broaden its applicability in various workflows. Immobilized enzymes facilitate catalyst re-use, adaptability to automation or high-throughput applications and removal of the enzyme without heat inactivation or reaction purification. In this report, we demonstrate a step-by-step procedure to carry out the bio-orthogonal immobilization of DNA modifying enzymes employing the self-labelling activity of the SNAP-tag to covalently conjugate the enzyme of interest to the solid support. We also demonstrate how modifying the surface functionality of the support can improve the activity of the immobilized enzyme. Finally, the utility of immobilized DNA-modifying enzymes is depicted through sequential processing of genomic DNA libraries for Illumina next-generation sequencing (NGS), resulting in improved read coverage across AT-rich sequences. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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18 pages, 4079 KiB  
Article
Two-Step Production of Neofructo-Oligosaccharides Using Immobilized Heterologous Aspergillus terreus 1F-Fructosyltransferase Expressed in Kluyveromyces lactis and Native Xanthophyllomyces dendrorhous G6-Fructosyltransferase
by Jan Philipp Burghardt, Markus Baas, Doreen Gerlach and Peter Czermak
Catalysts 2019, 9(8), 673; https://doi.org/10.3390/catal9080673 - 07 Aug 2019
Cited by 10 | Viewed by 4554
Abstract
Fructo-oligosaccharides (FOS) are prebiotic low-calorie sweeteners that are synthesized by the transfer of fructose units from sucrose by enzymes known as fructosyltransferases. If these enzymes generate β-(2,6) glycosidic bonds, the resulting oligosaccharides belong to the neoseries (neoFOS). Here, we characterized the properties of [...] Read more.
Fructo-oligosaccharides (FOS) are prebiotic low-calorie sweeteners that are synthesized by the transfer of fructose units from sucrose by enzymes known as fructosyltransferases. If these enzymes generate β-(2,6) glycosidic bonds, the resulting oligosaccharides belong to the neoseries (neoFOS). Here, we characterized the properties of three different fructosyltransferases using a design of experiments approach based on response surface methodology with a D-optimal design. The reaction time, pH, temperature, and substrate concentration were used as parameters to predict three responses: The total enzyme activity, the concentration of neoFOS and the neoFOS yield relative to the initial concentration of sucrose. We also conducted immobilization studies to establish a cascade reaction for neoFOS production with two different fructosyltransferases, achieving a total FOS yield of 47.02 ± 3.02%. The resulting FOS mixture included 53.07 ± 1.66 mM neonystose (neo-GF3) and 20.8 ± 1.91 mM neo-GF4. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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12 pages, 1920 KiB  
Article
Preparation of Cross-Linked Enzyme Aggregates (CLEAs) of an Inulosucrase Mutant for the Enzymatic Synthesis of Inulin-Type Fructooligosaccharides
by Thanapon Charoenwongpaiboon, Rath Pichyangkura, Robert A. Field and Manchumas Hengsakul Prousoontorn
Catalysts 2019, 9(8), 641; https://doi.org/10.3390/catal9080641 - 27 Jul 2019
Cited by 12 | Viewed by 3488
Abstract
Fructooligosaccharides are well-known carbohydrate molecules that exhibit good probiotic activity and are widely used as sweeteners. Inulin-type fructooligosaccharides (IFOs) can be synthesized from sucrose using inulosucrase. In this study, cross-linked enzyme aggregates (CLEAs) of Lactobacillus reuteri 121 inulosucrase (R483A-LrInu) were prepared and used [...] Read more.
Fructooligosaccharides are well-known carbohydrate molecules that exhibit good probiotic activity and are widely used as sweeteners. Inulin-type fructooligosaccharides (IFOs) can be synthesized from sucrose using inulosucrase. In this study, cross-linked enzyme aggregates (CLEAs) of Lactobacillus reuteri 121 inulosucrase (R483A-LrInu) were prepared and used as a biocatalyst for IFOs production. Under optimum conditions, R483A-LrInu CLEAs retained 42% of original inulosucrase activity. Biochemical characterization demonstrated that the optimum pH of inulosucrase changed from 5 to 4 after immobilization, while the optimum temperature was unchanged. Furthermore, the pH stability and thermostability of the R483A-LrInu CLEAs was significantly improved. IFOs product characterization indicated that the product specificity of the enzyme was impacted by CLEA generation, producing a narrower range of IFOs than the soluble enzyme. In addition, the R483A-LrInu CLEAs showed operational stability in the batch synthesis of IFOs. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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12 pages, 1835 KiB  
Article
Combined Cross-Linked Enzyme Aggregates of Monoamine Oxidase and Putrescine Oxidase as a Bifunctional Biocatalyst for Determination of Biogenic Amines in Foods
by Tianxiang Yang, Young-Jong Kim, Jetendra Kumar Roy and Young-Wan Kim
Catalysts 2019, 9(7), 579; https://doi.org/10.3390/catal9070579 - 30 Jun 2019
Cited by 2 | Viewed by 2522
Abstract
In order to determine total biogenic amines in fermented foods, the combined cross-linked enzyme aggregates of a monoamine oxidase and a putrescine oxidase (combi-CLEAs) and the cross-linked enzyme aggregates (CLEAs) of the fused enzyme of two amine oxidases (MonoAmine Putrescien Oxidase, MAPO) were [...] Read more.
In order to determine total biogenic amines in fermented foods, the combined cross-linked enzyme aggregates of a monoamine oxidase and a putrescine oxidase (combi-CLEAs) and the cross-linked enzyme aggregates (CLEAs) of the fused enzyme of two amine oxidases (MonoAmine Putrescien Oxidase, MAPO) were prepared. The effects of various parameters were examined to optimize the CLEAs formation. Biochemical characterization and stability of free and the CLEAs enzymes were performed. Through optimization of the CLEAs formation condition, the combi-CLEAs and the CLEAs-MAPO were prepared with 82% and 78% of residual activities relative to the activities of the subjected enzymes were in a preparative scale. The optimal pH for tyramine-activities of the CLEAs enzymes were shifted to relatively basic pH, leading to synchronization of the optimal performances of combi-CLEAs over pH for tyramine and putrescine. In addition, thermostability of the CLEAs enzymes were improved with almost double half-lives at 65 °C in comparison to the free enzymes. The catalytic efficiencies of combi-CLEAs for tyramine, histamine and putrescine were reduced by 41%, 56%, and 31%, respectively, and the inhibition potency by the substrate was reduced by two-fold in comparison of the mixed free enzymes. In conclusion, combi-CLEAs are a promising catalyst with the improved stability and the same optimum pH for dual activities in enzymatic determination of biogenic amines in foods. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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11 pages, 1654 KiB  
Article
Fabrication and Optimization of a Lipase Immobilized Enzymatic Membrane Bioreactor based on Polysulfone Gradient-Pore Hollow Fiber Membrane
by Peng-Cheng Chen, Zhen Ma, Xue-Yan Zhu, Da-Jing Chen and Xiao-Jun Huang
Catalysts 2019, 9(6), 495; https://doi.org/10.3390/catal9060495 - 28 May 2019
Cited by 13 | Viewed by 2607
Abstract
Enzymatic membrane bioreactors (EMBRs) possess the characteristic of combining catalysis with separation, and therefore have promising application potentials. In order to achieve a high-performance EMBR, membrane property, as well as operating parameters, should give special cause for concerns. In this work, an EMBR [...] Read more.
Enzymatic membrane bioreactors (EMBRs) possess the characteristic of combining catalysis with separation, and therefore have promising application potentials. In order to achieve a high-performance EMBR, membrane property, as well as operating parameters, should give special cause for concerns. In this work, an EMBR based on hollow fiber polysulfone microfiltration membranes with radial gradient pore structure was fabricated and enzyme immobilization was achieved through pressure-driven filtration. Lipase from Candida rugosa was used for immobilization and EMBR performance was studied with the enzymatic hydrolysis of glycerol triacetate as a model reaction. The influences of membrane pore diameter, substrate feed direction as well as operational parameters of operation pressure, substrate concentration, and temperature on the EMBR activity were investigated with the production of hydrolysates kinetically fitted. The complete EMBR system showed the highest activity of 1.07 × 104 U⋅g−1. The results in this work indicate future efforts for improvement in EMBR. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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17 pages, 2185 KiB  
Article
Preparation of Crosslinked Enzyme Aggregates of a Thermostable Cyclodextrin Glucosyltransferase from Thermoanaerobacter sp. Critical Effect of the Crosslinking Agent
by Mayerlenis Jimenez Rojas, Murilo Amaral-Fonseca, Gisella Maria Zanin, Roberto Fernandez-Lafuente, Raquel de Lima Camargo Giordano and Paulo Waldir Tardioli
Catalysts 2019, 9(2), 120; https://doi.org/10.3390/catal9020120 - 30 Jan 2019
Cited by 30 | Viewed by 4642
Abstract
Crosslinked enzyme aggregates (CLEAs) of a thermostable cyclodextrin glucosyltransferase (CGTase) from Thermoanaerobacter sp. have been prepared for the production of cyclodextrins (CDs). Different parameters in the precipitation (nature and concentration of precipitant) and crosslinking steps (time of reaction with cross-linker, nature and concentration [...] Read more.
Crosslinked enzyme aggregates (CLEAs) of a thermostable cyclodextrin glucosyltransferase (CGTase) from Thermoanaerobacter sp. have been prepared for the production of cyclodextrins (CDs). Different parameters in the precipitation (nature and concentration of precipitant) and crosslinking steps (time of reaction with cross-linker, nature and concentration of the crosslinker) were evaluated on the production of CLEAs of CGTase. Among the seven studied precipitants, acetone with a 75% (v/v) concentration produced the aggregates of CGTase with higher activity, which retained 97% of the initial activity. Concerning the cross-linker (glutaraldehyde, starch–aldehyde, and pectin–aldehyde), starch–aldehyde produced the most active CLEAs. The use of bovine serum albumin as co-feeder decreased the expressed activity. Addition of polyethylenimine at the end of cross-linking step prevented the leakage of the enzyme and the subsequent Schiff’s bases reduction with sodium borohydride permitted to maintain 24% of the initial activity even with the large dextrin as substrate. The optimal conditions for the immobilization process required were defined as 75% (v/v) acetone as precipitation reagent for 1 h at 20 °C, 20 mM starch–aldehyde as crosslinking reagent for 2 h at 20 °C, treatment with 1 mg/mL of polyethylenimine for 5 min, reduction with 1 mg/mL of sodium borohydride. The CLEAs of CGTase were active catalyst (similarly to the free enzyme) in the production of cyclodextrins at 50 °C and pH 6.0 for 6 h reaction, maintaining intact their structures. Besides this, after five cycles of 3 h the total cyclodextrin yield was 80% of the initial value (first batch, with around 45% CD yield). Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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11 pages, 1511 KiB  
Article
Immobilization of Arylmalonate Decarboxylase
by Kristína Markošová, Jana Husarčíková, Monika Halásová, Robert Kourist, Michal Rosenberg, Radek Stloukal, Ludmila Zajoncová and Martin Rebroš
Catalysts 2018, 8(12), 603; https://doi.org/10.3390/catal8120603 - 02 Dec 2018
Cited by 2 | Viewed by 2842
Abstract
Arylmalonate decarboxylase (AMD) is a monomeric enzyme of only 26 kDa. A recombinant AMDase from Bordetella bronchiseptica was expressed in Escherichia coli and the enzyme was immobilized using different techniques: entrapment in polyvinyl alcohol (PVA) gel (LentiKats®), covalent binding onto magnetic [...] Read more.
Arylmalonate decarboxylase (AMD) is a monomeric enzyme of only 26 kDa. A recombinant AMDase from Bordetella bronchiseptica was expressed in Escherichia coli and the enzyme was immobilized using different techniques: entrapment in polyvinyl alcohol (PVA) gel (LentiKats®), covalent binding onto magnetic microparticles (MMP, PERLOZA s.r.o., Lovosice, Czech Republic) and double-immobilization (MMP-LentiKats®) using the previous two methods. The double-immobilized AMDase was stable in 8 repeated biocatalytic reactions. This combined immobilization technique has the potential to be applied to different small proteins. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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23 pages, 6711 KiB  
Article
Rice Husk as an Inexpensive Renewable Immobilization Carrier for Biocatalysts Employed in the Food, Cosmetic and Polymer Sectors
by Marco Cespugli, Simone Lotteria, Luciano Navarini, Valentina Lonzarich, Lorenzo Del Terra, Francesca Vita, Marina Zweyer, Giovanna Baldini, Valerio Ferrario, Cynthia Ebert and Lucia Gardossi
Catalysts 2018, 8(10), 471; https://doi.org/10.3390/catal8100471 - 19 Oct 2018
Cited by 32 | Viewed by 5218
Abstract
The high cost and environmental impact of fossil-based organic carriers represent a critical bottleneck to their use in large-scale industrial processes. The present study demonstrates the applicability of rice husk as inexpensive renewable carrier for the immobilization of enzymes applicable sectors where the [...] Read more.
The high cost and environmental impact of fossil-based organic carriers represent a critical bottleneck to their use in large-scale industrial processes. The present study demonstrates the applicability of rice husk as inexpensive renewable carrier for the immobilization of enzymes applicable sectors where the covalent anchorage of the protein is a pre-requisite for preventing protein contamination while assuring the recyclability. Rice husk was oxidized and then functionalized with a di-amino spacer. The morphological characterization shed light on the properties that affect the functionalization processes. Lipase B from Candida antarctica (CaLB) and two commercial asparaginases were immobilized covalently achieving higher immobilization yield than previously reported. All enzymes were immobilized also on commercial epoxy methacrylic resins and the CaLB immobilized on rice husk demonstrated a higher efficiency in the solvent-free polycondensation of dimethylitaconate. CaLB on rice husk appears particularly suitable for applications in highly viscous processes because of the unusual combination of its low density and remarkable mechanical robustness. In the case of the two asparaginases, the biocatalyst immobilized on rice husk performed in aqueous solution at least as efficiently as the enzyme immobilized on methacrylic resins, although the rice husk loaded a lower amount of protein. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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10 pages, 3239 KiB  
Article
Preparation of a Flower-Like Immobilized D-Psicose 3-Epimerase with Enhanced Catalytic Performance
by Lu Zheng, Yining Sun, Jing Wang, He Huang, Xin Geng, Yi Tong and Zhi Wang
Catalysts 2018, 8(10), 468; https://doi.org/10.3390/catal8100468 - 18 Oct 2018
Cited by 32 | Viewed by 4729
Abstract
In this present study, we proposed a smart biomineralization method for creating hybrid organic–inorganic nanoflowers using a Co2+-dependent enzyme (D-psicose 3-epimerase; DPEase) as the organic component and cobalt phosphate as the inorganic component. The prepared nanoflowers have many separated [...] Read more.
In this present study, we proposed a smart biomineralization method for creating hybrid organic–inorganic nanoflowers using a Co2+-dependent enzyme (D-psicose 3-epimerase; DPEase) as the organic component and cobalt phosphate as the inorganic component. The prepared nanoflowers have many separated petals that have a nanometer size. Under optimum conditions (60 °C and pH of 8.5), the nanoflower can display its maximum activity (36.2 U/mg), which is about 7.2-fold higher than free DPEase. Furthermore, the immobilized DPEase presents enhanced pH and thermal stabilities. The DPEase-nanoflower maintained about 90% of its activity after six reaction cycles, highlighting its excellent reusability. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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15 pages, 1323 KiB  
Article
Stabilization of Enzymes by Multipoint Covalent Attachment on Aldehyde-Supports: 2-Picoline Borane as an Alternative Reducing Agent
by Alejandro H. Orrego, Maria Romero-Fernández, María Del Carmen Millán-Linares, María Del Mar Yust, José M. Guisán and Javier Rocha-Martin
Catalysts 2018, 8(8), 333; https://doi.org/10.3390/catal8080333 - 15 Aug 2018
Cited by 38 | Viewed by 4934
Abstract
Enzyme immobilization by multipoint covalent attachment on supports activated with aliphatic aldehyde groups (e.g., glyoxyl agarose) has proven to be an excellent immobilization technique for enzyme stabilization. Borohydride reduction of immobilized enzymes is necessary to convert enzyme–support linkages into stable secondary amino groups [...] Read more.
Enzyme immobilization by multipoint covalent attachment on supports activated with aliphatic aldehyde groups (e.g., glyoxyl agarose) has proven to be an excellent immobilization technique for enzyme stabilization. Borohydride reduction of immobilized enzymes is necessary to convert enzyme–support linkages into stable secondary amino groups and to convert the remaining aldehyde groups on the support into hydroxy groups. However, the use of borohydride can adversely affect the structure–activity of some immobilized enzymes. For this reason, 2-picoline borane is proposed here as an alternative milder reducing agent, especially, for those enzymes sensitive to borohydride reduction. The immobilization-stabilization parameters of five enzymes from different sources and nature (from monomeric to multimeric enzymes) were compared with those obtained by conventional methodology. The most interesting results were obtained for bacterial (R)-mandelate dehydrogenase (ManDH). Immobilized ManDH reduced with borohydride almost completely lost its catalytic activity (1.5% of expressed activity). In contrast, using 2-picoline borane and blocking the remaining aldehyde groups on the support with glycine allowed for a conjugate with a significant activity of 19.5%. This improved biocatalyst was 357-fold more stable than the soluble enzyme at 50 °C and pH 7. The results show that this alternative methodology can lead to more stable and active biocatalysts. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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Review

Jump to: Research

29 pages, 1396 KiB  
Review
More than a Confinement: “Soft” and “Hard” Enzyme Entrapment Modulates Biological Catalyst Function
by Grazia Cottone, Sergio Giuffrida, Stefano Bettati, Stefano Bruno, Barbara Campanini, Marialaura Marchetti, Stefania Abbruzzetti, Cristiano Viappiani, Antonio Cupane, Andrea Mozzarelli and Luca Ronda
Catalysts 2019, 9(12), 1024; https://doi.org/10.3390/catal9121024 - 04 Dec 2019
Cited by 11 | Viewed by 3221
Abstract
Catalysis makes chemical and biochemical reactions kinetically accessible. From a technological point of view, organic, inorganic, and biochemical catalysis is relevant for several applications, from industrial synthesis to biomedical, material, and food sciences. A heterogeneous catalyst, i.e., a catalyst confined in a different [...] Read more.
Catalysis makes chemical and biochemical reactions kinetically accessible. From a technological point of view, organic, inorganic, and biochemical catalysis is relevant for several applications, from industrial synthesis to biomedical, material, and food sciences. A heterogeneous catalyst, i.e., a catalyst confined in a different phase with respect to the reagents’ phase, requires either its physical confinement in an immobilization matrix or its physical adsorption on a surface. In this review, we will focus on the immobilization of biological catalysts, i.e., enzymes, by comparing hard and soft immobilization matrices and their effect on the modulation of the catalysts’ function. Indeed, unlike smaller molecules, the catalytic activity of protein catalysts depends on their structure, conformation, local environment, and dynamics, properties that can be strongly affected by the immobilization matrices, which, therefore, not only provide physical confinement, but also modulate catalysis. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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20 pages, 819 KiB  
Review
Surface Display—An Alternative to Classic Enzyme Immobilization
by Mateja Lozančić, Amir Sk. Hossain, Vladimir Mrša and Renata Teparić
Catalysts 2019, 9(9), 728; https://doi.org/10.3390/catal9090728 - 28 Aug 2019
Cited by 20 | Viewed by 5658
Abstract
Enzyme immobilization to solid matrices often presents a challenge due to protein conformation sensitivity, desired enzyme purity, and requirements for the particular carrier properties and immobilization technique. Surface display of enzymes at the cell walls of microorganisms presents an alternative that has been [...] Read more.
Enzyme immobilization to solid matrices often presents a challenge due to protein conformation sensitivity, desired enzyme purity, and requirements for the particular carrier properties and immobilization technique. Surface display of enzymes at the cell walls of microorganisms presents an alternative that has been the focus of many research groups worldwide in different fields, such as biotechnology, energetics, pharmacology, medicine, and food technology. The range of systems by which a heterologous protein can be displayed at the cell surface allows the appropriate one to be found for almost every case. However, the efficiency of display systems is still quite low. The most frequently used yeast for the surface display of proteins is Saccharomyces cerevisiae. However, apart from its many advantages, Saccharomyces cerevisiae has some disadvantages, such as low robustness in industrial applications, hyperglycosylation of some heterologous proteins, and relatively low efficiency of surface display. Thus, in the recent years the display systems for alternative yeast hosts with better performances including Pichia pastoris, Hansenula polymorpha, Blastobotrys adeninivorans, Yarrowia lipolytica, Kluyveromyces marxianus, and others have been developed. Different strategies of surface display aimed to increase the amount of displayed protein, including new anchoring systems and new yeast hosts are reviewed in this paper. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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43 pages, 11738 KiB  
Review
Dextran Aldehyde in Biocatalysis: More Than a Mere Immobilization System
by Veymar G. Tacias-Pascacio, Claudia Ortiz, Nazzoly Rueda, Ángel Berenguer-Murcia, Niuris Acosta, Inmaculada Aranaz, Concepción Civera, Roberto Fernandez-Lafuente and Andrés R. Alcántara
Catalysts 2019, 9(7), 622; https://doi.org/10.3390/catal9070622 - 21 Jul 2019
Cited by 30 | Viewed by 8459
Abstract
Dextran aldehyde (dexOx), resulting from the periodate oxidative cleavage of 1,2-diol moiety inside dextran, is a polymer that is very useful in many areas, including as a macromolecular carrier for drug delivery and other biomedical applications. In particular, it has been widely used [...] Read more.
Dextran aldehyde (dexOx), resulting from the periodate oxidative cleavage of 1,2-diol moiety inside dextran, is a polymer that is very useful in many areas, including as a macromolecular carrier for drug delivery and other biomedical applications. In particular, it has been widely used for chemical engineering of enzymes, with the aim of designing better biocatalysts that possess improved catalytic properties, making them more stable and/or active for different catalytic reactions. This polymer possesses a very flexible hydrophilic structure, which becomes inert after chemical reduction; therefore, dexOx comes to be highly versatile in a biocatalyst design. This paper presents an overview of the multiple applications of dexOx in applied biocatalysis, e.g., to modulate the adsorption of biomolecules on carrier surfaces in affinity chromatography and biosensors design, to serve as a spacer arm between a ligand and the support in biomacromolecule immobilization procedures or to generate artificial microenvironments around the enzyme molecules or to stabilize multimeric enzymes by intersubunit crosslinking, among many other applications. Full article
(This article belongs to the Special Issue Immobilization of Enzymes)
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