Editorial

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Open AccessEditorial

Special Issue: Enzyme Immobilization 2016

by Roberto Fernandez-Lafuente

Molecules 2017, 22(4), 601; https://doi.org/10.3390/molecules22040601 - 08 Apr 2017

Cited by 10 | Viewed by 4533

The use of enzymes as industrial biocatalysts is currently a solution for many problems of modern organic chemistry, which tries to carry out the most complex reactions under the rules of green chemistry [1].[...] Full article

(This article belongs to the Special Issue Enzyme Immobilization 2016)

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Open AccessEditorial

Editorial: Special Issue — Enzyme Immobilization

by Roberto Fernandez-Lafuente

Molecules 2014, 19(12), 20671-20674; https://doi.org/10.3390/molecules191220671 - 10 Dec 2014

Cited by 6 | Viewed by 4749

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

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Open AccessArticle

Prevention of Bacterial Contamination of a Silica Matrix Containing Entrapped β-Galactosidase through the Action of Covalently Bound Lysozymes

by Heng Li, Shuai Li, Pu Tian, Zhuofu Wu and Zhengqiang Li

Molecules 2017, 22(3), 377; https://doi.org/10.3390/molecules22030377 - 28 Feb 2017

Cited by 17 | Viewed by 4934

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

by Daniela F. M. Turati, Wilson G. Morais Júnior, César R. F. Terrasan, Sonia Moreno-Perez, Benevides C. Pessela, Gloria Fernandez-Lorente, Jose M. Guisan and Eleonora C. Carmona

Molecules 2017, 22(2), 339; https://doi.org/10.3390/molecules22020339 - 22 Feb 2017

Cited by 22 | Viewed by 5833

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

by Pedro Torres and Francisco Batista-Viera

Molecules 2017, 22(2), 284; https://doi.org/10.3390/molecules22020284 - 22 Feb 2017

Cited by 22 | Viewed by 7128

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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Novel Magnetic Cross-Linked Cellulase Aggregates with a Potential Application in Lignocellulosic Biomass Bioconversion

by Junqi Jia, Weiwei Zhang, Zengjie Yang, Xianling Yang, Na Wang and Xiaoqi Yu

Molecules 2017, 22(2), 269; https://doi.org/10.3390/molecules22020269 - 10 Feb 2017

Cited by 85 | Viewed by 6616

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

by Vinicius Vescovi, Raquel L. C. Giordano, Adriano A. Mendes and Paulo W. Tardioli

Molecules 2017, 22(2), 212; https://doi.org/10.3390/molecules22020212 - 30 Jan 2017

Cited by 36 | Viewed by 5423

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

by Chunfang Li, Shuhui Jiang, Xinying Zhao and Hao Liang

Molecules 2017, 22(1), 179; https://doi.org/10.3390/molecules22010179 - 23 Jan 2017

Cited by 42 | Viewed by 8172

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

by Jose J. Virgen-Ortíz, Sara G. Pedrero, Laura Fernandez-Lopez, Nerea Lopez-Carrobles, Beatriz C. Gorines, Cristina Otero and Roberto Fernandez-Lafuente

Molecules 2017, 22(1), 91; https://doi.org/10.3390/molecules22010091 - 05 Jan 2017

Cited by 51 | Viewed by 5940

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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Immobilization of Neutral Protease from Bacillus subtilis for Regioselective Hydrolysis of Acetylated Nucleosides: Application to Capecitabine Synthesis

by Teodora Bavaro, Giulia Cattaneo, Immacolata Serra, Ilaria Benucci, Massimo Pregnolato and Marco Terreni

Molecules 2016, 21(12), 1621; https://doi.org/10.3390/molecules21121621 - 25 Nov 2016

Cited by 22 | Viewed by 6813

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

by Katherine Ahumada, Ana Martínez-Gil, Yerko Moreno-Simunovic, Andrés Illanes and Lorena Wilson

Molecules 2016, 21(11), 1485; https://doi.org/10.3390/molecules21111485 - 08 Nov 2016

Cited by 25 | Viewed by 5986

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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Immobilization of α-Amylase from Anoxybacillus sp. SK3-4 on ReliZyme and Immobead Supports

by Ummirul Mukminin Kahar, Mohd Helmi Sani, Kok-Gan Chan and Kian Mau Goh

Molecules 2016, 21(9), 1196; https://doi.org/10.3390/molecules21091196 - 09 Sep 2016

Cited by 23 | Viewed by 7817

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

by Qing Chang, Jia Huang, Yaobin Ding and Heqing Tang

Molecules 2016, 21(8), 1044; https://doi.org/10.3390/molecules21081044 - 10 Aug 2016

Cited by 56 | Viewed by 6283

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

by Hui Jian, Yingwu Wang, Yan Bai, Rong Li and Renjun Gao

Molecules 2016, 21(7), 895; https://doi.org/10.3390/molecules21070895 - 11 Jul 2016

Cited by 24 | Viewed by 5877

Abstract

Formylglycine-generating enzymes can selectively recognize and oxidize cysteine residues within the sulfatase sub motif at the terminus of proteins to form aldehyde-bearing formylglycine (FGly) residues, and are normally used in protein labeling. In this study, an aldehyde tag was introduced to proteins using [...] Read more.

Formylglycine-generating enzymes can selectively recognize and oxidize cysteine residues within the sulfatase sub motif at the terminus of proteins to form aldehyde-bearing formylglycine (FGly) residues, and are normally used in protein labeling. In this study, an aldehyde tag was introduced to proteins using formylglycine-generating enzymes encoded by a reconstructed set of the pET28a plasmid system for enzyme immobilization. The haloacid dehalogenase ST2570 from Sulfolobus tokodaii was used as a model enzyme. The C-terminal aldehyde-tagged ST2570 (ST2570CQ) exhibited significant enzymological properties, such as new free aldehyde groups, a high level of protein expression and improved enzyme activity. SBA-15 has widely been used as an immobilization support for its large surface and excellent thermal and chemical stability. It was functionalized with amino groups by aminopropyltriethoxysilane. The C-terminal aldehyde-tagged ST2570 was immobilized to SBA-15 by covalent binding. The site-specific immobilization of ST2570 avoided the chemical denaturation that occurs in general covalent immobilization and resulted in better fastening compared to physical adsorption. The site-specific immobilized ST2570 showed 3-fold higher thermal stability, 1.2-fold higher catalytic ability and improved operational stability than free ST2570. The site-specific immobilized ST2570 retained 60% of its original activity after seven cycles of batch operation, and it was superior to the ST2570 immobilized to SBA-15 by physical adsorption, which loses 40% of its original activity when used for the second time. It is remarkable that the site-specific immobilized ST2570 still retained 100% of its original activity after 10 cycles of reuse in the semi-continuous flow reactor. Overall, these results provide support for the industrial-scale production and application of site-specific, covalently immobilized ST2570. Full article

(This article belongs to the Special Issue Enzyme Immobilization 2016)

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Open AccessArticle

Construction of an Immobilized Thermophilic Esterase on Epoxy Support for Poly(ε-caprolactone) Synthesis

by Hui Ren, Zhen Xing, Jiebing Yang, Wei Jiang, Gang Zhang, Jun Tang and Quanshun Li

Molecules 2016, 21(6), 796; https://doi.org/10.3390/molecules21060796 - 18 Jun 2016

Cited by 11 | Viewed by 5681

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 (M_n < 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 M_n value of 3005 g/mol in a gram-scale reaction. Full article

(This article belongs to the Special Issue Enzyme Immobilization 2016)

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Open AccessArticle

Tailoring the Spacer Arm for Covalent Immobilization of Candida antarctica Lipase B—Thermal Stabilization by Bisepoxide-Activated Aminoalkyl Resins in Continuous-Flow Reactors

by Emese Abaházi, Dávid Lestál, Zoltán Boros and László Poppe

Molecules 2016, 21(6), 767; https://doi.org/10.3390/molecules21060767 - 13 Jun 2016

Cited by 35 | Viewed by 6635

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 [...] Read more.

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 (r_flow), enantiomer selectivity (E) and enantiomeric excess (ee) achievable with the best immobilized CaLB preparations were studied as a function of temperature in kinetic resolution of rac-1 performed in continuous-flow packed-bed bioreactors. The optimum temperature of the most active HA-HDGE CaLB in continuous-flow mode was 60 °C. Although CaLB immobilized on the glycerol diglycidyl ether (GDGE)-activated EA-resin was less active and less selective, a much higher optimum temperature (80 °C) was observed with this form in continuous-flow mode KR of rac-1. Full article

(This article belongs to the Special Issue Enzyme Immobilization 2016)

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Open AccessArticle

Stabilization of Candida antarctica Lipase B (CALB) Immobilized on Octyl Agarose by Treatment with Polyethyleneimine (PEI)

by Sara Peirce, Veymar G. Tacias-Pascacio, Maria Elena Russo, Antonio Marzocchella, José J. Virgen-Ortíz and Roberto Fernandez-Lafuente

Molecules 2016, 21(6), 751; https://doi.org/10.3390/molecules21060751 - 08 Jun 2016

Cited by 48 | Viewed by 6898

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 [...] Read more.

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

by Nazzoly Rueda, Tiago L. Albuquerque, Rocio Bartolome-Cabrero, Laura Fernandez-Lopez, Rodrigo Torres, Claudia Ortiz, Jose C. S. Dos Santos, Oveimar Barbosa and Roberto Fernandez-Lafuente

Molecules 2016, 21(5), 646; https://doi.org/10.3390/molecules21050646 - 16 May 2016

Cited by 64 | Viewed by 7300

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 [...] Read more.

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

by Chien-Tai Tsai and Anne S. Meyer

Molecules 2014, 19(12), 19390-19406; https://doi.org/10.3390/molecules191219390 - 25 Nov 2014

Cited by 54 | Viewed by 7784

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 [...] Read more.

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)

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Open AccessArticle

Substrate Specificity and Enzyme Recycling Using Chitosan Immobilized Laccase

by Everton Skoronski, Mylena Fernandes, Maria De Lourdes Borba Magalhães, Gustavo Felippe Da Silva, Jair Juarez João, Carlos Henrique Lemos Soares and Agenor Fúrigo Júnior

Molecules 2014, 19(10), 16794-16809; https://doi.org/10.3390/molecules191016794 - 17 Oct 2014

Cited by 24 | Viewed by 9246

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 [...] Read more.

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

by Qing Chang and Heqing Tang

Molecules 2014, 19(10), 15768-15782; https://doi.org/10.3390/molecules191015768 - 29 Sep 2014

Cited by 108 | Viewed by 11258

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 [...] Read more.

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)

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Open AccessArticle

Carbon Nanotubes as Supports for Inulinase Immobilization

by Tais B. Garlet, Caroline T. Weber, Rodrigo Klaic, Edson L. Foletto, Sergio L. Jahn, Marcio A. Mazutti and Raquel C. Kuhn

Molecules 2014, 19(9), 14615-14624; https://doi.org/10.3390/molecules190914615 - 15 Sep 2014

Cited by 26 | Viewed by 5641

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 [...] Read more.

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)

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Open AccessArticle

Influence of the Morphology of Core-Shell Supports on the Immobilization of Lipase B from Candida antarctica

by Martina C. C. Pinto, Denise M. G. Freire and José Carlos Pinto

Molecules 2014, 19(8), 12509-12530; https://doi.org/10.3390/molecules190812509 - 18 Aug 2014

Cited by 38 | Viewed by 6861

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 [...] Read more.

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

by Carlos Luna, Cristóbal Verdugo, Enrique D. Sancho, Diego Luna, Juan Calero, Alejandro Posadillo, Felipa M. Bautista and Antonio A. Romero

Molecules 2014, 19(8), 11419-11439; https://doi.org/10.3390/molecules190811419 - 04 Aug 2014

Cited by 23 | Viewed by 8779

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, [...] Read more.

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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936 KiB

Open AccessArticle

Nucleoside 2'-Deoxyribosyltransferase from Psychrophilic Bacterium Bacillus psychrosaccharolyticus — Preparation of an Immobilized Biocatalyst for the Enzymatic Synthesis of Therapeutic Nucleosides

by Alba Fresco-Taboada, Immacolata Serra, Jesús Fernández-Lucas, Carmen Acebal, Miguel Arroyo, Marco Terreni and Isabel De la Mata

Molecules 2014, 19(8), 11231-11249; https://doi.org/10.3390/molecules190811231 - 31 Jul 2014

Cited by 23 | Viewed by 7190

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 [...] Read more.

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)

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Open AccessArticle

Preparation of Polyphosphazene Hydrogels for Enzyme Immobilization

by Yue-Cheng Qian, Peng-Cheng Chen, Gui-Jin He, Xiao-Jun Huang and Zhi-Kang Xu

Molecules 2014, 19(7), 9850-9863; https://doi.org/10.3390/molecules19079850 - 08 Jul 2014

Cited by 25 | Viewed by 7043

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, [...] Read more.

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)

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Open AccessArticle

Glutaraldehyde Cross-Linking of Immobilized Thermophilic Esterase on Hydrophobic Macroporous Resin for Application in Poly(ε-caprolactone) Synthesis

by Min Wang, Hui Shi, Di Wu, Haobo Han, Jianxu Zhang, Zhen Xing, Shuang Wang and Quanshun Li

Molecules 2014, 19(7), 9838-9849; https://doi.org/10.3390/molecules19079838 - 08 Jul 2014

Cited by 19 | Viewed by 6992

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 [...] Read more.

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 M_n 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)

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Open AccessArticle

Additives Enhancing the Catalytic Properties of Lipase from Burkholderia cepacia Immobilized on Mixed-Function-Grafted Mesoporous Silica Gel

by Emese Abaházi, Zoltán Boros and László Poppe

Molecules 2014, 19(7), 9818-9837; https://doi.org/10.3390/molecules19079818 - 08 Jul 2014

Cited by 39 | Viewed by 7143

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 ( [...] Read more.

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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457 KiB

Open AccessArticle

Combined Effects of Ultrasound and Immobilization Protocol on Butyl Acetate Synthesis Catalyzed by CALB

by Joana S. Alves, Cristina Garcia-Galan, Mirela F. Schein, Alexandre M. Silva, Oveimar Barbosa, Marco A. Z. Ayub, Roberto Fernandez-Lafuente and Rafael C. Rodrigues

Molecules 2014, 19(7), 9562-9576; https://doi.org/10.3390/molecules19079562 - 07 Jul 2014

Cited by 43 | Viewed by 6586

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 [...] Read more.

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

by Rubens A. Silva, Ana M. Carmona-Ribeiro and Denise F. S. Petri

Molecules 2014, 19(6), 8610-8628; https://doi.org/10.3390/molecules19068610 - 24 Jun 2014

Cited by 11 | Viewed by 7978

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 [...] Read more.

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 (K_M), but also the highest V_max and k_cat 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 K_M 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)

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Open AccessArticle

Immobilization of Trichoderma harzianum α-Amylase on Treated Wool: Optimization and Characterization

by Saleh A. Mohamed, Jalaluddin A. Khan, Omar A. M. Al-Bar and Reda M. El-Shishtawy

Molecules 2014, 19(6), 8027-8038; https://doi.org/10.3390/molecules19068027 - 13 Jun 2014

Cited by 40 | Viewed by 7395

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 [...] Read more.

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

by Cristina Garcia-Galan, Oveimar Barbosa, Karel Hernandez, Jose C. S. dos Santos, Rafael C. Rodrigues and Roberto Fernandez-Lafuente

Molecules 2014, 19(6), 7629-7645; https://doi.org/10.3390/molecules19067629 - 10 Jun 2014

Cited by 62 | Viewed by 6809

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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Fabrication of Implantable, Enzyme-Immobilized Glutamate Sensors for the Monitoring of Glutamate Concentration Changes in Vitro and in Vivo

by Tina T.-C. Tseng, Cheng-Fu Chang and Wen-Chin Chan

Molecules 2014, 19(6), 7341-7355; https://doi.org/10.3390/molecules19067341 - 05 Jun 2014

Cited by 28 | Viewed by 7495

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)

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Open AccessArticle

Mesoporous Silicas with Tunable Morphology for the Immobilization of Laccase

by Victoria Gascón, Isabel Díaz, Carlos Márquez-Álvarez and Rosa M. Blanco

Molecules 2014, 19(6), 7057-7071; https://doi.org/10.3390/molecules19067057 - 30 May 2014

Cited by 45 | Viewed by 7530

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

by Tzong-Yuan Juang, Shao-Ju Kan, Yi-Yu Chen, Yi-Lin Tsai, Min-Guan Lin and Long-Liu Lin

Molecules 2014, 19(4), 4997-5012; https://doi.org/10.3390/molecules19044997 - 22 Apr 2014

Cited by 25 | Viewed by 6813

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 Fe^2+/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 Fe^2+/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)

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Open AccessArticle

Laccase Immobilized on a PAN/Adsorbents Composite Nanofibrous Membrane for Catechol Treatment by a Biocatalysis/Adsorption Process

by Qingqing Wang, Jing Cui, Guohui Li, Jinning Zhang, Dawei Li, Fenglin Huang and Qufu Wei

Molecules 2014, 19(3), 3376-3388; https://doi.org/10.3390/molecules19033376 - 19 Mar 2014

Cited by 59 | Viewed by 7752

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)

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Open AccessFeature PaperReview

Agarose and Its Derivatives as Supports for Enzyme Immobilization

by Paolo Zucca, Roberto Fernandez-Lafuente and Enrico Sanjust

Molecules 2016, 21(11), 1577; https://doi.org/10.3390/molecules21111577 - 19 Nov 2016

Cited by 225 | Viewed by 25813

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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Enzyme Engineering for In Situ Immobilization

by Fabian B. H. Rehm, Shuxiong Chen and Bernd H. A. Rehm

Molecules 2016, 21(10), 1370; https://doi.org/10.3390/molecules21101370 - 14 Oct 2016

Cited by 82 | Viewed by 11785

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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Open AccessReview

Immobilization of Glycoside Hydrolase Families GH1, GH13, and GH70: State of the Art and Perspectives

by Natália G. Graebin, Jéssie Da N. Schöffer, Diandra De Andrades, Plinho F. Hertz, Marco A. Z. Ayub and Rafael C. Rodrigues

Molecules 2016, 21(8), 1074; https://doi.org/10.3390/molecules21081074 - 17 Aug 2016

Cited by 47 | Viewed by 10051

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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Inorganic Materials as Supports for Covalent Enzyme Immobilization: Methods and Mechanisms

by Paolo Zucca and Enrico Sanjust

Molecules 2014, 19(9), 14139-14194; https://doi.org/10.3390/molecules190914139 - 09 Sep 2014

Cited by 362 | Viewed by 23799

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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Trends in Protein-Based Biosensor Assemblies for Drug Screening and Pharmaceutical Kinetic Studies

by Ana M. Gonçalves, Augusto Q. Pedro, Fátima M. Santos, Luís M. Martins, Cláudio J. Maia, João A. Queiroz and Luís A. Passarinha

Molecules 2014, 19(8), 12461-12485; https://doi.org/10.3390/molecules190812461 - 18 Aug 2014

Cited by 31 | Viewed by 7402

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)

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Application of Iron Magnetic Nanoparticles in Protein Immobilization

by Jiakun Xu, Jingjing Sun, Yuejun Wang, Jun Sheng, Fang Wang and Mi Sun

Molecules 2014, 19(8), 11465-11486; https://doi.org/10.3390/molecules190811465 - 04 Aug 2014

Cited by 233 | Viewed by 16085

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)

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Immobilization as a Strategy for Improving Enzyme Properties-Application to Oxidoreductases

by Urszula Guzik, Katarzyna Hupert-Kocurek and Danuta Wojcieszyńska

Molecules 2014, 19(7), 8995-9018; https://doi.org/10.3390/molecules19078995 - 27 Jun 2014

Cited by 423 | Viewed by 17425

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)

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Open AccessReview

Polyhydroyxalkanoate Synthase Fusions as a Strategy for Oriented Enzyme Immobilisation

by David O. Hooks, Mark Venning-Slater, Jinping Du and Bernd H. A. Rehm

Molecules 2014, 19(6), 8629-8643; https://doi.org/10.3390/molecules19068629 - 24 Jun 2014

Cited by 29 | Viewed by 7436

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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Open AccessCommentary

Solid-State Metalloproteins—An Alternative to Immobilisation

by Trevor D. Rapson

Molecules 2016, 21(7), 919; https://doi.org/10.3390/molecules21070919 - 14 Jul 2016

Cited by 6 | Viewed by 4116

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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