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Special Issue "Enzyme Immobilization 2016"

A special issue of Molecules (ISSN 1420-3049). This special issue belongs to the section "Molecular Diversity".

Deadline for manuscript submissions: closed (1 December 2016)

Special Issue Editor

Guest Editor
Prof. Dr. Roberto Fernandez-Lafuente

Institute of catalysis and petrochemsitry-CSIC, Campus UAM-CSIC Cantoblanco, 28049 Madrid, Spain
Website | E-Mail
Fax: +34 91 585 4760
Interests: enzyme immobilization; stabilization and purification; biocatalyst and biosensors design; bioprocesses optimization

Special Issue Information

Dear Colleagues,

After the success of the first Special Issue edited by Molecules in 2014, I am very happy to announce the launching of a second issue on this topic. The immobilization of enzymes and proteins is a seemingly ancient research area that still attracts great interest. The initial interest in enzyme immobilization was motivated by a desire to simplify the reuse of these expensive biocatalysts. Nowadays, the research in this area has focused in its application to overcome many enzyme limitations, for example improving enzyme stability, activity, selectivity or specificity, reducing inhibition problems, and even coupling immobilization and purification.

Two years after the publication of the first issue in May 2014, where many interesting immobilization protocols were reported, still the control of the orientation of the enzyme on the support surface and of the intensity of the support-enzyme interactions is not properly solved. Coupling of genetic and chemical tools has been utilized to produce enzymes that may be better immobilized on tailor made supports. Each day more researches couple immobilization to any other strategy for enzyme stabilization and enhancement of their properties.

Thus, the efforts to design strategies involving the coupled use of immobilization with microbiological (e.g., the use of thermophilic enzymes), chemical or genetic modifications are of special interest. Enzymes are co-immobilized to catalyze cascade reactions; however, this process may produce additional complications, no always co-immobilization will be recommended. This Special Issue invites submissions (i.e., research or review papers) discussing the design of new immobilization protocols, especially when the control of the enzyme orientation is intended by design of the support, or modification of the enzyme. The use of the immobilized enzymes to take advantage of the catalytic improved properties will be also suitable for this second special issue. Papers related to the modification of immobilized enzymes, or to the modification of the support surface after enzyme immobilization, are also welcome.

Dr. Roberto Fernandez-Lafuente
Guest Editor

Manuscript Submission Information

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Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Molecules is an international peer-reviewed open access monthly journal published by MDPI.

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Keywords

  • controlled enzyme immobilization and co-immobilization
  • heterofunctional supports
  • improved enzyme properties via immobilization
  • chemical or genetic modification and immobilization
  • immobilization of enzymes from thermophilic microorganisms

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Published Papers (21 papers)

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Editorial

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Open AccessEditorial Special Issue: Enzyme Immobilization 2016
Molecules 2017, 22(4), 601; https://doi.org/10.3390/molecules22040601
Received: 5 April 2017 / Revised: 6 April 2017 / Accepted: 6 April 2017 / Published: 8 April 2017
Cited by 2 | PDF Full-text (176 KB) | HTML Full-text | XML Full-text
Abstract
The use of enzymes as industrial biocatalysts is currently a solution for many problems of modern organic chemistry, which tries to carry out the most complex reactions under the rules of green chemistry [1].[...] Full article
(This article belongs to the Special Issue Enzyme Immobilization 2016)

Research

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Open AccessArticle Prevention of Bacterial Contamination of a Silica Matrix Containing Entrapped β-Galactosidase through the Action of Covalently Bound Lysozymes
Molecules 2017, 22(3), 377; https://doi.org/10.3390/molecules22030377
Received: 15 January 2017 / Revised: 24 February 2017 / Accepted: 25 February 2017 / Published: 28 February 2017
Cited by 2 | PDF Full-text (9298 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
β-galactosidase was successfully encapsulated within an amino-functionalised silica matrix using a “fish-in-net” approach and molecular imprinting technique followed by covalent binding of lysozyme via a glutaraldehyde-based method. Transmission electron microscopy (TEM), X-ray diffraction (XRD), scanning electron microscopy (SEM), and Fourier transform infrared (FTIR)
[...] Read more.
β-galactosidase was successfully encapsulated within an amino-functionalised silica matrix using a “fish-in-net” approach and molecular imprinting technique followed by covalent binding of lysozyme via a glutaraldehyde-based method. Transmission electron microscopy (TEM), X-ray diffraction (XRD), scanning electron microscopy (SEM), and Fourier transform infrared (FTIR) spectroscopy were used to characterise the silica matrix hosting the two enzymes. Both encapsulated β-galactosidase and bound lysozyme exhibited high enzymatic activities and outstanding operational stability in model reactions. Moreover, enzyme activities of the co-immobilised enzymes did not obviously change relative to enzymes immobilised separately. In antibacterial tests, bound lysozyme exhibited 95.5% and 89.6% growth inhibition of Staphylococcus aureus ATCC (American type culture collection) 653 and Escherichia coli ATCC 1122, respectively. In milk treated with co-immobilised enzymes, favourable results were obtained regarding reduction of cell viability and high lactose hydrolysis rate. In addition, when both co-immobilised enzymes were employed to treat milk, high operational and storage stabilities were observed. The results demonstrate that the use of co-immobilised enzymes holds promise as an industrial strategy for producing low lactose milk to benefit people with lactose intolerance. Full article
(This article belongs to the Special Issue Enzyme Immobilization 2016)
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Open AccessArticle Immobilization of Lipase from Penicillium sp. Section Gracilenta (CBMAI 1583) on Different Hydrophobic Supports: Modulation of Functional Properties
Molecules 2017, 22(2), 339; https://doi.org/10.3390/molecules22020339
Received: 10 December 2016 / Revised: 14 February 2017 / Accepted: 14 February 2017 / Published: 22 February 2017
Cited by 5 | PDF Full-text (944 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Lipases are promising enzymes that catalyze the hydrolysis of triacylglycerol ester bonds at the oil/water interface. Apart from allowing biocatalyst reuse, immobilization can also affect enzyme structure consequently influencing its activity, selectivity, and stability. The lipase from Penicillium sp. section Gracilenta (CBMAI 1583)
[...] Read more.
Lipases are promising enzymes that catalyze the hydrolysis of triacylglycerol ester bonds at the oil/water interface. Apart from allowing biocatalyst reuse, immobilization can also affect enzyme structure consequently influencing its activity, selectivity, and stability. The lipase from Penicillium sp. section Gracilenta (CBMAI 1583) was successfully immobilized on supports bearing butyl, phenyl, octyl, octadecyl, and divinylbenzyl hydrophobic moieties wherein lipases were adsorbed through the highly hydrophobic opened active site. The highest activity in aqueous medium was observed for the enzyme adsorbed on octyl support, with a 150% hyperactivation regarding the soluble enzyme activity, and the highest adsorption strength was verified with the most hydrophobic support (octadecyl Sepabeads), requiring 5% Triton X-100 to desorb the enzyme from the support. Most of the derivatives presented improved properties such as higher stability to pH, temperature, and organic solvents than the covalently immobilized CNBr derivative (prepared under very mild experimental conditions and thus a reference mimicking free-enzyme behavior). A 30.8- and 46.3-fold thermostabilization was achieved in aqueous medium, respectively, by the octyl Sepharose and Toyopearl butyl derivatives at 60 °C, in relation to the CNBr derivative. The octyl- and phenyl-agarose derivatives retained 50% activity after four and seven cycles of p-nitrophenyl palmitate hydrolysis, respectively. Different derivatives exhibited different properties regarding their properties for fish oil hydrolysis in aqueous medium and ethanolysis in anhydrous medium. The most active derivative in ethanolysis of fish oil was the enzyme adsorbed on a surface covered by divinylbenzyl moieties and it was 50-fold more active than the enzyme adsorbed on octadecyl support. Despite having identical mechanisms of immobilization, different hydrophobic supports seem to promote different shapes of the adsorbed open active site of the lipase and hence different functional properties. Full article
(This article belongs to the Special Issue Enzyme Immobilization 2016)
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Open AccessArticle Immobilized Trienzymatic System with Enhanced Stabilization for the Biotransformation of Lactose
Molecules 2017, 22(2), 284; https://doi.org/10.3390/molecules22020284
Received: 29 November 2016 / Revised: 7 February 2017 / Accepted: 8 February 2017 / Published: 22 February 2017
Cited by 1 | PDF Full-text (1467 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
The use of ketohexose isomerases is a powerful tool in lactose whey processing, but these enzymes can be very sensitive and expensive. Development of immobilized/stabilized biocatalysts could be a further option to improve the process. In this work, β-galactosidase from Bacillus circulans,
[...] Read more.
The use of ketohexose isomerases is a powerful tool in lactose whey processing, but these enzymes can be very sensitive and expensive. Development of immobilized/stabilized biocatalysts could be a further option to improve the process. In this work, β-galactosidase from Bacillus circulans, l-arabinose (d-galactose) isomerase from Enterococcus faecium, and d-xylose (d-glucose) isomerase from Streptomyces rubiginosus were immobilized individually onto Eupergit C and Eupergit C 250 L. Immobilized activity yields were over 90% in all cases. With the purpose of increasing thermostability of derivatives, two post-immobilization treatments were performed: alkaline incubation to favor the formation of additional covalent linkages, and blocking of excess oxirane groups by reacting with glycine. The greatest thermostability was achieved when alkaline incubation was carried out for 24 h, producing l-arabinose isomerase-Eupergit C derivatives with a half-life of 379 h and d-xylose isomerase-Eupergit C derivatives with a half-life of 554 h at 50 °C. Preliminary assays using immobilized and stabilized biocatalysts sequentially to biotransform lactose at pH 7.0 and 50 °C demonstrated improved performances as compared with soluble enzymes. Further improvements in ketohexose productivities were achieved when the three single-immobilizates were incubated simultaneously with lactose in a mono-reactor system. Full article
(This article belongs to the Special Issue Enzyme Immobilization 2016)
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Open AccessArticle Novel Magnetic Cross-Linked Cellulase Aggregates with a Potential Application in Lignocellulosic Biomass Bioconversion
Molecules 2017, 22(2), 269; https://doi.org/10.3390/molecules22020269
Received: 12 December 2016 / Accepted: 2 February 2017 / Published: 10 February 2017
Cited by 10 | PDF Full-text (2808 KB) | HTML Full-text | XML Full-text
Abstract
The utilization of renewable biomass resources to produce high-value chemicals by enzymatic processes is beneficial for alternative energy production, due to the accelerating depletion of fossil fuels. As immobilization techniques can improve enzyme stability and reusability, a novel magnetic cross-linked cellulase aggregate has
[...] Read more.
The utilization of renewable biomass resources to produce high-value chemicals by enzymatic processes is beneficial for alternative energy production, due to the accelerating depletion of fossil fuels. As immobilization techniques can improve enzyme stability and reusability, a novel magnetic cross-linked cellulase aggregate has been developed and applied for biomass bioconversion. The crosslinked aggregates could purify and immobilize enzymes in a single operation, and could then be combined with magnetic nanoparticles (MNPs), which provides easy separation of the materials. The immobilized cellulase showed a better activity at a wider temperature range and pH values than that of the free cellulase. After six cycles of consecutive reuse, the immobilized cellulase performed successful magnetic separation and retained 74% of its initial activity when carboxylmethyl cellulose (CMC) was used as the model substrate. Furthermore, the structure and morphology of the immobilized cellulase were studied by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Moreover, the immobilized cellulase was shown to hydrolyze bamboo biomass with a yield of 21%, and was re-used in biomass conversion up to four cycles with 38% activity retention, which indicated that the immobilized enzyme has good potential for biomass applications. Full article
(This article belongs to the Special Issue Enzyme Immobilization 2016)
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Open AccessArticle Immobilized Lipases on Functionalized Silica Particles as Potential Biocatalysts for the Synthesis of Fructose Oleate in an Organic Solvent/Water System
Molecules 2017, 22(2), 212; https://doi.org/10.3390/molecules22020212
Received: 26 December 2016 / Accepted: 24 January 2017 / Published: 30 January 2017
Cited by 11 | PDF Full-text (1558 KB) | HTML Full-text | XML Full-text
Abstract
Lipases from Thermomyces lanuginosus (TLL) and Pseudomonas fluorescens (PFL) wereimmobilized on functionalized silica particles aiming their use in the synthesis of fructose oleate in a tert‐butyl alcohol/water system. Silica particles were chemically modified with octyl (OS), octyl plus glutaraldehyde (OSGlu), octyl plus
[...] Read more.
Lipases from Thermomyces lanuginosus (TLL) and Pseudomonas fluorescens (PFL) wereimmobilized on functionalized silica particles aiming their use in the synthesis of fructose oleate in a tert‐butyl alcohol/water system. Silica particles were chemically modified with octyl (OS), octyl plus glutaraldehyde (OSGlu), octyl plus glyoxyl(OSGlx), and octyl plus epoxy groups(OSEpx). PFL was hyperactivated on all functionalized supports (more than 100% recovered activity) using low protein loading (1 mg/g), however, for TLL, this phenomenon was observed only using octyl‐silica (OS). All prepared biocatalysts exhibited high stability by incubating in tert‐butyl alcohol (half‐lives around 50 h at 65 °C). The biocatalysts prepared using OS and OSGlu as supports showed excellent performance in the synthesis of fructose oleate. High estersynthesis was observed when a small amount of water (1%, v/v) was added to the organic phase, allowing an ester productivity until five times (0.88–0.96 g/L.h) higher than in the absence of water (0.18–0.34 g/L.h) under fixed enzyme concentration (0.51 IU/g of solvent). Maximum ester productivity (16.1–18.1 g/L.h) was achieved for 30 min of reaction catalyzed by immobilized lipases on OS and OSGlu at 8.4 IU/mL of solvent. Operational stability tests showed satisfactory stability after four consecutive cycles of reaction. Full article
(This article belongs to the Special Issue Enzyme Immobilization 2016)
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Open AccessCommunication Co-Immobilization of Enzymes and Magnetic Nanoparticles by Metal-Nucleotide Hydrogelnanofibers for Improving Stability and Recycling
Molecules 2017, 22(1), 179; https://doi.org/10.3390/molecules22010179
Received: 13 November 2016 / Revised: 13 January 2017 / Accepted: 17 January 2017 / Published: 23 January 2017
Cited by 11 | PDF Full-text (1865 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
In this paper we report a facile method for preparing co-immobilized enzyme and magnetic nanoparticles (MNPs) using metal coordinated hydrogel nanofibers. Candida rugosa lipase (CRL) was selected as guest protein. For good aqueous dispersity, low price and other unique properties, citric acid-modified magnetic
[...] Read more.
In this paper we report a facile method for preparing co-immobilized enzyme and magnetic nanoparticles (MNPs) using metal coordinated hydrogel nanofibers. Candida rugosa lipase (CRL) was selected as guest protein. For good aqueous dispersity, low price and other unique properties, citric acid-modified magnetic iron oxide nanoparticles (CA-Fe3O4 NPs) have been widely used for immobilizing enzymes. As a result, the relative activity of CA-Fe3O4@Zn/AMP nanofiber-immobilized CRL increased by 8-fold at pH 10.0 and nearly 1-fold in a 50 °C water bath after 30 min, compared to free CRL. Moreover, the immobilized CRL had excellent long-term storage stability (nearly 80% releative activity after storage for 13 days). This work indicated that metal-nucleotide nanofibers could efficiently co-immobilize enzymes and MNPs simultaneously, and improve the stability of biocatalysts. Full article
(This article belongs to the Special Issue Enzyme Immobilization 2016)
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Open AccessArticle Desorption of Lipases Immobilized on Octyl-Agarose Beads and Coated with Ionic Polymers after Thermal Inactivation. Stronger Adsorption of Polymers/Unfolded Protein Composites
Received: 24 November 2016 / Revised: 2 January 2017 / Accepted: 3 January 2017 / Published: 5 January 2017
Cited by 12 | PDF Full-text (1388 KB) | HTML Full-text | XML Full-text
Abstract
Lipases from Candida antarctica (isoform B) and Rhizomucor miehei (CALB and RML) have been immobilized on octyl-agarose (OC) and further coated with polyethylenimine (PEI) and dextran sulfate (DS). The enzymes just immobilized on OC supports could be easily released from the support using
[...] Read more.
Lipases from Candida antarctica (isoform B) and Rhizomucor miehei (CALB and RML) have been immobilized on octyl-agarose (OC) and further coated with polyethylenimine (PEI) and dextran sulfate (DS). The enzymes just immobilized on OC supports could be easily released from the support using 2% SDS at pH 7, both intact or after thermal inactivation (in fact, after inactivation most enzyme molecules were already desorbed). The coating with PEI and DS greatly reduced the enzyme release during thermal inactivation and improved enzyme stability. However, using OC-CALB/RML-PEI-DS, the full release of the immobilized enzyme to reuse the support required more drastic conditions: a pH value of 3, a buffer concentration over 2 M, and temperatures above 45 °C. However, even these conditions were not able to fully release the thermally inactivated enzyme molecules from the support, being necessary to increase the buffer concentration to 4 M sodium phosphate and decrease the pH to 2.5. The formation of unfolded protein/polymers composites seems to be responsible for this strong interaction between the octyl and some anionic groups of OC supports. The support could be reused five cycles using these conditions with similar loading capacity of the support and stability of the immobilized enzyme. Full article
(This article belongs to the Special Issue Enzyme Immobilization 2016)
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Open AccessArticle Immobilization of Neutral Protease from Bacillus subtilis for Regioselective Hydrolysis of Acetylated Nucleosides: Application to Capecitabine Synthesis
Molecules 2016, 21(12), 1621; https://doi.org/10.3390/molecules21121621
Received: 10 September 2016 / Revised: 19 November 2016 / Accepted: 21 November 2016 / Published: 25 November 2016
Cited by 5 | PDF Full-text (2030 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
This paper describes the immobilization of the neutral protease from Bacillus subtilis and its application in the regioselective hydrolysis of acetylated nucleosides, including building blocks useful for the preparation of anticancer products. Regarding the immobilization study, different results have been obtained depending on
[...] Read more.
This paper describes the immobilization of the neutral protease from Bacillus subtilis and its application in the regioselective hydrolysis of acetylated nucleosides, including building blocks useful for the preparation of anticancer products. Regarding the immobilization study, different results have been obtained depending on the immobilization procedure. Epoxy hydrophobic carriers gave a poorly stable derivative that released almost 50% of the immobilized protein under the required reaction conditions. On the contrary, covalent immobilization on a differently activated hydrophilic carrier (agarose) resulted in very stable enzyme derivatives. In an attempt to explain the obtained enzyme immobilization results, the hypothetical localization of lysines on the enzyme surface was predicted in a 3D structure model of B. subtilis protease N built in silico by using the structure of Staphylococcus aureus metalloproteinase as the template. The immobilized enzyme shown a high regioselectivity in the hydrolysis of different peracetylated nucleosides. A stable enzyme derivative was obtained and successfully used in the development of efficient preparative bioprocesses for the hydrolysis of acetylated nucleosides, giving new intermediates for the synthesis of capecitabine in high yield. Full article
(This article belongs to the Special Issue Enzyme Immobilization 2016)
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Open AccessArticle Aroma Release in Wine Using Co-Immobilized Enzyme Aggregates
Molecules 2016, 21(11), 1485; https://doi.org/10.3390/molecules21111485
Received: 31 August 2016 / Revised: 18 October 2016 / Accepted: 19 October 2016 / Published: 8 November 2016
Cited by 6 | PDF Full-text (1464 KB) | HTML Full-text | XML Full-text
Abstract
Aroma is a remarkable factor of quality and consumer preference in wine, representing a distinctive feature of the product. Most aromatic compounds in varietals are in the form of glycosidic precursors, which are constituted by a volatile aglycone moiety linked to a glucose
[...] Read more.
Aroma is a remarkable factor of quality and consumer preference in wine, representing a distinctive feature of the product. Most aromatic compounds in varietals are in the form of glycosidic precursors, which are constituted by a volatile aglycone moiety linked to a glucose residue by an O-glycosidic bond; glucose is often linked to another sugar (arabinose, rhamnose or apiose). The use of soluble β-glycosidases for aroma liberation implies the addition of a precipitating agent to remove it from the product and precludes its reuse after one batch. An attractive option from a technological perspective that will aid in removing such constraints is the use of immobilized glycosidases. Immobilization by aggregation and crosslinking is a simple strategy producing enzyme catalysts of very high specific activity, being an attractive option to conventional immobilization to solid inert supports. The purpose of this work was the evaluation of co-immobilized β-glycosidases crosslinked aggregates produced from the commercial preparation AR2000, which contains the enzymes involved in the release of aromatic terpenes in Muscat wine (α-l-arabinofuranosidase and β-d-glucopyranosidase). To do so, experiments were conducted with co-immobilized crosslinked enzyme aggregates (combi-CLEAs), and with the soluble enzymes, using an experiment without enzyme addition as control. Stability of the enzymes at the conditions of winemaking was assessed and the volatiles composition of wine was determined by SPE-GC-MS. Stability of enzymes in combi-CLEAs was much higher than in soluble form, 80% of the initial activity remaining after 60 days in contact with the wine; at the same conditions, the soluble enzymes had lost 80% of their initial activities after 20 days. Such higher stabilities will allow prolonged use of the enzyme catalyst reducing its impact in the cost of winemaking. Wine treated with combi-CLEAs was the one exhibiting the highest concentration of total terpenes (18% higher than the control) and the highest concentrations of linalool (20% higher), nerol (20% higher) and geraniol (100% higher), which are the most important terpenes in determining Muscat typicity. Co-immobilized enzymes were highly stable at winemaking conditions, so their reutilization is possible and technologically attractive by reducing the impact of enzyme cost on winemaking cost. Full article
(This article belongs to the Special Issue Enzyme Immobilization 2016)
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Open AccessArticle Immobilization of α-Amylase from Anoxybacillus sp. SK3-4 on ReliZyme and Immobead Supports
Molecules 2016, 21(9), 1196; https://doi.org/10.3390/molecules21091196
Received: 21 July 2016 / Revised: 4 September 2016 / Accepted: 5 September 2016 / Published: 9 September 2016
Cited by 7 | PDF Full-text (8657 KB) | HTML Full-text | XML Full-text
Abstract
α-Amylase from Anoxybacillus sp. SK3-4 (ASKA) is a thermostable enzyme that produces a high level of maltose from starches. A truncated ASKA (TASKA) variant with improved expression and purification efficiency was characterized in an earlier study. In this work, TASKA was purified and
[...] Read more.
α-Amylase from Anoxybacillus sp. SK3-4 (ASKA) is a thermostable enzyme that produces a high level of maltose from starches. A truncated ASKA (TASKA) variant with improved expression and purification efficiency was characterized in an earlier study. In this work, TASKA was purified and immobilized through covalent attachment on three epoxide (ReliZyme EP403/M, Immobead IB-150P, and Immobead IB-150A) and an amino-epoxide (ReliZyme HFA403/M) activated supports. Several parameters affecting immobilization were analyzed, including the pH, temperature, and quantity (mg) of enzyme added per gram of support. The influence of the carrier surface properties, pore sizes, and lengths of spacer arms (functional groups) on biocatalyst performances were studied. Free and immobilized TASKAs were stable at pH 6.0–9.0 and active at pH 8.0. The enzyme showed optimal activity and considerable stability at 60 °C. Immobilized TASKA retained 50% of its initial activity after 5–12 cycles of reuse. Upon degradation of starches and amylose, only immobilized TASKA on ReliZyme HFA403/M has comparable hydrolytic ability with the free enzyme. To the best of our knowledge, this is the first report of an immobilization study of an α-amylase from Anoxybacillus spp. and the first report of α-amylase immobilization using ReliZyme and Immobeads as supports. Full article
(This article belongs to the Special Issue Enzyme Immobilization 2016)
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Open AccessArticle Catalytic Oxidation of Phenol and 2,4-Dichlorophenol by Using Horseradish Peroxidase Immobilized on Graphene Oxide/Fe3O4
Molecules 2016, 21(8), 1044; https://doi.org/10.3390/molecules21081044
Received: 12 April 2016 / Revised: 30 July 2016 / Accepted: 3 August 2016 / Published: 10 August 2016
Cited by 9 | PDF Full-text (1956 KB) | HTML Full-text | XML Full-text
Abstract
Graphene oxide/Fe3O4 (GO/Fe3O4) nanoparticles were synthesized by an ultrasonic-assisted reverse co-precipitation method, and then horseradish peroxidase (HRP) was covalently immobilized onto GO/Fe3O4 with 1-ethyl-3-(3-dimethyaminopropyl)carbodiimide (EDC) as a cross-linking agent. In order to enhance
[...] Read more.
Graphene oxide/Fe3O4 (GO/Fe3O4) nanoparticles were synthesized by an ultrasonic-assisted reverse co-precipitation method, and then horseradish peroxidase (HRP) was covalently immobilized onto GO/Fe3O4 with 1-ethyl-3-(3-dimethyaminopropyl)carbodiimide (EDC) as a cross-linking agent. In order to enhance the phenol removal efficiency and prevent the inactivation of the enzyme, the polyethylene glycol with highly hydrophilicity was added in this reaction, because the adsorption capacity for the polymer by degradation was stronger than the HRP. The results showed that the immobilized enzyme removed over 95% of phenol from aqueous solution. The catalytic condition was extensively optimized among the range of pH, mass ratio of PEG/phenol as well as initial concentration of immobilized enzyme and H2O2. The HRP immobilized on GO/Fe3O4 composite could be easily separated under a magnetic field from the reaction solution and reused. Full article
(This article belongs to the Special Issue Enzyme Immobilization 2016)
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Open AccessArticle Site-Specific, Covalent Immobilization of Dehalogenase ST2570 Catalyzed by Formylglycine-Generating Enzymes and Its Application in Batch and Semi-Continuous Flow Reactors
Molecules 2016, 21(7), 895; https://doi.org/10.3390/molecules21070895
Received: 2 April 2016 / Revised: 3 July 2016 / Accepted: 5 July 2016 / Published: 11 July 2016
Cited by 10 | PDF Full-text (1879 KB) | HTML Full-text | XML Full-text
Abstract
Formylglycine-generating enzymes can selectively recognize and oxidize cysteine residues within the sulfatase sub motif at the terminus of proteins to form aldehyde-bearing formylglycine (FGly) residues, and are normally used in protein labeling. In this study, an aldehyde tag was introduced to proteins using
[...] Read more.
Formylglycine-generating enzymes can selectively recognize and oxidize cysteine residues within the sulfatase sub motif at the terminus of proteins to form aldehyde-bearing formylglycine (FGly) residues, and are normally used in protein labeling. In this study, an aldehyde tag was introduced to proteins using formylglycine-generating enzymes encoded by a reconstructed set of the pET28a plasmid system for enzyme immobilization. The haloacid dehalogenase ST2570 from Sulfolobus tokodaii was used as a model enzyme. The C-terminal aldehyde-tagged ST2570 (ST2570CQ) exhibited significant enzymological properties, such as new free aldehyde groups, a high level of protein expression and improved enzyme activity. SBA-15 has widely been used as an immobilization support for its large surface and excellent thermal and chemical stability. It was functionalized with amino groups by aminopropyltriethoxysilane. The C-terminal aldehyde-tagged ST2570 was immobilized to SBA-15 by covalent binding. The site-specific immobilization of ST2570 avoided the chemical denaturation that occurs in general covalent immobilization and resulted in better fastening compared to physical adsorption. The site-specific immobilized ST2570 showed 3-fold higher thermal stability, 1.2-fold higher catalytic ability and improved operational stability than free ST2570. The site-specific immobilized ST2570 retained 60% of its original activity after seven cycles of batch operation, and it was superior to the ST2570 immobilized to SBA-15 by physical adsorption, which loses 40% of its original activity when used for the second time. It is remarkable that the site-specific immobilized ST2570 still retained 100% of its original activity after 10 cycles of reuse in the semi-continuous flow reactor. Overall, these results provide support for the industrial-scale production and application of site-specific, covalently immobilized ST2570. Full article
(This article belongs to the Special Issue Enzyme Immobilization 2016)
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Open AccessArticle Construction of an Immobilized Thermophilic Esterase on Epoxy Support for Poly(ε-caprolactone) Synthesis
Molecules 2016, 21(6), 796; https://doi.org/10.3390/molecules21060796
Received: 4 May 2016 / Revised: 14 June 2016 / Accepted: 16 June 2016 / Published: 18 June 2016
Cited by 2 | PDF Full-text (1809 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Developing an efficient immobilized enzyme is of great significance for improving the operational stability of enzymes in poly(ε-caprolactone) synthesis. In this paper, a thermophilic esterase AFEST from the archaeon Archaeoglobus fulgidus was successfully immobilized on the epoxy support Sepabeads EC-EP via covalent attachment,
[...] Read more.
Developing an efficient immobilized enzyme is of great significance for improving the operational stability of enzymes in poly(ε-caprolactone) synthesis. In this paper, a thermophilic esterase AFEST from the archaeon Archaeoglobus fulgidus was successfully immobilized on the epoxy support Sepabeads EC-EP via covalent attachment, and the immobilized enzyme was then employed as a biocatalyst for poly(ε-caprolactone) synthesis. The enzyme loading and recovered activity of immobilized enzyme was measured to be 72 mg/g and 10.4 U/mg using p-nitrophenyl caprylate as the substrate at 80 °C, respectively. Through the optimization of reaction conditions (enzyme concentration, temperature, reaction time and medium), poly(ε-caprolactone) was obtained with 100% monomer conversion and low number-average molecular weight (Mn < 1300 g/mol). Further, the immobilized enzyme exhibited excellent reusability, with monomer conversion values exceeding 75% during 15 batch reactions. Finally, poly(ε-caprolactone) was enzymatically synthesized with an isolated yield of 75% and Mn value of 3005 g/mol in a gram-scale reaction. Full article
(This article belongs to the Special Issue Enzyme Immobilization 2016)
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Open AccessArticle Tailoring the Spacer Arm for Covalent Immobilization of Candida antarctica Lipase B—Thermal Stabilization by Bisepoxide-Activated Aminoalkyl Resins in Continuous-Flow Reactors
Molecules 2016, 21(6), 767; https://doi.org/10.3390/molecules21060767
Received: 10 May 2016 / Revised: 7 June 2016 / Accepted: 8 June 2016 / Published: 13 June 2016
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Abstract
An efficient and easy-to-perform method was developed for immobilization of CaLB on mesoporous aminoalkyl polymer supports by bisepoxide activation. Polyacrylate resins (100–300 µm; ~50 nm pores) with different aminoalkyl functional groups (ethylamine: EA and hexylamine: HA) were modified with bisepoxides differing in
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An efficient and easy-to-perform method was developed for immobilization of CaLB on mesoporous aminoalkyl polymer supports by bisepoxide activation. Polyacrylate resins (100–300 µm; ~50 nm pores) with different aminoalkyl functional groups (ethylamine: EA and hexylamine: HA) were modified with bisepoxides differing in the length, rigidity and hydrophobicity of the units linking the two epoxy functions. After immobilization, the different CaLB preparations were evaluated using the lipase-catalyzed kinetic resolution (KR) of racemic 1-phenylethanol (rac-1) in batch mode and in a continuous-flow reactor as well. Catalytic activity, enantiomer selectivity, recyclability, and the mechanical and long-term stability of CaLB immobilized on the various supports were tested. The most active CaLB preparation (on HA-resin activated with 1,6-hexanediol diglycidyl ether—HDGE) retained 90% of its initial activity after 13 consecutive reaction cycles or after 12 month of storage at 4 °C. The specific rate (rflow), enantiomer selectivity (E) and enantiomeric excess (ee) achievable with the best immobilized CaLB preparations were studied as a function of temperature in kinetic resolution of rac-1 performed in continuous-flow packed-bed bioreactors. The optimum temperature of the most active HA-HDGE CaLB in continuous-flow mode was 60 °C. Although CaLB immobilized on the glycerol diglycidyl ether (GDGE)-activated EA-resin was less active and less selective, a much higher optimum temperature (80 °C) was observed with this form in continuous-flow mode KR of rac-1. Full article
(This article belongs to the Special Issue Enzyme Immobilization 2016)
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Open AccessArticle Stabilization of Candida antarctica Lipase B (CALB) Immobilized on Octyl Agarose by Treatment with Polyethyleneimine (PEI)
Molecules 2016, 21(6), 751; https://doi.org/10.3390/molecules21060751
Received: 13 May 2016 / Revised: 1 June 2016 / Accepted: 6 June 2016 / Published: 8 June 2016
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Abstract
Lipase B from Candida antarctica (CALB) was immobilized on octyl agarose (OC) and physically modified with polyethyleneimine (PEI) in order to confer a strong ion exchange character to the enzyme and thus enable the immobilization of other enzymes on its surface. The enzyme
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Lipase B from Candida antarctica (CALB) was immobilized on octyl agarose (OC) and physically modified with polyethyleneimine (PEI) in order to confer a strong ion exchange character to the enzyme and thus enable the immobilization of other enzymes on its surface. The enzyme activity was fully maintained during the coating and the thermal stability was marginally improved. The enzyme release from the support by incubation in the non-ionic detergent Triton X-100 was more difficult after the PEI-coating, suggesting that some intermolecular physical crosslinking had occurred, making this desorption more difficult. Thermal stability was marginally improved, but the stability of the OCCALB-PEI was significantly better than that of OCCALB during inactivation in mixtures of aqueous buffer and organic cosolvents. SDS-PAGE analysis of the inactivated biocatalyst showed the OCCALB released some enzyme to the medium during inactivation, and this was partially prevented by coating with PEI. This effect was obtained without preventing the possibility of reuse of the support by incubation in 2% ionic detergents. That way, this modified CALB not only has a strong anion exchange nature, while maintaining the activity, but it also shows improved stability under diverse reaction conditions without affecting the reversibility of the immobilization. Full article
(This article belongs to the Special Issue Enzyme Immobilization 2016)
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Open AccessArticle Reversible Immobilization of Lipases on Heterofunctional Octyl-Amino Agarose Beads Prevents Enzyme Desorption
Molecules 2016, 21(5), 646; https://doi.org/10.3390/molecules21050646
Received: 16 March 2016 / Revised: 28 April 2016 / Accepted: 9 May 2016 / Published: 16 May 2016
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Abstract
Two different heterofunctional octyl-amino supports have been prepared using ethylenediamine and hexylendiamine (OCEDA and OCHDA) and utilized to immobilize five lipases (lipases A (CALA) and B (CALB) from Candida antarctica, lipases from Thermomyces lanuginosus (TLL), from Rhizomucor miehei (RML) and from Candida
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Two different heterofunctional octyl-amino supports have been prepared using ethylenediamine and hexylendiamine (OCEDA and OCHDA) and utilized to immobilize five lipases (lipases A (CALA) and B (CALB) from Candida antarctica, lipases from Thermomyces lanuginosus (TLL), from Rhizomucor miehei (RML) and from Candida rugosa (CRL) and the phospholipase Lecitase Ultra (LU). Using pH 5 and 50 mM sodium acetate, the immobilizations proceeded via interfacial activation on the octyl layer, after some ionic bridges were established. These supports did not release enzyme when incubated at Triton X-100 concentrations that released all enzyme molecules from the octyl support. The octyl support produced significant enzyme hyperactivation, except for CALB. However, the activities of the immobilized enzymes were usually slightly higher using the new supports than the octyl ones. Thermal and solvent stabilities of LU and TLL were significantly improved compared to the OC counterparts, while in the other enzymes the stability decreased in most cases (depending on the pH value). As a general rule, OCEDA had lower negative effects on the stability of the immobilized enzymes than OCHDA and while in solvent inactivation the enzyme molecules remained attached to the support using the new supports and were released using monofunctional octyl supports, in thermal inactivations this only occurred in certain cases. Full article
(This article belongs to the Special Issue Enzyme Immobilization 2016)
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Review

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Open AccessFeature PaperReview Agarose and Its Derivatives as Supports for Enzyme Immobilization
Molecules 2016, 21(11), 1577; https://doi.org/10.3390/molecules21111577
Received: 27 September 2016 / Revised: 12 November 2016 / Accepted: 16 November 2016 / Published: 19 November 2016
Cited by 27 | PDF Full-text (2984 KB) | HTML Full-text | XML Full-text
Abstract
Agarose is a polysaccharide obtained from some seaweeds, with a quite particular structure that allows spontaneous gelation. Agarose-based beads are highly porous, mechanically resistant, chemically and physically inert, and sharply hydrophilic. These features—that could be further improved by means of covalent cross-linking—render them
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Agarose is a polysaccharide obtained from some seaweeds, with a quite particular structure that allows spontaneous gelation. Agarose-based beads are highly porous, mechanically resistant, chemically and physically inert, and sharply hydrophilic. These features—that could be further improved by means of covalent cross-linking—render them particularly suitable for enzyme immobilization with a wide range of derivatization methods taking advantage of chemical modification of a fraction of the polymer hydroxyls. The main properties of the polymer are described here, followed by a review of cross-linking and derivatization methods. Some recent, innovative procedures to optimize the catalytic activity and operational stability of the obtained preparations are also described, together with multi-enzyme immobilized systems and the main guidelines to exploit their performances. Full article
(This article belongs to the Special Issue Enzyme Immobilization 2016)
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Open AccessReview Enzyme Engineering for In Situ Immobilization
Molecules 2016, 21(10), 1370; https://doi.org/10.3390/molecules21101370
Received: 30 August 2016 / Revised: 5 October 2016 / Accepted: 5 October 2016 / Published: 14 October 2016
Cited by 17 | PDF Full-text (1250 KB) | HTML Full-text | XML Full-text
Abstract
Enzymes are used as biocatalysts in a vast range of industrial applications. Immobilization of enzymes to solid supports or their self-assembly into insoluble particles enhances their applicability by strongly improving properties such as stability in changing environments, re-usability and applicability in continuous biocatalytic
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Enzymes are used as biocatalysts in a vast range of industrial applications. Immobilization of enzymes to solid supports or their self-assembly into insoluble particles enhances their applicability by strongly improving properties such as stability in changing environments, re-usability and applicability in continuous biocatalytic processes. The possibility of co-immobilizing various functionally related enzymes involved in multistep synthesis, conversion or degradation reactions enables the design of multifunctional biocatalyst with enhanced performance compared to their soluble counterparts. This review provides a brief overview of up-to-date in vitro immobilization strategies while focusing on recent advances in enzyme engineering towards in situ self-assembly into insoluble particles. In situ self-assembly approaches include the bioengineering of bacteria to abundantly form enzymatically active inclusion bodies such as enzyme inclusions or enzyme-coated polyhydroxyalkanoate granules. These one-step production strategies for immobilized enzymes avoid prefabrication of the carrier as well as chemical cross-linking or attachment to a support material while the controlled oriented display strongly enhances the fraction of accessible catalytic sites and hence functional enzymes. Full article
(This article belongs to the Special Issue Enzyme Immobilization 2016)
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Open AccessReview Immobilization of Glycoside Hydrolase Families GH1, GH13, and GH70: State of the Art and Perspectives
Molecules 2016, 21(8), 1074; https://doi.org/10.3390/molecules21081074
Received: 27 July 2016 / Revised: 11 August 2016 / Accepted: 12 August 2016 / Published: 17 August 2016
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Abstract
Glycoside hydrolases (GH) are enzymes capable to hydrolyze the glycosidic bond between two carbohydrates or even between a carbohydrate and a non-carbohydrate moiety. Because of the increasing interest for industrial applications of these enzymes, the immobilization of GH has become an important development
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Glycoside hydrolases (GH) are enzymes capable to hydrolyze the glycosidic bond between two carbohydrates or even between a carbohydrate and a non-carbohydrate moiety. Because of the increasing interest for industrial applications of these enzymes, the immobilization of GH has become an important development in order to improve its activity, stability, as well as the possibility of its reuse in batch reactions and in continuous processes. In this review, we focus on the broad aspects of immobilization of enzymes from the specific GH families. A brief introduction on methods of enzyme immobilization is presented, discussing some advantages and drawbacks of this technology. We then review the state of the art of enzyme immobilization of families GH1, GH13, and GH70, with special attention on the enzymes β-glucosidase, α-amylase, cyclodextrin glycosyltransferase, and dextransucrase. In each case, the immobilization protocols are evaluated considering their positive and negative aspects. Finally, the perspectives on new immobilization methods are briefly presented. Full article
(This article belongs to the Special Issue Enzyme Immobilization 2016)
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Open AccessCommentary Solid-State Metalloproteins—An Alternative to Immobilisation
Molecules 2016, 21(7), 919; https://doi.org/10.3390/molecules21070919
Received: 12 May 2016 / Revised: 7 July 2016 / Accepted: 8 July 2016 / Published: 14 July 2016
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Abstract
This commentary outlines a protein engineering approach as an alternative to immobilisation developed in our laboratory. We use a recombinant silk protein into which metal active sites can be incorporated to produce solid-state metalloprotein materials. The silk protein directly coordinates to the metal
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This commentary outlines a protein engineering approach as an alternative to immobilisation developed in our laboratory. We use a recombinant silk protein into which metal active sites can be incorporated to produce solid-state metalloprotein materials. The silk protein directly coordinates to the metal centres providing control over their reactivity akin to that seen in naturally occurring metalloproteins. These solid-state materials are remarkably stable at a range of temperatures and different solvent conditions. I discuss the genesis of this approach and highlight areas where such solid-state materials could find application. Full article
(This article belongs to the Special Issue Enzyme Immobilization 2016)
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