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Special Issue "Enzyme Immobilization"

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

Deadline for manuscript submissions: closed (15 May 2014)

Special Issue Editor

Guest Editor
Prof. Dr. Roberto Fernandez-Lafuente (Website)

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

Special Issue Information

Dear Collegues,

The immobilization of enzymes and proteins is a seemingly ancient research area that still attracts great interest. The initial interest in enzyme immobilization was motivated by a desire to simplify the reuse of these expensive biocatalysts. However, at present, immobilization has developed into a very powerful tool that has been used to overcome many enzyme limitations. Applications include improving enzyme stability, activity, selectivity or specificity, reducing inhibition problems, and even coupling immobilization and purification, etc.

To date, many immobilization protocols have been reported. However, research is still the cornerstone of obtaining full control of the orientation of the enzyme on the support surface, and of the intensity of the support-enzyme interactions. Moreover, immobilization should not be considered an alternative to any other enzyme improvement strategy. For example, genetic and chemical tools may be utilized to produce a better enzyme that maintain improved properties after immobilization, or to improve immobilization; in both situations, alternative techniques produce an improved biocatalyst.

Thus, the efforts to design strategies involving the coupled use of immobilization with microbiological (e.g., the use of thermophilic enzymes), chemical or genetic modifications are of special interest. Enzymes are co-immobilized to catalyze cascade reactions; however, this process may produce additional complications. This special issue invites submissions (i.e., research or review papers) discussing the design of new immobilization protocols, especially when the control of the enzyme orientation is intended by design of the support, or modification of the enzyme. Papers related to the modification of immobilized enzymes, or to the modification of the support surface after enzyme immobilization, are also welcome.

Dr. Roberto Fernandez-Lafuente
Guest Editor

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

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Keywords

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

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Editorial

Jump to: Research, Review

Open AccessEditorial Editorial: Special Issue — Enzyme Immobilization
Molecules 2014, 19(12), 20671-20674; doi:10.3390/molecules191220671
Received: 5 December 2014 / Accepted: 8 December 2014 / Published: 10 December 2014
Cited by 2 | PDF Full-text (645 KB) | HTML Full-text | XML Full-text
Abstract
Immobilization of enzymes and proteins is a requirement for many industrial enzyme applications, as this facilitates enzyme recovery and reuse. Bearing in mind this necessity, the coupling of immobilization to the improvement of other enzyme features has been pursued by many researchers, [...] Read more.
Immobilization of enzymes and proteins is a requirement for many industrial enzyme applications, as this facilitates enzyme recovery and reuse. Bearing in mind this necessity, the coupling of immobilization to the improvement of other enzyme features has been pursued by many researchers, and nowadays immobilization is recognized as a tool to improve not only stability, but also enzyme selectivity, specificity, resistance to inhibition or chemical modifiers, etc. To achieve these overall improvements of enzymes’ properties via immobilization, it is necessary to both develop new immobilization systems suitable for these purposes, and to achieve a deeper knowledge of the mechanisms of interaction between enzymes and activated solids. That way, immobilization of enzymes, far being an old-fashioned methodology to just reuse these expensive biocatalysts, is a tool of continuous interest that requires a continuous effort to be exploited in all its potential. This special issue collects 23 papers reporting advances in the field of immobilization of enzymes.[...] Full article
(This article belongs to the Special Issue Enzyme Immobilization)

Research

Jump to: Editorial, Review

Open AccessArticle Enzymatic Cellulose Hydrolysis: Enzyme Reusability and Visualization of β-Glucosidase Immobilized in Calcium Alginate
Molecules 2014, 19(12), 19390-19406; doi:10.3390/molecules191219390
Received: 31 July 2014 / Revised: 15 November 2014 / Accepted: 17 November 2014 / Published: 25 November 2014
Cited by 4 | PDF Full-text (2491 KB) | HTML Full-text | XML Full-text
Abstract
The high cellulase enzyme dosages required for hydrolysis of cellulose is a major cost challenge in lignocellulosic ethanol production. One method to decrease the enzyme dosage and increase biocatalytic productivity is to re-use β-glucosidase (BG) via immobilization. In the present research, glutaraldehyde [...] 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)
Open AccessArticle Substrate Specificity and Enzyme Recycling Using Chitosan Immobilized Laccase
Molecules 2014, 19(10), 16794-16809; doi:10.3390/molecules191016794
Received: 16 May 2014 / Revised: 22 September 2014 / Accepted: 8 October 2014 / Published: 17 October 2014
Cited by 4 | PDF Full-text (389 KB) | HTML Full-text | XML Full-text
Abstract
The immobilization of laccase (Aspergillus sp.) on chitosan by cross-linking and its application in bioconversion of phenolic compounds in batch reactors were studied. Investigation was performed using laccase immobilized via chemical cross-linking due to the higher enzymatic operational stability of this [...] 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)
Figures

Open AccessArticle Immobilization of Horseradish Peroxidase on NH2-Modified Magnetic Fe3O4/SiO2 Particles and Its Application in Removal of 2,4-Dichlorophenol
Molecules 2014, 19(10), 15768-15782; doi:10.3390/molecules191015768
Received: 14 May 2014 / Revised: 9 August 2014 / Accepted: 7 September 2014 / Published: 29 September 2014
Cited by 20 | PDF Full-text (1766 KB) | HTML Full-text | XML Full-text
Abstract
Fe3O4 nanoparticles were prepared by a co-precipitation method with the assistance of ultrasound irradiation, and then coated with silica generated by hydrolysis and condensation of tetraethoxysilane. The silica-coated Fe3O4 nanoparticles were further modified with 3-aminopropyltriethoxysilane, resulting [...] 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)
Open AccessArticle Carbon Nanotubes as Supports for Inulinase Immobilization
Molecules 2014, 19(9), 14615-14624; doi:10.3390/molecules190914615
Received: 16 May 2014 / Revised: 16 August 2014 / Accepted: 22 August 2014 / Published: 15 September 2014
Cited by 6 | PDF Full-text (731 KB) | HTML Full-text | XML Full-text
Abstract
The commercial inulinase obtained from Aspergillus niger was non-covalently immobilized on multiwalled carbon nanotubes (MWNT-COOH). The immobilization conditions for the carbon nanotubes were defined by the central composite rotational design (CCRD). The effects of enzyme concentration (0.8%–1.7% v/v) and adsorbent:adsorbate ratio (1:460–1:175) [...] 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)
Open AccessArticle Influence of the Morphology of Core-Shell Supports on the Immobilization of Lipase B from Candida antarctica
Molecules 2014, 19(8), 12509-12530; doi:10.3390/molecules190812509
Received: 19 May 2014 / Revised: 28 July 2014 / Accepted: 1 August 2014 / Published: 18 August 2014
Cited by 4 | PDF Full-text (1146 KB) | HTML Full-text | XML Full-text
Abstract
Core-shell polymer particles with different properties were produced through combined suspension-emulsion polymerizations and employed as supports for immobilization of lipase B from Candida antarctica. In order to evaluate how the morphology of the particles affects the immobilization parameters, empirical models were [...] 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)
Figures

Open AccessArticle Biocatalytic Behaviour of Immobilized Rhizopus oryzae Lipase in the 1,3-Selective Ethanolysis of Sunflower Oil to Obtain a Biofuel Similar to Biodiesel
Molecules 2014, 19(8), 11419-11439; doi:10.3390/molecules190811419
Received: 5 May 2014 / Revised: 22 July 2014 / Accepted: 22 July 2014 / Published: 4 August 2014
Cited by 3 | PDF Full-text (1249 KB) | HTML Full-text | XML Full-text
Abstract
A new biofuel similar to biodiesel was obtained in the 1,3-selective transesterification reaction of sunflower oil with ethanol using as biocatalyst a Rhizopus oryzae lipase (ROL) immobilized on Sepiolite, an inorganic support. The studied lipase was a low cost powdered enzyme preparation, [...] 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)
Figures

Open AccessArticle Nucleoside 2'-Deoxyribosyltransferase from Psychrophilic Bacterium Bacillus psychrosaccharolyticus — Preparation of an Immobilized Biocatalyst for the Enzymatic Synthesis of Therapeutic Nucleosides
Molecules 2014, 19(8), 11231-11249; doi:10.3390/molecules190811231
Received: 11 June 2014 / Revised: 23 July 2014 / Accepted: 24 July 2014 / Published: 31 July 2014
Cited by 3 | PDF Full-text (936 KB) | HTML Full-text | XML Full-text
Abstract
Nucleoside 2'-deoxyribosyltransferase (NDT) from the psychrophilic bacterium Bacillus psychrosaccharolyticus CECT 4074 has been cloned and produced for the first time. A preliminary characterization of the recombinant protein indicates that the enzyme is an NDT type II since it catalyzes the transfer of [...] 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)
Open AccessArticle Additives Enhancing the Catalytic Properties of Lipase from Burkholderia cepacia Immobilized on Mixed-Function-Grafted Mesoporous Silica Gel
Molecules 2014, 19(7), 9818-9837; doi:10.3390/molecules19079818
Received: 16 May 2014 / Revised: 23 June 2014 / Accepted: 2 July 2014 / Published: 8 July 2014
Cited by 8 | PDF Full-text (517 KB) | HTML Full-text | XML Full-text
Abstract
Effects of various additives on the lipase from Burkholderia cepacia (BcL) immobilized on mixed-function-grafted mesoporous silica gel support by hydrophobic adsorption and covalent attachment were investigated. Catalytic properties of the immobilized biocatalysts were characterized in kinetic resolution of racemic 1-phenylethanol [...] 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)
Figures

Open AccessArticle Glutaraldehyde Cross-Linking of Immobilized Thermophilic Esterase on Hydrophobic Macroporous Resin for Application in Poly(ε-caprolactone) Synthesis
Molecules 2014, 19(7), 9838-9849; doi:10.3390/molecules19079838
Received: 21 May 2014 / Revised: 3 July 2014 / Accepted: 4 July 2014 / Published: 8 July 2014
Cited by 2 | PDF Full-text (330 KB) | HTML Full-text | XML Full-text
Abstract
The immobilized thermophilic esterase from Archaeoglobus fulgidus was successfully constructed through the glutaraldehyde-mediated covalent coupling after its physical adsorption on a hydrophobic macroporous resin, Sepabeads EC-OD. Through 0.05% glutaraldehyde treatment, the prevention of enzyme leaching and the maintenance of catalytic activity could [...] 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 Mn values lower than 1010 g/mol. Furthermore, the cross‑linked immobilized thermophilic esterase exhibited an excellent operational stability, with monomer conversion values exceeding 90% over the course of 12 batch reactions, still more than 80% after 16 batch reactions. Full article
(This article belongs to the Special Issue Enzyme Immobilization)
Open AccessArticle Preparation of Polyphosphazene Hydrogels for Enzyme Immobilization
Molecules 2014, 19(7), 9850-9863; doi:10.3390/molecules19079850
Received: 19 May 2014 / Revised: 23 June 2014 / Accepted: 24 June 2014 / Published: 8 July 2014
Cited by 3 | PDF Full-text (490 KB) | HTML Full-text | XML Full-text
Abstract
We report on the synthesis and application of a new hydrogel based on a methacrylate substituted polyphosphazene. Through ring-opening polymerization and nucleophilic substitution, poly[bis(methacrylate)phosphazene] (PBMAP) was successfully synthesized from hexachlorocyclotriphosphazene. By adding PBMAP to methacrylic acid solution and then treating with UV [...] 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)
Open AccessArticle Combined Effects of Ultrasound and Immobilization Protocol on Butyl Acetate Synthesis Catalyzed by CALB
Molecules 2014, 19(7), 9562-9576; doi:10.3390/molecules19079562
Received: 26 May 2014 / Revised: 30 June 2014 / Accepted: 1 July 2014 / Published: 7 July 2014
Cited by 4 | PDF Full-text (457 KB) | HTML Full-text | XML Full-text
Abstract
It is well established that the performance of lipase B from Candida antarctica (CALB) as catalyst for esterification reactions may be improved by the use of ultrasound technology or by its immobilization on styrene-divinylbenzene beads (MCI-CALB). The present research evaluated the synthesis [...] 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
Molecules 2014, 19(6), 8610-8628; doi:10.3390/molecules19068610
Received: 4 May 2014 / Revised: 20 June 2014 / Accepted: 20 June 2014 / Published: 24 June 2014
Cited by 4 | PDF Full-text (565 KB) | HTML Full-text | XML Full-text
Abstract
Poly(ethylene glycol) (PEG)-decorated polystyrene (PS) nanoparticles with mean hydrodynamic diameter (D) and zeta–potential (ζ) of (286 ± 15) nm and (−50 ± 5) mV, respectively, were modified by the adsorption of Congo red (CR). The PS/PEG/CR particles presented [...] 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 (KM), but also the highest Vmax and kcat values. Moreover, it could be recycled seven times, losing a maximum 10% or 30% of the original enzymatic activity at 40 °C or 25 °C, respectively. Although lipases immobilized onto PS/PEG particles presented the smallest KM values, the reactions were comparatively the slowest and recycling was not possible. Hydrolysis reactions performed in the temperature range of 25 °C to 60 °C with free lipases and lipases immobilized onto PS/PEG/CR particles presented an optimal temperature at 40 °C. At 60 °C free lipases and lipases immobilized onto PS/PEG/CR presented ~80% and ~50% of the activity measured at 40 °C, indicating good thermal stability. Bioconjugation effects between CR and lipase were evidenced by circular dichroism spectroscopy and spectrophotometry. CR molecules mediate the open state conformation of the lipase lid and favor the substrate approaching. Full article
(This article belongs to the Special Issue Enzyme Immobilization)
Open AccessArticle Immobilization of Trichoderma harzianum α-Amylase on Treated Wool: Optimization and Characterization
Molecules 2014, 19(6), 8027-8038; doi:10.3390/molecules19068027
Received: 8 March 2014 / Revised: 4 June 2014 / Accepted: 6 June 2014 / Published: 13 June 2014
Cited by 5 | PDF Full-text (541 KB) | HTML Full-text | XML Full-text
Abstract
α-Amylase from Trichoderma harzianum was covalently immobilized on activated wool by cyanuric chloride. Immobilized α-amylase exhibited 75% of its initial activity after 10 runs. The soluble and immobilized α-amylases exhibited maximum activity at pH values 6.0 and 6.5, respectively. The immobilized enzyme [...] 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
Molecules 2014, 19(6), 7629-7645; doi:10.3390/molecules19067629
Received: 3 April 2014 / Revised: 3 June 2014 / Accepted: 4 June 2014 / Published: 10 June 2014
Cited by 12 | PDF Full-text (240 KB) | HTML Full-text | XML Full-text
Abstract
A commercial and very hydrophobic styrene-divinylbenzene matrix, MCI GEL® CHP20P, has been compared to octyl-Sepharose® beads as support to immobilize three different enzymes: lipases from Thermomyces lanuginosus (TLL) and from Rhizomucor miehie (RML) and Lecitase® Ultra, a commercial artificial [...] Read more.
A commercial and very hydrophobic styrene-divinylbenzene matrix, MCI GEL® CHP20P, has been compared to octyl-Sepharose® beads as support to immobilize three different enzymes: lipases from Thermomyces lanuginosus (TLL) and from Rhizomucor miehie (RML) and Lecitase® Ultra, a commercial artificial phospholipase. The immobilization mechanism on both supports was similar: interfacial activation of the enzymes versus the hydrophobic surface of the supports. Immobilization rate and loading capacity is much higher using MCI GEL® CHP20P compared to octyl-Sepharose® (87.2 mg protein/g of support using TLL, 310 mg/g using RML and 180 mg/g using Lecitase® Ultra). The thermal stability of all new preparations is much lower than that of the standard octyl-Sepharose® immobilized preparations, while the opposite occurs when the inactivations were performed in the presence of organic co-solvents. Regarding the hydrolytic activities, the results were strongly dependent on the substrate and pH of measurement. Octyl-Sepharose® immobilized enzymes were more active versus p-NPB than the enzymes immobilized on MCI GEL® CHP20P, while RML became 700-fold less active versus methyl phenylacetate. Thus, the immobilization of a lipase on this matrix needs to be empirically evaluated, since it may present very positive effects in some cases while in other cases it may have very negative ones. Full article
(This article belongs to the Special Issue Enzyme Immobilization)
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Open AccessArticle Fabrication of Implantable, Enzyme-Immobilized Glutamate Sensors for the Monitoring of Glutamate Concentration Changes in Vitro and in Vivo
Molecules 2014, 19(6), 7341-7355; doi:10.3390/molecules19067341
Received: 17 March 2014 / Revised: 30 May 2014 / Accepted: 3 June 2014 / Published: 5 June 2014
Cited by 6 | PDF Full-text (651 KB) | HTML Full-text | XML Full-text
Abstract
Glutamate sensors based on the immobilization of glutamate oxidase (GlutOx) were prepared by adsorption on electrodeposited chitosan (Method 1) and by crosslinking with glutaraldehyde (Method 2) on micromachined platinum microelectrodes. It was observed that glutamate sensors prepared by [...] Read more.
Glutamate sensors based on the immobilization of glutamate oxidase (GlutOx) were prepared by adsorption on electrodeposited chitosan (Method 1) and by crosslinking with glutaraldehyde (Method 2) on micromachined platinum microelectrodes. It was observed that glutamate sensors prepared by Method 1 have faster response time (<2 s) and lower detection limit (2.5 ± 1.1 μM) compared to that prepared by Method 2 (response time: <5 sec and detection limit: 6.5 ± 1.7 μM); glutamate sensors prepared by Method 2 have a larger linear detection range (20–352 μM) and higher sensitivity (86.8 ± 8.8 nA·μM−1·cm−2, N = 12) compared to those prepared by Method 1 (linear detection range: 20–217 μM and sensitivity: 34.9 ± 4.8 nA·μM−1·cm−2, N = 8). The applicability of the glutamate sensors in vivo was also demonstrated. The glutamate sensors were implanted into the rat brain to monitor the stress-induced extracellular glutamate release in the hypothalamus of the awake, freely moving rat. Full article
(This article belongs to the Special Issue Enzyme Immobilization)
Open AccessArticle Mesoporous Silicas with Tunable Morphology for the Immobilization of Laccase
Molecules 2014, 19(6), 7057-7071; doi:10.3390/molecules19067057
Received: 8 May 2014 / Revised: 20 May 2014 / Accepted: 23 May 2014 / Published: 30 May 2014
Cited by 9 | PDF Full-text (1170 KB) | HTML Full-text | XML Full-text
Abstract
Siliceous ordered mesoporous materials (OMM) are gaining interest as supports for enzyme immobilization due to their uniform pore size, large surface area, tunable pore network and the introduction of organic components to mesoporous structure. We used SBA-15 type silica materials, which exhibit [...] Read more.
Siliceous ordered mesoporous materials (OMM) are gaining interest as supports for enzyme immobilization due to their uniform pore size, large surface area, tunable pore network and the introduction of organic components to mesoporous structure. We used SBA-15 type silica materials, which exhibit a regular 2D hexagonal packing of cylindrical mesopores of uniform size, for non-covalent immobilization of laccase. Synthesis conditions were adjusted in order to obtain supports with different particle shape, where those with shorter channels had higher loading capacity. Despite the similar isoelectric points of silica and laccase and the close match between the size of laccase and the pore dimensions of these SBA-15 materials, immobilization was achieved with very low leaching. Surface modification of macro-/mesoporous amorphous silica by grafting of amine moieties was proved to significantly increase the isoelectric point of this support and improve the immobilization yield. Full article
(This article belongs to the Special Issue Enzyme Immobilization)
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Open AccessArticle Surface-Functionalized Hyperbranched Poly(Amido Acid) Magnetic Nanocarriers for Covalent Immobilization of a Bacterial γ-Glutamyltranspeptidase
Molecules 2014, 19(4), 4997-5012; doi:10.3390/molecules19044997
Received: 11 March 2014 / Revised: 15 April 2014 / Accepted: 17 April 2014 / Published: 22 April 2014
Cited by 9 | PDF Full-text (2201 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
In this study, we synthesized water-soluble hyperbranched poly(amido acid)s (HBPAAs) featuring multiple terminal CO2H units and internal tertiary amino and amido moieties and then used them in conjunction with an in situ Fe2+/Fe3+ co-precipitation process to prepare [...] Read more.
In this study, we synthesized water-soluble hyperbranched poly(amido acid)s (HBPAAs) featuring multiple terminal CO2H units and internal tertiary amino and amido moieties and then used them in conjunction with an in situ Fe2+/Fe3+ co-precipitation process to prepare organic/magnetic nanocarriers comprising uniformly small magnetic iron oxide nanoparticles (NP) incorporated within the globular HBPAAs. Transmission electron microscopy revealed that the HBPAA-γ-Fe2O3 NPs had dimensions of 6–11 nm, significantly smaller than those of the pristine γ-Fe2O3 (20–30 nm). Subsequently, we covalently immobilized a bacterial γ-glutamyltranspeptidase (BlGGT) upon the HBPAA-γ-Fe2O3 nanocarriers through the formation of amide linkages in the presence of a coupling agent. Magnetization curves of the HBPAA-γ-Fe2O3/BlGGT composites measured at 300 K suggested superparamagnetic characteristics, with a saturation magnetization of 52 emu g−1. The loading capacity of BlGGT on the HBPAA-γ-Fe2O3 nanocarriers was 16 mg g−1 support; this sample provided a 48% recovery of the initial activity. The immobilized enzyme could be recycled 10 times with 32% retention of the initial activity; it had stability comparable with that of the free enzyme during a storage period of 63 days. The covalent immobilization and stability of the enzyme and the magnetization provided by the HBPAA-γ-Fe2O3 NPs suggests that this approach could be an economical means of depositing bioactive enzymes upon nanocarriers for BlGGT-mediated bio-catalysis. Full article
(This article belongs to the Special Issue Enzyme Immobilization)
Open AccessArticle Laccase Immobilized on a PAN/Adsorbents Composite Nanofibrous Membrane for Catechol Treatment by a Biocatalysis/Adsorption Process
Molecules 2014, 19(3), 3376-3388; doi:10.3390/molecules19033376
Received: 20 January 2014 / Revised: 6 March 2014 / Accepted: 6 March 2014 / Published: 19 March 2014
Cited by 9 | PDF Full-text (1930 KB) | HTML Full-text | XML Full-text
Abstract
The treatment of catechol via biocatalysis and adsorption with a commercial laccase immobilized on polyacrylonitrile/montmorillonite/graphene oxide (PAN/MMT/GO) composite nanofibers was evaluated with a homemade nanofibrous membrane reactor. The properties in this process of the immobilized laccase on PAN, PAN/MMT as well as [...] Read more.
The treatment of catechol via biocatalysis and adsorption with a commercial laccase immobilized on polyacrylonitrile/montmorillonite/graphene oxide (PAN/MMT/GO) composite nanofibers was evaluated with a homemade nanofibrous membrane reactor. The properties in this process of the immobilized laccase on PAN, PAN/MMT as well as PAN/MMT/GO with different weight ratios of MMT and GO were investigated. These membranes were successfully applied for removal of catechol from an aqueous solution. Scanning electron microscope images revealed different morphologies of the enzyme aggregates on different supports. After incorporation of MMT or MMT/GO, the optimum pH showed an alkaline shift to 4, compared to 3.5 for laccase immobilized on pure PAN nanofibers. The optimum temperature was at 55 °C for all the immobilized enzymes. Besides, the addition of GO improved the operational stability and storage stability. A 39% ± 2.23% chemical oxygen demand (COD) removal from the catechol aqueous solution was achieved. Experimental results suggested that laccase, PAN, adsorbent nanoparticles (MMT/GO) can be combined together for catechol treatment in industrial applications. Full article
(This article belongs to the Special Issue Enzyme Immobilization)

Review

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Open AccessReview Inorganic Materials as Supports for Covalent Enzyme Immobilization: Methods and Mechanisms
Molecules 2014, 19(9), 14139-14194; doi:10.3390/molecules190914139
Received: 5 July 2014 / Revised: 9 August 2014 / Accepted: 22 August 2014 / Published: 9 September 2014
Cited by 43 | PDF Full-text (1733 KB) | HTML Full-text | XML Full-text
Abstract
Several inorganic materials are potentially suitable for enzymatic covalent immobilization, by means of several different techniques. Such materials must meet stringent criteria to be suitable as solid matrices: complete insolubility in water, reasonable mechanical strength and chemical resistance under the operational conditions, [...] Read more.
Several inorganic materials are potentially suitable for enzymatic covalent immobilization, by means of several different techniques. Such materials must meet stringent criteria to be suitable as solid matrices: complete insolubility in water, reasonable mechanical strength and chemical resistance under the operational conditions, the capability to form manageable particles with high surface area, reactivity towards derivatizing/functionalizing agents. Non-specific protein adsorption should be always considered when planning covalent immobilization on inorganic solids. A huge mass of experimental work has shown that silica, silicates, borosilicates and aluminosilicates, alumina, titania, and other oxides, are the materials of choice when attempting enzyme immobilizations on inorganic supports. More recently, some forms of elemental carbon, silicon, and certain metals have been also proposed for certain applications. With regard to the derivatization/functionalization techniques, the use of organosilanes through silanization is undoubtedly the most studied and the most applied, although inorganic bridge formation and acylation with selected acyl halides have been deeply studied. In the present article, the most common inorganic supports for covalent immobilization of the enzymes are reviewed, with particular focus on their advantages and disadvantages in terms of enzyme loadings, operational stability, undesired adsorption, and costs. Mechanisms and methods for covalent immobilization are also discussed, focusing on the most widespread activating approaches (such as glutaraldehyde, cyanogen bromide, divinylsulfone, carbodiimides, carbonyldiimidazole, sulfonyl chlorides, chlorocarbonates, N-hydroxysuccinimides). Full article
(This article belongs to the Special Issue Enzyme Immobilization)
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Open AccessReview Trends in Protein-Based Biosensor Assemblies for Drug Screening and Pharmaceutical Kinetic Studies
Molecules 2014, 19(8), 12461-12485; doi:10.3390/molecules190812461
Received: 28 May 2014 / Revised: 2 August 2014 / Accepted: 5 August 2014 / Published: 18 August 2014
Cited by 3 | PDF Full-text (1143 KB) | HTML Full-text | XML Full-text
Abstract
The selection of natural and chemical compounds for potential applications in new pharmaceutical formulations constitutes a time-consuming procedure in drug screening. To overcome this issue, new devices called biosensors, have already demonstrated their versatility and capacity for routine clinical diagnosis. Designed to [...] Read more.
The selection of natural and chemical compounds for potential applications in new pharmaceutical formulations constitutes a time-consuming procedure in drug screening. To overcome this issue, new devices called biosensors, have already demonstrated their versatility and capacity for routine clinical diagnosis. Designed to perform analytical analysis for the detection of a particular analyte, biosensors based on the coupling of proteins to amperometric and optical devices have shown the appropriate selectivity, sensibility and accuracy. During the last years, the exponential demand for pharmacokinetic studies in the early phases of drug development, along with the need of lower molecular weight detection, have led to new biosensor structure materials with innovative immobilization strategies. The result has been the development of smaller, more reproducible biosensors with lower detection limits, and with a drastic reduction in the required sample volumes. Therefore in order to describe the main achievements in biosensor fields, the present review has the main aim of summarizing the essential strategies used to generate these specific devices, that can provide, under physiological conditions, a credible molecule profile and assess specific pharmacokinetic parameters. Full article
(This article belongs to the Special Issue Enzyme Immobilization)
Open AccessReview Application of Iron Magnetic Nanoparticles in Protein Immobilization
Molecules 2014, 19(8), 11465-11486; doi:10.3390/molecules190811465
Received: 23 May 2014 / Revised: 9 July 2014 / Accepted: 9 July 2014 / Published: 4 August 2014
Cited by 27 | PDF Full-text (1045 KB) | HTML Full-text | XML Full-text
Abstract
Due to their properties such as superparamagnetism, high surface area, large surface-to-volume ratio, easy separation under external magnetic fields, iron magnetic nanoparticles have attracted much attention in the past few decades. Various modification methods have been developed to produce biocompatible magnetic nanoparticles [...] Read more.
Due to their properties such as superparamagnetism, high surface area, large surface-to-volume ratio, easy separation under external magnetic fields, iron magnetic nanoparticles have attracted much attention in the past few decades. Various modification methods have been developed to produce biocompatible magnetic nanoparticles for protein immobilization. This review provides an updated and integrated focus on the fabrication and characterization of suitable magnetic iron nanoparticle-based nano-active materials for protein immobilization. Full article
(This article belongs to the Special Issue Enzyme Immobilization)
Open AccessReview Immobilization as a Strategy for Improving Enzyme Properties-Application to Oxidoreductases
Molecules 2014, 19(7), 8995-9018; doi:10.3390/molecules19078995
Received: 12 May 2014 / Revised: 13 June 2014 / Accepted: 16 June 2014 / Published: 27 June 2014
Cited by 61 | PDF Full-text (858 KB) | HTML Full-text | XML Full-text
Abstract
The main objective of the immobilization of enzymes is to enhance the economics of biocatalytic processes. Immobilization allows one to re-use the enzyme for an extended period of time and enables easier separation of the catalyst from the product. Additionally, immobilization improves [...] Read more.
The main objective of the immobilization of enzymes is to enhance the economics of biocatalytic processes. Immobilization allows one to re-use the enzyme for an extended period of time and enables easier separation of the catalyst from the product. Additionally, immobilization improves many properties of enzymes such as performance in organic solvents, pH tolerance, heat stability or the functional stability. Increasing the structural rigidity of the protein and stabilization of multimeric enzymes which prevents dissociation-related inactivation. In the last decade, several papers about immobilization methods have been published. In our work, we present a relation between the influence of immobilization on the improvement of the properties of selected oxidoreductases and their commercial value. We also present our view on the role that different immobilization methods play in the reduction of enzyme inhibition during biotechnological processes. Full article
(This article belongs to the Special Issue Enzyme Immobilization)
Open AccessReview Polyhydroyxalkanoate Synthase Fusions as a Strategy for Oriented Enzyme Immobilisation
Molecules 2014, 19(6), 8629-8643; doi:10.3390/molecules19068629
Received: 16 May 2014 / Revised: 19 June 2014 / Accepted: 19 June 2014 / Published: 24 June 2014
Cited by 5 | PDF Full-text (279 KB) | HTML Full-text | XML Full-text
Abstract
Polyhydroxyalkanoate (PHA) is a carbon storage polymer produced by certain bacteria in unbalanced nutrient conditions. The PHA forms spherical inclusions surrounded by granule associate proteins including the PHA synthase (PhaC). Recently, the intracellular formation of PHA granules with covalently attached synthase from [...] 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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