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176 KiB

Open AccessEditorial

Special Issue: Enzyme Immobilization 2016

by Roberto Fernandez-Lafuente

Molecules 2017, 22(4), 601; https://doi.org/10.3390/molecules22040601 - 8 Apr 2017

Cited by 10 | Viewed by 4794

Abstract

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

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

Research

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9298 KiB

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 5327

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 23 | Viewed by 6241

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 23 | Viewed by 7664

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

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 91 | Viewed by 7077

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 38 | Viewed by 5803

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 45 | Viewed by 8700

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 - 5 Jan 2017

Cited by 56 | Viewed by 6413

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

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 23 | Viewed by 7248

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 - 8 Nov 2016

Cited by 27 | Viewed by 6480

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

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 - 9 Sep 2016

Cited by 26 | Viewed by 8268

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 61 | Viewed by 6703

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 6341

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 12 | Viewed by 6048

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 38 | Viewed by 7168

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 - 8 Jun 2016

Cited by 48 | Viewed by 7621

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 66 | Viewed by 7829

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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Review

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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 245 | Viewed by 28857

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 88 | Viewed by 12736

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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1376 KiB

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 52 | Viewed by 10777

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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Other

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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 4377

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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