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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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Editorial

Jump to: Research, Review, Other

Open AccessEditorial Special Issue: Enzyme Immobilization 2016
Molecules 2017, 22(4), 601; doi:10.3390/molecules22040601
Received: 5 April 2017 / Revised: 6 April 2017 / Accepted: 6 April 2017 / Published: 8 April 2017
PDF Full-text (176 KB) | HTML Full-text | XML Full-text
(This article belongs to the Special Issue Enzyme Immobilization 2016)
Open AccessEditorial Editorial: Special Issue — Enzyme Immobilization
Molecules 2014, 19(12), 20671-20674; doi:10.3390/molecules191220671
Received: 5 December 2014 / Accepted: 8 December 2014 / Published: 10 December 2014
Cited by 3 | PDF Full-text (645 KB) | HTML Full-text | XML Full-text
Abstract
Immobilization of enzymes and proteins is a requirement for many industrial enzyme applications, as this facilitates enzyme recovery and reuse. Bearing in mind this necessity, the coupling of immobilization to the improvement of other enzyme features has been pursued by many researchers, and
[...] Read more.
Immobilization of enzymes and proteins is a requirement for many industrial enzyme applications, as this facilitates enzyme recovery and reuse. Bearing in mind this necessity, the coupling of immobilization to the improvement of other enzyme features has been pursued by many researchers, and nowadays immobilization is recognized as a tool to improve not only stability, but also enzyme selectivity, specificity, resistance to inhibition or chemical modifiers, etc. To achieve these overall improvements of enzymes’ properties via immobilization, it is necessary to both develop new immobilization systems suitable for these purposes, and to achieve a deeper knowledge of the mechanisms of interaction between enzymes and activated solids. That way, immobilization of enzymes, far being an old-fashioned methodology to just reuse these expensive biocatalysts, is a tool of continuous interest that requires a continuous effort to be exploited in all its potential. This special issue collects 23 papers reporting advances in the field of immobilization of enzymes.[...] Full article
(This article belongs to the Special Issue Enzyme Immobilization)

Research

Jump to: Editorial, Review, Other

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; doi:10.3390/molecules22030377
Received: 15 January 2017 / Revised: 24 February 2017 / Accepted: 25 February 2017 / Published: 28 February 2017
Cited by 1 | 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; doi:10.3390/molecules22020339
Received: 10 December 2016 / Revised: 14 February 2017 / Accepted: 14 February 2017 / Published: 22 February 2017
Cited by 3 | 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; doi: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; doi:10.3390/molecules22020269
Received: 12 December 2016 / Accepted: 2 February 2017 / Published: 10 February 2017
Cited by 3 | 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; doi:10.3390/molecules22020212
Received: 26 December 2016 / Accepted: 24 January 2017 / Published: 30 January 2017
Cited by 7 | 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; doi:10.3390/molecules22010179
Received: 13 November 2016 / Revised: 13 January 2017 / Accepted: 17 January 2017 / Published: 23 January 2017
Cited by 4 | 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
Molecules 2017, 22(1), 91; doi:10.3390/molecules22010091
Received: 24 November 2016 / Revised: 2 January 2017 / Accepted: 3 January 2017 / Published: 5 January 2017
Cited by 7 | 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; doi:10.3390/molecules21121621
Received: 10 September 2016 / Revised: 19 November 2016 / Accepted: 21 November 2016 / Published: 25 November 2016
Cited by 3 | 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; doi:10.3390/molecules21111485
Received: 31 August 2016 / Revised: 18 October 2016 / Accepted: 19 October 2016 / Published: 8 November 2016
Cited by 5 | 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; doi:10.3390/molecules21091196
Received: 21 July 2016 / Revised: 4 September 2016 / Accepted: 5 September 2016 / Published: 9 September 2016
Cited by 5 | 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; doi:10.3390/molecules21081044
Received: 12 April 2016 / Revised: 30 July 2016 / Accepted: 3 August 2016 / Published: 10 August 2016
Cited by 4 | 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; doi:10.3390/molecules21070895
Received: 2 April 2016 / Revised: 3 July 2016 / Accepted: 5 July 2016 / Published: 11 July 2016
Cited by 9 | 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)
Open AccessArticle Construction of an Immobilized Thermophilic Esterase on Epoxy Support for Poly(ε-caprolactone) Synthesis
Molecules 2016, 21(6), 796; doi:10.3390/molecules21060796
Received: 4 May 2016 / Revised: 14 June 2016 / Accepted: 16 June 2016 / Published: 18 June 2016
Cited by 1 | 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)
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; doi:10.3390/molecules21060767
Received: 10 May 2016 / Revised: 7 June 2016 / Accepted: 8 June 2016 / Published: 13 June 2016
Cited by 6 | PDF Full-text (3148 KB) | HTML Full-text | XML Full-text
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)
Open AccessArticle Stabilization of Candida antarctica Lipase B (CALB) Immobilized on Octyl Agarose by Treatment with Polyethyleneimine (PEI)
Molecules 2016, 21(6), 751; doi:10.3390/molecules21060751
Received: 13 May 2016 / Revised: 1 June 2016 / Accepted: 6 June 2016 / Published: 8 June 2016
Cited by 15 | PDF Full-text (2855 KB) | HTML Full-text | XML Full-text
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; doi:10.3390/molecules21050646
Received: 16 March 2016 / Revised: 28 April 2016 / Accepted: 9 May 2016 / Published: 16 May 2016
Cited by 18 | PDF Full-text (4510 KB) | HTML Full-text | XML Full-text
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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Open AccessArticle Enzymatic Cellulose Hydrolysis: Enzyme Reusability and Visualization of β-Glucosidase Immobilized in Calcium Alginate
Molecules 2014, 19(12), 19390-19406; doi:10.3390/molecules191219390
Received: 31 July 2014 / Revised: 15 November 2014 / Accepted: 17 November 2014 / Published: 25 November 2014
Cited by 15 | PDF Full-text (2491 KB) | HTML Full-text | XML Full-text
Abstract
The high cellulase enzyme dosages required for hydrolysis of cellulose is a major cost challenge in lignocellulosic ethanol production. One method to decrease the enzyme dosage and increase biocatalytic productivity is to re-use β-glucosidase (BG) via immobilization. In the present research, glutaraldehyde cross-linked
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The high cellulase enzyme dosages required for hydrolysis of cellulose is a major cost challenge in lignocellulosic ethanol production. One method to decrease the enzyme dosage and increase biocatalytic productivity is to re-use β-glucosidase (BG) via immobilization. In the present research, glutaraldehyde cross-linked BG was entrapped in calcium alginate gel particles. More than 60% of the enzyme activity could be recovered under optimized conditions, and glutaraldehyde cross-linking decreased leakage of BG from the calcium alginate particles. The immobilized BG aggregates were visualized by confocal laser scanning microscopy (CLSM). The CLSM images, which we believe are the first to be published, corroborate that more BG aggregates were entrapped in the matrix when the enzymes were cross-linked by glutaraldehyde as opposed to when they are not cross-linked. The particles with the immobilized BG were recycled for cellulase catalyzed hydrolysis of Avicel. No significant loss in BG activity was observed for up to 20 rounds of reaction recycle steps of the BG particles of 48 h each, verifying a significant stabilization of the BG by immobilization. Similar high glucose yields were obtained by one round of enzymatic hydrolysis of hydrothermally pretreated barley straw during a 72 h reaction with immobilized BG and free BG. Full article
(This article belongs to the Special Issue Enzyme Immobilization)
Open AccessArticle Substrate Specificity and Enzyme Recycling Using Chitosan Immobilized Laccase
Molecules 2014, 19(10), 16794-16809; doi:10.3390/molecules191016794
Received: 16 May 2014 / Revised: 22 September 2014 / Accepted: 8 October 2014 / Published: 17 October 2014
Cited by 9 | PDF Full-text (389 KB) | HTML Full-text | XML Full-text
Abstract
The immobilization of laccase (Aspergillus sp.) on chitosan by cross-linking and its application in bioconversion of phenolic compounds in batch reactors were studied. Investigation was performed using laccase immobilized via chemical cross-linking due to the higher enzymatic operational stability of this method
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The immobilization of laccase (Aspergillus sp.) on chitosan by cross-linking and its application in bioconversion of phenolic compounds in batch reactors were studied. Investigation was performed using laccase immobilized via chemical cross-linking due to the higher enzymatic operational stability of this method as compared to immobilization via physical adsorption. To assess the influence of different substrate functional groups on the enzyme’s catalytic efficiency, substrate specificity was investigated using chitosan-immobilized laccase and eighteen different phenol derivatives. It was observed that 4-nitrophenol was not oxidized, while 2,5-xylenol, 2,6-xylenol, 2,3,5-trimethylphenol, syringaldazine, 2,6-dimetoxyphenol and ethylphenol showed reaction yields up 90% at 40 °C. The kinetic of process, enzyme recyclability and operational stability were studied. In batch reactors, it was not possible to reuse the enzyme when it was applied to syringaldazne bioconversion. However, when the enzyme was applied to bioconversion of 2,6-DMP, the activity was stable for eight reaction batches. Full article
(This article belongs to the Special Issue Enzyme Immobilization)
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Open AccessArticle Immobilization of Horseradish Peroxidase on NH2-Modified Magnetic Fe3O4/SiO2 Particles and Its Application in Removal of 2,4-Dichlorophenol
Molecules 2014, 19(10), 15768-15782; doi:10.3390/molecules191015768
Received: 14 May 2014 / Revised: 9 August 2014 / Accepted: 7 September 2014 / Published: 29 September 2014
Cited by 37 | PDF Full-text (1766 KB) | HTML Full-text | XML Full-text
Abstract
Fe3O4 nanoparticles were prepared by a co-precipitation method with the assistance of ultrasound irradiation, and then coated with silica generated by hydrolysis and condensation of tetraethoxysilane. The silica-coated Fe3O4 nanoparticles were further modified with 3-aminopropyltriethoxysilane, resulting in
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Fe3O4 nanoparticles were prepared by a co-precipitation method with the assistance of ultrasound irradiation, and then coated with silica generated by hydrolysis and condensation of tetraethoxysilane. The silica-coated Fe3O4 nanoparticles were further modified with 3-aminopropyltriethoxysilane, resulting in anchoring of primary amine groups on the surface of the particles. Horseradish peroxidase (HRP) was then immobilized on the magnetic core-shell particles by using glutaraldehyde as a crosslinking agent. Immobilization conditions were optimized to obtain the highest relative activity of the immobilized enzyme. It was found the durability of the immobilized enzyme to heating and pH variation were improved in comparison with free HRP. The apparent Michaelis constants of the immobilized HRP and free HRP with substrate were compared, showing that the enzyme activity of the immobilized HRP was close to that of free HRP. The HRP immobilized particles, as an enzyme catalyst, were used to activate H2O2 for degrading 2,4-dichlorophenol. The rapid degradation of 2,4-dichlorophenol indicated that the immobilized enzyme has potential applications for removing organic pollutants. Full article
(This article belongs to the Special Issue Enzyme Immobilization)
Open AccessArticle Carbon Nanotubes as Supports for Inulinase Immobilization
Molecules 2014, 19(9), 14615-14624; doi:10.3390/molecules190914615
Received: 16 May 2014 / Revised: 16 August 2014 / Accepted: 22 August 2014 / Published: 15 September 2014
Cited by 11 | PDF Full-text (731 KB) | HTML Full-text | XML Full-text
Abstract
The commercial inulinase obtained from Aspergillus niger was non-covalently immobilized on multiwalled carbon nanotubes (MWNT-COOH). The immobilization conditions for the carbon nanotubes were defined by the central composite rotational design (CCRD). The effects of enzyme concentration (0.8%–1.7% v/v) and adsorbent:adsorbate ratio (1:460–1:175) on
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The commercial inulinase obtained from Aspergillus niger was non-covalently immobilized on multiwalled carbon nanotubes (MWNT-COOH). The immobilization conditions for the carbon nanotubes were defined by the central composite rotational design (CCRD). The effects of enzyme concentration (0.8%–1.7% v/v) and adsorbent:adsorbate ratio (1:460–1:175) on the enzyme immobilization were studied. The adsorbent:adsorbate ratio variable has positive effect and the enzyme concentration has a negative effect on the inulinase immobilization (U/g) response at the 90% significance level. These results show that the lower the enzyme concentration and the higher the adsorbent:adsorbate ratio, better is the immobilization. According to the results, it is possible to observe that the carbon nanotubes present an effective inulinase adsorption. Fast adsorption in about six minutes and a loading capacity of 51,047 U/g support using a 1.3% (v/v) inulinase concentration and a 1:460 adsorbent:adsorbate ratio was observed. The effects of temperature on the immobilized enzyme activity were evaluated, showing better activity at 50 °C. The immobilized enzyme maintained 100% of its activity during five weeks at room temperature. The immobilization strategy with MWNT-COOH was defined by the experimental design, showing that inulinase immobilization is a promising biotechnological application of carbon nanotubes. Full article
(This article belongs to the Special Issue Enzyme Immobilization)
Open AccessArticle Influence of the Morphology of Core-Shell Supports on the Immobilization of Lipase B from Candida antarctica
Molecules 2014, 19(8), 12509-12530; doi:10.3390/molecules190812509
Received: 19 May 2014 / Revised: 28 July 2014 / Accepted: 1 August 2014 / Published: 18 August 2014
Cited by 7 | PDF Full-text (1146 KB) | HTML Full-text | XML Full-text
Abstract
Core-shell polymer particles with different properties were produced through combined suspension-emulsion polymerizations and employed as supports for immobilization of lipase B from Candida antarctica. In order to evaluate how the morphology of the particles affects the immobilization parameters, empirical models were developed
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Core-shell polymer particles with different properties were produced through combined suspension-emulsion polymerizations and employed as supports for immobilization of lipase B from Candida antarctica. In order to evaluate how the morphology of the particles affects the immobilization parameters, empirical models were developed to describe the performance of the biocatalysts as a function of the specific area, volume of pores and average pore diameter of the supports. It was observed that the average pore sizes did not affect the enzymatic activities in the analyzed range of pore sizes. It was also observed that the increase of the specific area (and of the volume of pores) led to higher enzyme loadings, also leading to an increase in the esterification activity, as expected. However, when the specific area (and volume of pores) increased, the hydrolytic activity and the retention of hydrolytic activity of the biocatalysts decreased, indicating the existence of diffusional limitations for some hydrolytic reactions, probably because of the high reaction rates. Full article
(This article belongs to the Special Issue Enzyme Immobilization)
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Open AccessArticle Biocatalytic Behaviour of Immobilized Rhizopus oryzae Lipase in the 1,3-Selective Ethanolysis of Sunflower Oil to Obtain a Biofuel Similar to Biodiesel
Molecules 2014, 19(8), 11419-11439; doi:10.3390/molecules190811419
Received: 5 May 2014 / Revised: 22 July 2014 / Accepted: 22 July 2014 / Published: 4 August 2014
Cited by 8 | PDF Full-text (1249 KB) | HTML Full-text | XML Full-text
Abstract
A new biofuel similar to biodiesel was obtained in the 1,3-selective transesterification reaction of sunflower oil with ethanol using as biocatalyst a Rhizopus oryzae lipase (ROL) immobilized on Sepiolite, an inorganic support. The studied lipase was a low cost powdered enzyme preparation, Biolipase-R,
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A new biofuel similar to biodiesel was obtained in the 1,3-selective transesterification reaction of sunflower oil with ethanol using as biocatalyst a Rhizopus oryzae lipase (ROL) immobilized on Sepiolite, an inorganic support. The studied lipase was a low cost powdered enzyme preparation, Biolipase-R, from Biocon-Spain, a multipurpose additive used in food industry. In this respect, it is developed a study to optimize the immobilization procedure of these lipases on Sepiolite. Covalent immobilization was achieved by the development of an inorganic-organic hybrid linker formed by a functionalized hydrocarbon chain with a pendant benzaldehyde, bonded to the AlPO4 support surface. Thus, the covalent immobilization of lipases on amorphous AlPO4/sepiolite (20/80 wt %) support was evaluated by using two different linkers (p-hydroxybenzaldehyde and benzylamine-terephthalic aldehyde, respectively). Besides, the catalytic behavior of lipases after physical adsorption on the demineralized sepiolite was also evaluated. Obtained results indicated that covalent immobilization with the p-hydroxybenzaldehyde linker gave the best biocatalytic behavior. Thus, this covalently immobilized lipase showed a remarkable stability as well as an excellent capacity of reutilization (more than five successive reuses) without a significant loss of its initial catalytic activity. This could allow a more efficient fabrication of biodiesel minimizing the glycerol waste production. Full article
(This article belongs to the Special Issue Enzyme Immobilization)
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Open AccessArticle Nucleoside 2'-Deoxyribosyltransferase from Psychrophilic Bacterium Bacillus psychrosaccharolyticus — Preparation of an Immobilized Biocatalyst for the Enzymatic Synthesis of Therapeutic Nucleosides
Molecules 2014, 19(8), 11231-11249; doi:10.3390/molecules190811231
Received: 11 June 2014 / Revised: 23 July 2014 / Accepted: 24 July 2014 / Published: 31 July 2014
Cited by 9 | PDF Full-text (936 KB) | HTML Full-text | XML Full-text
Abstract
Nucleoside 2'-deoxyribosyltransferase (NDT) from the psychrophilic bacterium Bacillus psychrosaccharolyticus CECT 4074 has been cloned and produced for the first time. A preliminary characterization of the recombinant protein indicates that the enzyme is an NDT type II since it catalyzes the transfer of 2'-deoxyribose
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Nucleoside 2'-deoxyribosyltransferase (NDT) from the psychrophilic bacterium Bacillus psychrosaccharolyticus CECT 4074 has been cloned and produced for the first time. A preliminary characterization of the recombinant protein indicates that the enzyme is an NDT type II since it catalyzes the transfer of 2'-deoxyribose between purines and pyrimidines. The enzyme (BpNDT) displays a high activity and stability in a broad range of pH and temperature. In addition, different approaches for the immobilization of BpNDT onto several supports have been studied in order to prepare a suitable biocatalyst for the one-step industrial enzymatic synthesis of different therapeutic nucleosides. Best results were obtained by adsorbing the enzyme on PEI-functionalized agarose and subsequent cross-linking with aldehyde-dextran (20 kDa and 70% oxidation degree). The immobilized enzyme could be recycled for at least 30 consecutive cycles in the synthesis of 2'-deoxyadenosine from 2'-deoxyuridine and adenine at 37 °C and pH 8.0, with a 25% loss of activity. High conversion yield of trifluridine (64.4%) was achieved in 2 h when 20 mM of 2'-deoxyuridine and 10 mM 5-trifluorothymine were employed in the transglycosylation reaction catalyzed by immobilized BpNDT at 37 °C and pH 7.5. Full article
(This article belongs to the Special Issue Enzyme Immobilization)
Open AccessArticle Additives Enhancing the Catalytic Properties of Lipase from Burkholderia cepacia Immobilized on Mixed-Function-Grafted Mesoporous Silica Gel
Molecules 2014, 19(7), 9818-9837; doi:10.3390/molecules19079818
Received: 16 May 2014 / Revised: 23 June 2014 / Accepted: 2 July 2014 / Published: 8 July 2014
Cited by 17 | PDF Full-text (517 KB) | HTML Full-text | XML Full-text
Abstract
Effects of various additives on the lipase from Burkholderia cepacia (BcL) immobilized on mixed-function-grafted mesoporous silica gel support by hydrophobic adsorption and covalent attachment were investigated. Catalytic properties of the immobilized biocatalysts were characterized in kinetic resolution of racemic 1-phenylethanol (
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Effects of various additives on the lipase from Burkholderia cepacia (BcL) immobilized on mixed-function-grafted mesoporous silica gel support by hydrophobic adsorption and covalent attachment were investigated. Catalytic properties of the immobilized biocatalysts were characterized in kinetic resolution of racemic 1-phenylethanol (rac-1a) and 1-(thiophen-2-yl)ethan-1-ol (rac-1b). Screening of more than 40 additives showed significantly enhanced productivity of immobilized BcL with several additives such as PEGs, oleic acid and polyvinyl alcohol. Effects of substrate concentration and temperature between 0–100 °C on kinetic resolution of rac-1a were studied with the best adsorbed BcLs containing PEG 20 k or PVA 18–88 additives in continuous-flow packed-bed reactor. The optimum temperature of lipase activity for BcL co-immobilized with PEG 20k found at around 30 °C determined in the continuous-flow system increased remarkably to around 80 °C for BcL co-immobilized with PVA 18–88. Full article
(This article belongs to the Special Issue Enzyme Immobilization)
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Open AccessArticle Glutaraldehyde Cross-Linking of Immobilized Thermophilic Esterase on Hydrophobic Macroporous Resin for Application in Poly(ε-caprolactone) Synthesis
Molecules 2014, 19(7), 9838-9849; doi:10.3390/molecules19079838
Received: 21 May 2014 / Revised: 3 July 2014 / Accepted: 4 July 2014 / Published: 8 July 2014
Cited by 6 | PDF Full-text (330 KB) | HTML Full-text | XML Full-text
Abstract
The immobilized thermophilic esterase from Archaeoglobus fulgidus was successfully constructed through the glutaraldehyde-mediated covalent coupling after its physical adsorption on a hydrophobic macroporous resin, Sepabeads EC-OD. Through 0.05% glutaraldehyde treatment, the prevention of enzyme leaching and the maintenance of catalytic activity could be
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The immobilized thermophilic esterase from Archaeoglobus fulgidus was successfully constructed through the glutaraldehyde-mediated covalent coupling after its physical adsorption on a hydrophobic macroporous resin, Sepabeads EC-OD. Through 0.05% glutaraldehyde treatment, the prevention of enzyme leaching and the maintenance of catalytic activity could be simultaneously realized. Using the enzymatic ring-opening polymerization of ε-caprolactone as a model, effects of organic solvents and reaction temperature on the monomer conversion and product molecular weight were systematically investigated. After the optimization of reaction conditions, products were obtained with 100% monomer conversion and Mn values lower than 1010 g/mol. Furthermore, the cross‑linked immobilized thermophilic esterase exhibited an excellent operational stability, with monomer conversion values exceeding 90% over the course of 12 batch reactions, still more than 80% after 16 batch reactions. Full article
(This article belongs to the Special Issue Enzyme Immobilization)
Open AccessArticle Preparation of Polyphosphazene Hydrogels for Enzyme Immobilization
Molecules 2014, 19(7), 9850-9863; doi:10.3390/molecules19079850
Received: 19 May 2014 / Revised: 23 June 2014 / Accepted: 24 June 2014 / Published: 8 July 2014
Cited by 8 | PDF Full-text (490 KB) | HTML Full-text | XML Full-text
Abstract
We report on the synthesis and application of a new hydrogel based on a methacrylate substituted polyphosphazene. Through ring-opening polymerization and nucleophilic substitution, poly[bis(methacrylate)phosphazene] (PBMAP) was successfully synthesized from hexachlorocyclotriphosphazene. By adding PBMAP to methacrylic acid solution and then treating with UV light,
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We report on the synthesis and application of a new hydrogel based on a methacrylate substituted polyphosphazene. Through ring-opening polymerization and nucleophilic substitution, poly[bis(methacrylate)phosphazene] (PBMAP) was successfully synthesized from hexachlorocyclotriphosphazene. By adding PBMAP to methacrylic acid solution and then treating with UV light, we could obtain a cross-linked polyphosphazene network, which showed an ultra-high absorbency for distilled water. Lipase from Candida rugosa was used as the model lipase for entrapment immobilization in the hydrogel. The influence of methacrylic acid concentration on immobilization efficiency was studied. Results showed that enzyme loading reached a maximum of 24.02 mg/g with an activity retention of 67.25% when the methacrylic acid concentration was 20% (w/w). Full article
(This article belongs to the Special Issue Enzyme Immobilization)
Open AccessArticle Combined Effects of Ultrasound and Immobilization Protocol on Butyl Acetate Synthesis Catalyzed by CALB
Molecules 2014, 19(7), 9562-9576; doi:10.3390/molecules19079562
Received: 26 May 2014 / Revised: 30 June 2014 / Accepted: 1 July 2014 / Published: 7 July 2014
Cited by 17 | PDF Full-text (457 KB) | HTML Full-text | XML Full-text
Abstract
It is well established that the performance of lipase B from Candida antarctica (CALB) as catalyst for esterification reactions may be improved by the use of ultrasound technology or by its immobilization on styrene-divinylbenzene beads (MCI-CALB). The present research evaluated the synthesis of
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It is well established that the performance of lipase B from Candida antarctica (CALB) as catalyst for esterification reactions may be improved by the use of ultrasound technology or by its immobilization on styrene-divinylbenzene beads (MCI-CALB). The present research evaluated the synthesis of butyl acetate using MCI-CALB under ultrasonic energy, comparing the results against those obtained using the commercial preparation, Novozym 435. The optimal conditions were determined using response surface methodology (RSM) evaluating the following parameters: reaction temperature, substrate molar ratio, amount of biocatalyst, and added water. The optimal conditions for butyl acetate synthesis catalyzed by MCI-CALB were: temperature, 48.8 °C; substrate molar ratio, 3.46:1 alcohol:acid; amount of biocatalyst, 7.5%; and added water 0.28%, both as substrate mass. Under these conditions, 90% of conversion was reached in 1.5 h. In terms of operational stability, MCI-CALB was reused in seven cycles while keeping 70% of its initial activity under ultrasonic energy. The support pore size and resistance are key points for the enzyme activity and stability under mechanical stirring. The use of ultrasound improved both activity and stability because of better homogeneity and reduced mechanical stress to the immobilized system. Full article
(This article belongs to the Special Issue Enzyme Immobilization)
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Open AccessArticle Catalytic Behavior of Lipase Immobilized onto Congo Red and PEG-Decorated Particles
Molecules 2014, 19(6), 8610-8628; doi:10.3390/molecules19068610
Received: 4 May 2014 / Revised: 20 June 2014 / Accepted: 20 June 2014 / Published: 24 June 2014
Cited by 6 | PDF Full-text (565 KB) | HTML Full-text | XML Full-text
Abstract
Poly(ethylene glycol) (PEG)-decorated polystyrene (PS) nanoparticles with mean hydrodynamic diameter (D) and zeta–potential (ζ) of (286 ± 15) nm and (−50 ± 5) mV, respectively, were modified by the adsorption of Congo red (CR). The PS/PEG/CR particles presented D
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Poly(ethylene glycol) (PEG)-decorated polystyrene (PS) nanoparticles with mean hydrodynamic diameter (D) and zeta–potential (ζ) of (286 ± 15) nm and (−50 ± 5) mV, respectively, were modified by the adsorption of Congo red (CR). The PS/PEG/CR particles presented D and ζ values of (290 ± 19) nm and (−36 ± 5) mV, respectively. The adsorption of lipase onto PS/PEG or PS/PEG/CR particles at (24 ± 1) °C and pH 7 changed the mean D value to (380 ± 20) and (405 ± 11) nm, respectively, and ζ value to (−32 ± 4) mV and (−25 ± 2) mV, respectively. The kinetic parameters of the hydrolysis of p-nitrophenyl butyrate were determined for free lipase, lipase immobilized onto PS/PEG and PS/PEG/CR particles. Lipase on PS/PEG/CR presented the largest Michaelis-Menten constant (KM), but also the highest Vmax and kcat values. Moreover, it could be recycled seven times, losing a maximum 10% or 30% of the original enzymatic activity at 40 °C or 25 °C, respectively. Although lipases immobilized onto PS/PEG particles presented the smallest KM values, the reactions were comparatively the slowest and recycling was not possible. Hydrolysis reactions performed in the temperature range of 25 °C to 60 °C with free lipases and lipases immobilized onto PS/PEG/CR particles presented an optimal temperature at 40 °C. At 60 °C free lipases and lipases immobilized onto PS/PEG/CR presented ~80% and ~50% of the activity measured at 40 °C, indicating good thermal stability. Bioconjugation effects between CR and lipase were evidenced by circular dichroism spectroscopy and spectrophotometry. CR molecules mediate the open state conformation of the lipase lid and favor the substrate approaching. Full article
(This article belongs to the Special Issue Enzyme Immobilization)
Open AccessArticle Immobilization of Trichoderma harzianum α-Amylase on Treated Wool: Optimization and Characterization
Molecules 2014, 19(6), 8027-8038; doi:10.3390/molecules19068027
Received: 8 March 2014 / Revised: 4 June 2014 / Accepted: 6 June 2014 / Published: 13 June 2014
Cited by 8 | PDF Full-text (541 KB) | HTML Full-text | XML Full-text
Abstract
α-Amylase from Trichoderma harzianum was covalently immobilized on activated wool by cyanuric chloride. Immobilized α-amylase exhibited 75% of its initial activity after 10 runs. The soluble and immobilized α-amylases exhibited maximum activity at pH values 6.0 and 6.5, respectively. The immobilized enzyme was
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α-Amylase from Trichoderma harzianum was covalently immobilized on activated wool by cyanuric chloride. Immobilized α-amylase exhibited 75% of its initial activity after 10 runs. The soluble and immobilized α-amylases exhibited maximum activity at pH values 6.0 and 6.5, respectively. The immobilized enzyme was more thermally stable than the soluble one. Various substrates were hydrolyzed by immobilized α-amylase with high efficiencies compared to those of soluble α-amylase. The inhibition of the immobilized α-amylase by metal ions was low as compared with soluble enzyme. On the basis of the results obtained, immobilized α-amylase could be employed in the saccharification of starch processing. Full article
(This article belongs to the Special Issue Enzyme Immobilization)
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Open AccessArticle Evaluation of Styrene-Divinylbenzene Beads as a Support to Immobilize Lipases
Molecules 2014, 19(6), 7629-7645; doi:10.3390/molecules19067629
Received: 3 April 2014 / Revised: 3 June 2014 / Accepted: 4 June 2014 / Published: 10 June 2014
Cited by 22 | PDF Full-text (240 KB) | HTML Full-text | XML Full-text
Abstract
A commercial and very hydrophobic styrene-divinylbenzene matrix, MCI GEL® CHP20P, has been compared to octyl-Sepharose® beads as support to immobilize three different enzymes: lipases from Thermomyces lanuginosus (TLL) and from Rhizomucor miehie (RML) and Lecitase® Ultra, a commercial artificial phospholipase.
[...] Read more.
A commercial and very hydrophobic styrene-divinylbenzene matrix, MCI GEL® CHP20P, has been compared to octyl-Sepharose® beads as support to immobilize three different enzymes: lipases from Thermomyces lanuginosus (TLL) and from Rhizomucor miehie (RML) and Lecitase® Ultra, a commercial artificial phospholipase. The immobilization mechanism on both supports was similar: interfacial activation of the enzymes versus the hydrophobic surface of the supports. Immobilization rate and loading capacity is much higher using MCI GEL® CHP20P compared to octyl-Sepharose® (87.2 mg protein/g of support using TLL, 310 mg/g using RML and 180 mg/g using Lecitase® Ultra). The thermal stability of all new preparations is much lower than that of the standard octyl-Sepharose® immobilized preparations, while the opposite occurs when the inactivations were performed in the presence of organic co-solvents. Regarding the hydrolytic activities, the results were strongly dependent on the substrate and pH of measurement. Octyl-Sepharose® immobilized enzymes were more active versus p-NPB than the enzymes immobilized on MCI GEL® CHP20P, while RML became 700-fold less active versus methyl phenylacetate. Thus, the immobilization of a lipase on this matrix needs to be empirically evaluated, since it may present very positive effects in some cases while in other cases it may have very negative ones. Full article
(This article belongs to the Special Issue Enzyme Immobilization)
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Open AccessArticle Fabrication of Implantable, Enzyme-Immobilized Glutamate Sensors for the Monitoring of Glutamate Concentration Changes in Vitro and in Vivo
Molecules 2014, 19(6), 7341-7355; doi:10.3390/molecules19067341
Received: 17 March 2014 / Revised: 30 May 2014 / Accepted: 3 June 2014 / Published: 5 June 2014
Cited by 10 | PDF Full-text (651 KB) | HTML Full-text | XML Full-text
Abstract
Glutamate sensors based on the immobilization of glutamate oxidase (GlutOx) were prepared by adsorption on electrodeposited chitosan (Method 1) and by crosslinking with glutaraldehyde (Method 2) on micromachined platinum microelectrodes. It was observed that glutamate sensors prepared by Method
[...] Read more.
Glutamate sensors based on the immobilization of glutamate oxidase (GlutOx) were prepared by adsorption on electrodeposited chitosan (Method 1) and by crosslinking with glutaraldehyde (Method 2) on micromachined platinum microelectrodes. It was observed that glutamate sensors prepared by Method 1 have faster response time (<2 s) and lower detection limit (2.5 ± 1.1 μM) compared to that prepared by Method 2 (response time: <5 sec and detection limit: 6.5 ± 1.7 μM); glutamate sensors prepared by Method 2 have a larger linear detection range (20–352 μM) and higher sensitivity (86.8 ± 8.8 nA·μM−1·cm−2, N = 12) compared to those prepared by Method 1 (linear detection range: 20–217 μM and sensitivity: 34.9 ± 4.8 nA·μM−1·cm−2, N = 8). The applicability of the glutamate sensors in vivo was also demonstrated. The glutamate sensors were implanted into the rat brain to monitor the stress-induced extracellular glutamate release in the hypothalamus of the awake, freely moving rat. Full article
(This article belongs to the Special Issue Enzyme Immobilization)
Open AccessArticle Mesoporous Silicas with Tunable Morphology for the Immobilization of Laccase
Molecules 2014, 19(6), 7057-7071; doi:10.3390/molecules19067057
Received: 8 May 2014 / Revised: 20 May 2014 / Accepted: 23 May 2014 / Published: 30 May 2014
Cited by 15 | PDF Full-text (1170 KB) | HTML Full-text | XML Full-text
Abstract
Siliceous ordered mesoporous materials (OMM) are gaining interest as supports for enzyme immobilization due to their uniform pore size, large surface area, tunable pore network and the introduction of organic components to mesoporous structure. We used SBA-15 type silica materials, which exhibit a
[...] Read more.
Siliceous ordered mesoporous materials (OMM) are gaining interest as supports for enzyme immobilization due to their uniform pore size, large surface area, tunable pore network and the introduction of organic components to mesoporous structure. We used SBA-15 type silica materials, which exhibit a regular 2D hexagonal packing of cylindrical mesopores of uniform size, for non-covalent immobilization of laccase. Synthesis conditions were adjusted in order to obtain supports with different particle shape, where those with shorter channels had higher loading capacity. Despite the similar isoelectric points of silica and laccase and the close match between the size of laccase and the pore dimensions of these SBA-15 materials, immobilization was achieved with very low leaching. Surface modification of macro-/mesoporous amorphous silica by grafting of amine moieties was proved to significantly increase the isoelectric point of this support and improve the immobilization yield. Full article
(This article belongs to the Special Issue Enzyme Immobilization)
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Open AccessArticle Surface-Functionalized Hyperbranched Poly(Amido Acid) Magnetic Nanocarriers for Covalent Immobilization of a Bacterial γ-Glutamyltranspeptidase
Molecules 2014, 19(4), 4997-5012; doi:10.3390/molecules19044997
Received: 11 March 2014 / Revised: 15 April 2014 / Accepted: 17 April 2014 / Published: 22 April 2014
Cited by 15 | PDF Full-text (2201 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
In this study, we synthesized water-soluble hyperbranched poly(amido acid)s (HBPAAs) featuring multiple terminal CO2H units and internal tertiary amino and amido moieties and then used them in conjunction with an in situ Fe2+/Fe3+ co-precipitation process to prepare organic/magnetic
[...] Read more.
In this study, we synthesized water-soluble hyperbranched poly(amido acid)s (HBPAAs) featuring multiple terminal CO2H units and internal tertiary amino and amido moieties and then used them in conjunction with an in situ Fe2+/Fe3+ co-precipitation process to prepare organic/magnetic nanocarriers comprising uniformly small magnetic iron oxide nanoparticles (NP) incorporated within the globular HBPAAs. Transmission electron microscopy revealed that the HBPAA-γ-Fe2O3 NPs had dimensions of 6–11 nm, significantly smaller than those of the pristine γ-Fe2O3 (20–30 nm). Subsequently, we covalently immobilized a bacterial γ-glutamyltranspeptidase (BlGGT) upon the HBPAA-γ-Fe2O3 nanocarriers through the formation of amide linkages in the presence of a coupling agent. Magnetization curves of the HBPAA-γ-Fe2O3/BlGGT composites measured at 300 K suggested superparamagnetic characteristics, with a saturation magnetization of 52 emu g−1. The loading capacity of BlGGT on the HBPAA-γ-Fe2O3 nanocarriers was 16 mg g−1 support; this sample provided a 48% recovery of the initial activity. The immobilized enzyme could be recycled 10 times with 32% retention of the initial activity; it had stability comparable with that of the free enzyme during a storage period of 63 days. The covalent immobilization and stability of the enzyme and the magnetization provided by the HBPAA-γ-Fe2O3 NPs suggests that this approach could be an economical means of depositing bioactive enzymes upon nanocarriers for BlGGT-mediated bio-catalysis. Full article
(This article belongs to the Special Issue Enzyme Immobilization)
Open AccessArticle Laccase Immobilized on a PAN/Adsorbents Composite Nanofibrous Membrane for Catechol Treatment by a Biocatalysis/Adsorption Process
Molecules 2014, 19(3), 3376-3388; doi:10.3390/molecules19033376
Received: 20 January 2014 / Revised: 6 March 2014 / Accepted: 6 March 2014 / Published: 19 March 2014
Cited by 16 | PDF Full-text (1930 KB) | HTML Full-text | XML Full-text
Abstract
The treatment of catechol via biocatalysis and adsorption with a commercial laccase immobilized on polyacrylonitrile/montmorillonite/graphene oxide (PAN/MMT/GO) composite nanofibers was evaluated with a homemade nanofibrous membrane reactor. The properties in this process of the immobilized laccase on PAN, PAN/MMT as well as PAN/MMT/GO
[...] Read more.
The treatment of catechol via biocatalysis and adsorption with a commercial laccase immobilized on polyacrylonitrile/montmorillonite/graphene oxide (PAN/MMT/GO) composite nanofibers was evaluated with a homemade nanofibrous membrane reactor. The properties in this process of the immobilized laccase on PAN, PAN/MMT as well as PAN/MMT/GO with different weight ratios of MMT and GO were investigated. These membranes were successfully applied for removal of catechol from an aqueous solution. Scanning electron microscope images revealed different morphologies of the enzyme aggregates on different supports. After incorporation of MMT or MMT/GO, the optimum pH showed an alkaline shift to 4, compared to 3.5 for laccase immobilized on pure PAN nanofibers. The optimum temperature was at 55 °C for all the immobilized enzymes. Besides, the addition of GO improved the operational stability and storage stability. A 39% ± 2.23% chemical oxygen demand (COD) removal from the catechol aqueous solution was achieved. Experimental results suggested that laccase, PAN, adsorbent nanoparticles (MMT/GO) can be combined together for catechol treatment in industrial applications. Full article
(This article belongs to the Special Issue Enzyme Immobilization)

Review

Jump to: Editorial, Research, Other

Open AccessFeature PaperReview Agarose and Its Derivatives as Supports for Enzyme Immobilization
Molecules 2016, 21(11), 1577; doi:10.3390/molecules21111577
Received: 27 September 2016 / Revised: 12 November 2016 / Accepted: 16 November 2016 / Published: 19 November 2016
Cited by 13 | 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
[...] Read more.
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; doi:10.3390/molecules21101370
Received: 30 August 2016 / Revised: 5 October 2016 / Accepted: 5 October 2016 / Published: 14 October 2016
Cited by 8 | 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
[...] Read more.
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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Figure 1

Open AccessReview Immobilization of Glycoside Hydrolase Families GH1, GH13, and GH70: State of the Art and Perspectives
Molecules 2016, 21(8), 1074; doi:10.3390/molecules21081074
Received: 27 July 2016 / Revised: 11 August 2016 / Accepted: 12 August 2016 / Published: 17 August 2016
Cited by 8 | PDF Full-text (1376 KB) | HTML Full-text | XML Full-text
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
[...] Read more.
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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Figure 1

Open AccessReview Inorganic Materials as Supports for Covalent Enzyme Immobilization: Methods and Mechanisms
Molecules 2014, 19(9), 14139-14194; doi:10.3390/molecules190914139
Received: 5 July 2014 / Revised: 9 August 2014 / Accepted: 22 August 2014 / Published: 9 September 2014
Cited by 64 | PDF Full-text (1733 KB) | HTML Full-text | XML Full-text
Abstract
Several inorganic materials are potentially suitable for enzymatic covalent immobilization, by means of several different techniques. Such materials must meet stringent criteria to be suitable as solid matrices: complete insolubility in water, reasonable mechanical strength and chemical resistance under the operational conditions, the
[...] Read more.
Several inorganic materials are potentially suitable for enzymatic covalent immobilization, by means of several different techniques. Such materials must meet stringent criteria to be suitable as solid matrices: complete insolubility in water, reasonable mechanical strength and chemical resistance under the operational conditions, the capability to form manageable particles with high surface area, reactivity towards derivatizing/functionalizing agents. Non-specific protein adsorption should be always considered when planning covalent immobilization on inorganic solids. A huge mass of experimental work has shown that silica, silicates, borosilicates and aluminosilicates, alumina, titania, and other oxides, are the materials of choice when attempting enzyme immobilizations on inorganic supports. More recently, some forms of elemental carbon, silicon, and certain metals have been also proposed for certain applications. With regard to the derivatization/functionalization techniques, the use of organosilanes through silanization is undoubtedly the most studied and the most applied, although inorganic bridge formation and acylation with selected acyl halides have been deeply studied. In the present article, the most common inorganic supports for covalent immobilization of the enzymes are reviewed, with particular focus on their advantages and disadvantages in terms of enzyme loadings, operational stability, undesired adsorption, and costs. Mechanisms and methods for covalent immobilization are also discussed, focusing on the most widespread activating approaches (such as glutaraldehyde, cyanogen bromide, divinylsulfone, carbodiimides, carbonyldiimidazole, sulfonyl chlorides, chlorocarbonates, N-hydroxysuccinimides). Full article
(This article belongs to the Special Issue Enzyme Immobilization)
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Open AccessReview Trends in Protein-Based Biosensor Assemblies for Drug Screening and Pharmaceutical Kinetic Studies
Molecules 2014, 19(8), 12461-12485; doi:10.3390/molecules190812461
Received: 28 May 2014 / Revised: 2 August 2014 / Accepted: 5 August 2014 / Published: 18 August 2014
Cited by 7 | PDF Full-text (1143 KB) | HTML Full-text | XML Full-text
Abstract
The selection of natural and chemical compounds for potential applications in new pharmaceutical formulations constitutes a time-consuming procedure in drug screening. To overcome this issue, new devices called biosensors, have already demonstrated their versatility and capacity for routine clinical diagnosis. Designed to perform
[...] Read more.
The selection of natural and chemical compounds for potential applications in new pharmaceutical formulations constitutes a time-consuming procedure in drug screening. To overcome this issue, new devices called biosensors, have already demonstrated their versatility and capacity for routine clinical diagnosis. Designed to perform analytical analysis for the detection of a particular analyte, biosensors based on the coupling of proteins to amperometric and optical devices have shown the appropriate selectivity, sensibility and accuracy. During the last years, the exponential demand for pharmacokinetic studies in the early phases of drug development, along with the need of lower molecular weight detection, have led to new biosensor structure materials with innovative immobilization strategies. The result has been the development of smaller, more reproducible biosensors with lower detection limits, and with a drastic reduction in the required sample volumes. Therefore in order to describe the main achievements in biosensor fields, the present review has the main aim of summarizing the essential strategies used to generate these specific devices, that can provide, under physiological conditions, a credible molecule profile and assess specific pharmacokinetic parameters. Full article
(This article belongs to the Special Issue Enzyme Immobilization)
Open AccessReview Application of Iron Magnetic Nanoparticles in Protein Immobilization
Molecules 2014, 19(8), 11465-11486; doi:10.3390/molecules190811465
Received: 23 May 2014 / Revised: 9 July 2014 / Accepted: 9 July 2014 / Published: 4 August 2014
Cited by 49 | PDF Full-text (1045 KB) | HTML Full-text | XML Full-text
Abstract
Due to their properties such as superparamagnetism, high surface area, large surface-to-volume ratio, easy separation under external magnetic fields, iron magnetic nanoparticles have attracted much attention in the past few decades. Various modification methods have been developed to produce biocompatible magnetic nanoparticles for
[...] Read more.
Due to their properties such as superparamagnetism, high surface area, large surface-to-volume ratio, easy separation under external magnetic fields, iron magnetic nanoparticles have attracted much attention in the past few decades. Various modification methods have been developed to produce biocompatible magnetic nanoparticles for protein immobilization. This review provides an updated and integrated focus on the fabrication and characterization of suitable magnetic iron nanoparticle-based nano-active materials for protein immobilization. Full article
(This article belongs to the Special Issue Enzyme Immobilization)
Open AccessReview Immobilization as a Strategy for Improving Enzyme Properties-Application to Oxidoreductases
Molecules 2014, 19(7), 8995-9018; doi:10.3390/molecules19078995
Received: 12 May 2014 / Revised: 13 June 2014 / Accepted: 16 June 2014 / Published: 27 June 2014
Cited by 111 | PDF Full-text (858 KB) | HTML Full-text | XML Full-text
Abstract
The main objective of the immobilization of enzymes is to enhance the economics of biocatalytic processes. Immobilization allows one to re-use the enzyme for an extended period of time and enables easier separation of the catalyst from the product. Additionally, immobilization improves many
[...] Read more.
The main objective of the immobilization of enzymes is to enhance the economics of biocatalytic processes. Immobilization allows one to re-use the enzyme for an extended period of time and enables easier separation of the catalyst from the product. Additionally, immobilization improves many properties of enzymes such as performance in organic solvents, pH tolerance, heat stability or the functional stability. Increasing the structural rigidity of the protein and stabilization of multimeric enzymes which prevents dissociation-related inactivation. In the last decade, several papers about immobilization methods have been published. In our work, we present a relation between the influence of immobilization on the improvement of the properties of selected oxidoreductases and their commercial value. We also present our view on the role that different immobilization methods play in the reduction of enzyme inhibition during biotechnological processes. Full article
(This article belongs to the Special Issue Enzyme Immobilization)
Open AccessReview Polyhydroyxalkanoate Synthase Fusions as a Strategy for Oriented Enzyme Immobilisation
Molecules 2014, 19(6), 8629-8643; doi:10.3390/molecules19068629
Received: 16 May 2014 / Revised: 19 June 2014 / Accepted: 19 June 2014 / Published: 24 June 2014
Cited by 14 | PDF Full-text (279 KB) | HTML Full-text | XML Full-text
Abstract
Polyhydroxyalkanoate (PHA) is a carbon storage polymer produced by certain bacteria in unbalanced nutrient conditions. The PHA forms spherical inclusions surrounded by granule associate proteins including the PHA synthase (PhaC). Recently, the intracellular formation of PHA granules with covalently attached synthase from Ralstonia
[...] Read more.
Polyhydroxyalkanoate (PHA) is a carbon storage polymer produced by certain bacteria in unbalanced nutrient conditions. The PHA forms spherical inclusions surrounded by granule associate proteins including the PHA synthase (PhaC). Recently, the intracellular formation of PHA granules with covalently attached synthase from Ralstonia eutropha has been exploited as a novel strategy for oriented enzyme immobilisation. Fusing the enzyme of interest to PHA synthase results in a bifunctional protein able to produce PHA granules and immobilise the active enzyme of choice to the granule surface. Functionalised PHA granules can be isolated from the bacterial hosts, such as Escherichia coli, and maintain enzymatic activity in a wide variety of assay conditions. This approach to oriented enzyme immobilisation has produced higher enzyme activities and product levels than non-oriented immobilisation techniques such as protein inclusion based particles. Here, enzyme immobilisation via PHA synthase fusion is reviewed in terms of the genetic designs, the choices of enzymes, the control of enzyme orientations, as well as their current and potential applications. Full article
(This article belongs to the Special Issue Enzyme Immobilization)
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Other

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Open AccessCommentary Solid-State Metalloproteins—An Alternative to Immobilisation
Molecules 2016, 21(7), 919; doi:10.3390/molecules21070919
Received: 12 May 2016 / Revised: 7 July 2016 / Accepted: 8 July 2016 / Published: 14 July 2016
Cited by 2 | PDF Full-text (729 KB) | HTML Full-text | XML Full-text
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
[...] Read more.
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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Planned Papers

The below list represents only planned manuscripts. Some of these manuscripts have not been received by the Editorial Office yet. Papers submitted to MDPI journals are subject to peer-review.

Type of the paper: article
Title: Immobilization of glycoside hydrolases families GH1, GH13 and GH70: state of art and perspectives
Authors: Natália G. Graebin, Jéssie da N. Schöffer, Diandra de Andrades, Manuela P. Klein, Plinho F. Hertz, Marco A. Z. Ayub, Rafael C. Rodrigues *
Affiliation: Biotechnology, Bioprocess, and Biocatalysis Group, Food Science and Technology Institute, Federal University of Rio Grande do Sul, Av. Bento Gonçalves 9500, PO Box 15090, ZC 91501-970 Porto Alegre, RS, Brazil
E-Mail:
Abstract: Glycoside hydrolases (GH) are a widespread group of enzymes that hydrolyzes the glycosidic bond between two carbohydrates or between a carbohydrate and a non-carbohydrate moiety. Due to the increasing interest for industrial application of these enzymes, immobilization of GH is an important feature to improve activity, stability, and possible reuses in batch reaction or in continuous processes. Thus, in this review we will focus in the immobilization of enzymes from specific GH families. Firstly, a brief introduction of enzyme immobilization methods will be presented, discussing some advantages and drawbacks. Following, it will be reviewed the studies of enzyme immobilization from families GH1, GH13 and GH70, focusing in the enzymes β-glucosidase, β-galactosidase, α-amylase, cyclodextrin glycosyltransferase and dextransucrase. In each case, the immobilization protocols will be evaluated considering their pros and cons. Finally, the perspectives for new immobilization methods, as well as industrial applications will be briefly presented.
Keywords: enzyme immobilization; glycoside hydrolases; β-glucosidase; β-galactosidase; amylase; dextransucrase.

Type of the paper: article
Tentative title: Site-Specific Immobilization Of Dehalogenase St2570 Mediated By Formylglycine-Generating Enzyme And Applications Of The Process In Batch And Semi-Continuous Flow Reactors
Authors: Hui Jian, Yingwu Wang*, Yan Bai, Rong Li, Renjun Gao*
Affiliations: Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, School of Life Science, Jilin University, Changchun 130012, China
Abstract: Formylglycine-generating enzymes can selectively recognise and oxidise cysteine residues within the sulphatase sub motif at the terminus of proteins to form aldehyde-bearing formylglycine (FGly) residues; these residues can form covalent bonds with amino functionalised fluoresceins or supports through the Schiff base reaction. In this experiment, aldehyde-tagged proteins were prepared using a previously designed set of the pET28a plasmid system. The C-terminal aldehyde-tagged protein (ST2570CQ) exhibits significant enzymological properties, such as generation of new free aldehyde groups, high level of protein expression and improved enzyme activity. The immobilization of ST2570CQ on SBA15 modified with 2% APTES ethanol solution (V/V) was performed under the following optimal conditions: 2 mg of ST2570CQ per 10 mg of SBA15-NH2 was incubated in 50 mM PB buffer (pH 6.5) at 10 °C for more than 3 h. The immobilized ST2570CQ shows threefold higher thermal stability, 1.2-fold higher catalytic ability and improved operational stability than free ST2570. The immobilized ST2570CQ retains 60% of its original activity after seven cycles of batch operation and 100% of its original activity after 10 cycles of reuse in the semi-continuous flow reactor. These results provide a basis for industrial-scale production of immobilized ST2570.
Key words: Immobilization; Dehalogenase; Formylglycine-generating enzyme; Sulfolobus tokodaii

Type of the paper: article
Tentative title: Reversible Immobilization Of Lipases On Heterofunctional Octyl-Amino Agarose Beads Prevents Enzyme Desorption
Authors: Nazzoly Rueda 1,2,+, Tiago L. Albuquerquea 3,+, Rocio Bartolome-Cabrero 1, Laura Fernandez-Lopez 1, Rodrigo Torres 2,4, Claudia Ortiz 5, Cleiton S. dos Santos 1,3, Oveimar Barbosa 6, Roberto Fernandez-Lafuente 1,*
Affiliations: 1 Departamento de Biocatalisis. ICP-CSIC, Campus UAM-CSIC Madrid. Spain.
2 Escuela de Química, Grupo de investigación en Bioquímica y Microbiología (GIBIM), Edificio Camilo Torres 210, Universidad Industrial de Santander, Bucaramanga, Colombia.
3 Departamento de Engenharia Química, Universidade Federal Do Ceará, Campus Do Pici, CEP 60455-760, Fortaleza, CE, Brazil.
4 Current address: Laboratorio de Biotecnología, Instituto Colombiano del Petróleo-Ecopetrol, Piedecuesta, Bucaramanga, Colombia.
5 Escuela de Microbiología, Universidad Industrial de Santander, Bucaramanga, Colombia
6 Departamento de Química, Facultad de Ciencias. Universidad del Tolima, Ibagué, Colombia
+ Both authors have evenly participated in this research
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 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 at Triton X-100 concentrations that released all enzyme from the octyl support. The activities of the immobilized enzymes were usually slightly higher using the new supports than the octyl ones (that, except for CALB, produced significant enzyme hyperactivation). Thermal and solvent stabilities of LU and TLL were significantly improved compared to the OC counterparts, while in the other enzymes the stability used to decrease (depending on the pH value). As a general rule, OCEDA had lower negative effects immobilized enzyme stability than OCHDA. While in solvent inactivation the enzyme molecules remained attached to the support using the new supports and not using monofunctional octyl supports, in thermal inactivation this only occurred in certain cases.

Type of the paper: Article
Title: Stabilization of CALB Immobilized on Octyl Agarose by Physical Intermolecular Crosslinking with PEI. Prevention of the Enzyme desorption
Authors: Sara Peirce 1,2, Veymar G. Tacias-Pascacio 1,3, Maria Elena Russo 4, Antonio Marzocchella 2, José J. Virgen-Ortíz 1,* and Roberto Fernandez-Lafuente 1,*
Affiliation: 1 Departamento de Biocatálisis. Instituto de Catálisis-CSIC, Campus UAM-CSIC Madrid, Spain
2 Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione Industriale. Universita' degli Studi di Napoli Federico II, Italy
3 Unidad de Investigación y Desarrollo en Alimentos. Instituto Tecnológico de Veracruz, Calzada Miguel A. de Quevedo 2779, 91897 Veracruz, Mexico
4 Istituto di Ricerche sulla Combustione–Consiglio Nazionale delle Ricerche, Napoli, Italy
E-Mails: juanvirgen@hotmail.com (J.J.V.-O.); rfl@icp.csic.es (R.F.-L.)
Abstract: Lipase B from Candida antarctica (CALB) was immobilized on octyl agarose (OC) and physically modified with PEI in order to confer a strong ion exchanger to the enzyme and that way, enable the immobilization of other enzymes over it. 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 been achieved making more difficult this desorption. 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 re-use of the support by incubation in 2% ionic detergents. That way, this modified CALB not only has a strong anion exchange nature, keeping the activity, but also improves its stability under diverse reaction conditions without losing the reversibility of the immobilization.
Keywords: reversible immobilization, interfacial adsorption, PEI modification, enzyme stabilization, enzyme physical intermolecular crosslinking

Type of the paper: Commentary
Title: Solid-state metalloproteins—An alternative to immobilisation
Author: Trevor D. Rapson
Affiliation: CSIRO, Black Mountain, Canberra, ACT, 2601, Australia
E-Mail: trevor.rapson@csiro.au
Abstract: Immobilisation is often the key step required to use an enzyme in an industrial processes. Ideally immobilisation will stabilise the enzyme without compromising its functional properties. While great progress has been made in this area, immobilisation continues to remain a bottleneck in the commercialisation process. This commentary outlines an alternative approach to immobilisation which uses a recombinant silk protein into which metal active sites can be incorporated to produce solid‐state materials which mimic metalloprotein function. These solid15 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.
Keywords: biocatalysis; industrial biotechnology; silk; biosensors; de novo engineering

Type of the paper: Article
Title: Tailoring the Spacer Arm for Covalent Immobilization of Candida antarctica Lipase B–Thermal Stabilization by Bisepoxide-activated Aminoalkyl Resins in Continuous-flow Reactors
Authors: Emese Abaházi 1, Dávid Lestál 1, Zoltán Boros 2 and László Poppe 1,2,*
Affiliation: 1 Budapest University of Technology and Economics
2 SynBiocat LLC
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 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 continuous-flow reactor as well. Catalytic activity, enantiomer selectivity, recyclability, 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.
Keywords: Lipase, immobilization, covalent attachment, kinetic resolution, thermal stabilization, continuous-flow reactor.

Type of the paper: Article
Title: Construction of an Immobilized Thermophilic Esterase on Epoxy Support for Poly(ε-caprolactone) Synthesis
Authors: Hui Ren 1, Zhen Xing 2, Jiebing Yang 2, Wei Jiang 3, Gang Zhang 3,*, Jun Tang 3,*,
Quanshun Li 2,*
Affiliation: 1 Department of Colorectal Surgery, the Second Hospital of Jilin University, Changchun 130041, China
2 Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, School of Life Sciences, Jilin University, Changchun 130012, China
3 Department of Polymer Science, College of Chemistry, Jilin University, Changchun 130012, China
E-Mails: quanshun@jlu.edu.cn (Q.L.); chemjtang@jlu.edu.cn (J.T.); gzhang@jlu.edu.cn (G.Z.)
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, and the immobilized enzyme was then employed as a biocatalyst for poly(ε-caprolactone) synthesis. The enzyme loading on support was measured to be 72 mg/g support after the immobilization. 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 (<1300 g/mol). Further, the immobilized enzyme exhibited excellent reusability, with monomer conversion values exceeding 75% during 15 batch reactions.
Keywords: thermophilic esterase; immobilization; epoxy support; ring-opening polymerization; poly(ε-caprolactone)

Type of the paper: Article
Title: Catalytic Oxidation of Phenolic Wastewater by Using Horseradish Peroxidase Immobilized on Graphene Oxide/Fe3O4
Authors: Qing Chang, Jia Huang, Yaobin Ding and Heqing Tang*
Affiliation: College of Resources and Environmental Science, South Central University for Nationalities, South-Central University for Nationalities, Wuhan 430074, China
E-Mail: tangheqing@mail.scuec.edu.cn
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 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.
Keywords: graphene oxide; magnetite; phenolic wastewater; immobilized enzyme; catalytic oxidation

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