Special Issue "Immobilized Biocatalysts"

A special issue of Catalysts (ISSN 2073-4344).

Deadline for manuscript submissions: 31 March 2018

Special Issue Editor

Guest Editor
Prof. Dr. Peter Grunwald

University of Hamburg, Department of Physical Chemistry, Grindelallee 117, 20146 Hamburg, Germany
Website | E-Mail
Interests: biocatalysis; enzymatic analysis; Environmental biotechnology

Special Issue Information

Dear Colleagues,

Biocatalysts—single enzymes or whole cells—have gained increasing importance in the last few decades as green alternatives to their chemical counterparts for a variety of industrial processes. They catalyze reactions with the advantage of superior chemo-, regio-, and stereo-specificity at mild conditions, thereby avoiding the production of larger amounts of waste. To prevent spoiling of the resulting products with protein the biocatalysts should be employed in an immobilized form. In contrast to the application of soluble enzymes, they enable a higher volume specific biocatalyst loading together with simplified downstream processing. In addition, they can be reused tantamount to reducing cost contribution to the final product. Immobilization may also contribute to an enhanced storage and operational stability compared to soluble biocatalysts.

Immobilized biocatalysts are—apart from application in the chemical/pharmaceutical industry—used as biosensors, in medical diagnoses, genomics and genome sequencing (next generation sequencing), for protein microarrays (tracking interactions and activities of proteins, drug screening, etc.), or enzyme biocomputing.

The activity (efficiency) of an immobilized biocatalyst depends on various properties of the carrier material such as particle size and shape, its chemical nature, density, porosity (mass transfer limitation of the compounds involved), pore-size distribution or the mechanical strength. In addition, suited immobilization supports aside from bearing functional groups on their surface should be hydrophilic, biocompatible, resistant to microbial attack, and readily available at a low cost. They are of inorganic or organic origin and include natural and synthetic polymers. Bioinspired scaffolds and nanostructured materials (nanoparticles (paramagnetic), nanofibers, nanotubes, graphene, nanocomposites) are also used for enzyme immobilization.

Traditional immobilization techniques comprise adsorption, covalent binding, crosslinking, and entrapment. Moreover, immobilization and chemical modification may be coupled with site-directed mutagenesis, and nanobiocatalysts are generated by biological assembly methods. For covalent, site-specific immobilization several chemical and enzymatic approaches have proven. A variety of surface analysis technologies exist to control enzyme immobilization.

Prof. Dr. Peter Grunwald
Guest Editor

Manuscript Submission Information

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Keywords

  • Immobilized Biocatalysts, application areas (industrial production of goods and chemicals, pharmaceuticals, biofuel) analytics, diagnoses, microarrays, biocomputing, biofuel cells, bioactive coatings
  • Immobilization techniques, -traditional, site-specific protein immobilization, enzyme-mediated immobilization, biological assembly methods, cascade reactions, protein scaffolds, self-assembly of enzyme nanostructures, multi-enzyme immobilization, immobilization and site-directed mutagenesis
  • Immobilization supports, -properties, (quantum dots, magnetic nanoparticles, nanorods, nanotubes, nanofibres, nanocomposites
  • inorganic and organic materials, natural and synthetic polymers), biosynthesis of nanoparticles
  • surface analysis technologies (atomic force spectroscopy, FRET, BRET, TEM, SPR, and others)
  • kinetics of immobilized biocatalysts.

Published Papers (8 papers)

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Research

Open AccessArticle Immobilization Effects on the Catalytic Properties of Two Fusarium Verticillioides Lipases: Stability, Hydrolysis, Transesterification and Enantioselectivity Improvement
Catalysts 2018, 8(2), 84; doi:10.3390/catal8020084
Received: 15 December 2017 / Revised: 30 January 2018 / Accepted: 2 February 2018 / Published: 16 February 2018
PDF Full-text (689 KB) | Supplementary Files
Abstract
Fusarium verticillioides lipases were purified in a “cascade” method using octadecyl Sepabeads and octyl Sepharose resins, which led to the isolation of two proteins with lipolytic activities. Lip 1 was purified after octyl Sepharose adsorption presenting 30.3 kDa and, Lip 2 presented 68.0
[...] Read more.
Fusarium verticillioides lipases were purified in a “cascade” method using octadecyl Sepabeads and octyl Sepharose resins, which led to the isolation of two proteins with lipolytic activities. Lip 1 was purified after octyl Sepharose adsorption presenting 30.3 kDa and, Lip 2 presented 68.0 kDa after octadecyl adsorption. These immobilization processes resulted in an increase of 3-fold in activity of each immobilized enzyme. These enzymes presented optima of pH of 5.0 and 6.0, respectively and temperature at 40 °C. They were thermostable at 40 °C and both remained more than 50% of its activity at the pH range of 5.0 to 7.0, with 180 min of incubation. The sardine oil hydrolysis showed higher EPA/DHA ratio. Concerning the ethanolysis reaction, Lip 2 showed higher conversion (5.5%) and both lipases showed activity in the release of the S enantiomers from 2-O-butyryl-2-phenylacetic acid (mandelic butyrate acid) and HPBE hydrolysis. Lip 2 also demonstrated capacity of transesterification. These applications made these enzymes attractive for industrial application. Full article
(This article belongs to the Special Issue Immobilized Biocatalysts)
Open AccessArticle Genetically Fused T4L Acts as a Shield in Covalent Enzyme Immobilisation Enhancing the Rescued Activity
Catalysts 2018, 8(1), 40; doi:10.3390/catal8010040
Received: 3 January 2018 / Revised: 15 January 2018 / Accepted: 16 January 2018 / Published: 20 January 2018
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Abstract
Enzyme immobilisation is a common strategy to increase enzymes resistance and reusability in a variety of excellent ‘green’ applications. However, the interaction with the solid support often leads to diminished specific activity, especially when non-specific covalent binding to the carrier takes place which
[...] Read more.
Enzyme immobilisation is a common strategy to increase enzymes resistance and reusability in a variety of excellent ‘green’ applications. However, the interaction with the solid support often leads to diminished specific activity, especially when non-specific covalent binding to the carrier takes place which affects the delicate architecture of the enzyme. Here we developed a broadly applicable strategy where the T4-lysozyme (T4L) is genetically fused at the N-terminus of different enzymes and used as inert protein spacer which directly attaches to the carrier preventing shape distortion of the catalyst. Halomonas elongata aminotransferase (HEWT), Bacillus subtilis engineered esterase (BS2m), and horse liver alcohol dehydrogenase (HLADH) were used as model enzymes to elucidate the benefits of the spacer. While HEWT and HLADH activity and expression were diminished by the fused T4L, both enzymes retained almost quantitative activity after immobilisation. In the case of BS2m, the protective effect of the T4L effectively was important and led to up to 10-fold improvement in the rescued activity. Full article
(This article belongs to the Special Issue Immobilized Biocatalysts)
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Open AccessCommunication Co-Detection of Dopamine and Glucose with High Temporal Resolution
Catalysts 2018, 8(1), 34; doi:10.3390/catal8010034
Received: 6 December 2017 / Revised: 11 January 2018 / Accepted: 15 January 2018 / Published: 19 January 2018
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Abstract
Neuronal activity and brain glucose metabolism are tightly coupled, where triggered neurotransmission leads to a higher demand for glucose. To better understand the regulation of neuronal activity and its relation to high-speed metabolism, development of analytical tools that can temporally resolve the transients
[...] Read more.
Neuronal activity and brain glucose metabolism are tightly coupled, where triggered neurotransmission leads to a higher demand for glucose. To better understand the regulation of neuronal activity and its relation to high-speed metabolism, development of analytical tools that can temporally resolve the transients of vesicular neurotransmitter release and fluctuations of metabolites such as glucose in the local vicinity of the activated neurons is needed. Here we present an amperometric biosensor design for rapid co-detection of glucose and the neurotransmitter dopamine. The sensor is based on the immobilization of an ultra-thin layer of glucose oxidase on to a gold-nanoparticle-covered carbon fiber microelectrode. Our electrode, by altering the potential applied at the sensor surface, allows for the high-speed recording of both glucose and dopamine. We demonstrate that, even though glucose is electrochemically detected indirectly through the enzymatic product and the electroactive dopamine is sensed directly, when exposing the sensor surface to a mixture of the two analytes, fluctuations in glucose and dopamine concentrations can be visualized with similar speed and at a millisecond time scale. Hence, by minimizing the enzyme coating thickness at the sensor surface, dual detection of glucose and dopamine can be realized at the same sensor surface and at time scales necessary for monitoring fast metabolic alterations during neurotransmission. Full article
(This article belongs to the Special Issue Immobilized Biocatalysts)
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Open AccessArticle “Deceived” Concentrated Immobilized Cells as Biocatalyst for Intensive Bacterial Cellulose Production from Various Sources
Catalysts 2018, 8(1), 33; doi:10.3390/catal8010033
Received: 21 December 2017 / Revised: 9 January 2018 / Accepted: 15 January 2018 / Published: 18 January 2018
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Abstract
A new biocatalyst in the form of Komagataeibacter xylinum B-12429 cells immobilized in poly(vinyl alcohol) cryogel for production of bacterial cellulose was demonstrated. Normally, the increased bacteria concentration causes an enlarged bacterial cellulose synthesis while cells push the polysaccharide out to pack themselves
[...] Read more.
A new biocatalyst in the form of Komagataeibacter xylinum B-12429 cells immobilized in poly(vinyl alcohol) cryogel for production of bacterial cellulose was demonstrated. Normally, the increased bacteria concentration causes an enlarged bacterial cellulose synthesis while cells push the polysaccharide out to pack themselves into this polymer and go into a stasis. Immobilization of cells into the poly(vinyl alcohol) cryogel allowed “deceiving” them: bacteria producing cellulose pushed it out, which further passed through the pores of cryogel matrix and was accumulated in the medium while not covering the cells; hence, the latter were deprived of a possible transition to inactivity and worked on the synthesis of bacterial cellulose even more actively. The repeated use of immobilized cells retaining 100% of their metabolic activity for at least 10 working cycles (60 days) was performed. The immobilized cells produce bacterial cellulose with crystallinity and porosity similar to polysaccharide of free cells, but having improved stiffness and tensile strength. Various media containing sugars and glycerol, based on hydrolysates of renewable biomass sources (aspen, Jerusalem artichoke, rice straw, microalgae) were successfully applied for bacterial cellulose production by immobilized cells, and the level of polysaccharide accumulation was 1.3–1.8-times greater than suspended cells could produce. Full article
(This article belongs to the Special Issue Immobilized Biocatalysts)
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Open AccessArticle Immobilization of Cellulase on a Functional Inorganic–Organic Hybrid Support: Stability and Kinetic Study
Catalysts 2017, 7(12), 374; doi:10.3390/catal7120374
Received: 7 November 2017 / Revised: 28 November 2017 / Accepted: 29 November 2017 / Published: 1 December 2017
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Abstract
Cellulase from Aspergillus niger was immobilized on a synthesized TiO2–lignin hybrid support. The enzyme was effectively deposited on the inorganic–organic hybrid matrix, mainly via physical interactions. The optimal initial immobilization parameters, selected for the highest relative activity, were pH 5.0, 6
[...] Read more.
Cellulase from Aspergillus niger was immobilized on a synthesized TiO2–lignin hybrid support. The enzyme was effectively deposited on the inorganic–organic hybrid matrix, mainly via physical interactions. The optimal initial immobilization parameters, selected for the highest relative activity, were pH 5.0, 6 h process duration, and an enzyme solution concentration of 5 mg/mL. Moreover, the effects of pH, temperature, and number of consecutive catalytic cycles and the storage stability of free and immobilized cellulase were evaluated and compared. Thermal and chemical stability were significantly improved, while after 3 h at a temperature of 50 °C and pH 6.0, the immobilized cellulase retained over 80% of its initial activity. In addition, the half-life of the immobilized cellulase (307 min) was five times that of the free enzyme (63 min). After ten repeated catalytic cycles, the immobilized biocatalyst retained over 90% of its initial catalytic properties. This study presents a protocol for the production of highly stable and reusable biocatalytic systems for practical application in the hydrolysis of cellulose. Full article
(This article belongs to the Special Issue Immobilized Biocatalysts)
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Open AccessArticle Approaching Immobilization of Enzymes onto Open Porous Basotect®
Catalysts 2017, 7(12), 359; doi:10.3390/catal7120359
Received: 17 October 2017 / Revised: 14 November 2017 / Accepted: 16 November 2017 / Published: 27 November 2017
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Abstract
For the first time, commercial macroporous melamine formaldehyde foam Basotect® (BT) was used as a basic carrier material for both adsorptive and covalent enzyme immobilization. In order to access inherent amino groups, the Basotect® surface was pretreated with hydrochloric acid. The
[...] Read more.
For the first time, commercial macroporous melamine formaldehyde foam Basotect® (BT) was used as a basic carrier material for both adsorptive and covalent enzyme immobilization. In order to access inherent amino groups, the Basotect® surface was pretreated with hydrochloric acid. The resulting material revealed 6 nmol of superficial amino groups per milligram Basotect®. Different optimized strategies for tethering the laccase from Trametes versicolor and the lipase from Thermomyces lanuginosus onto the pre-treated Basotect® surface were studied. Particularly, for covalent immobilization, two different strategies were pursued: lipase was tethered via a cross-linking method using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and laccase was bound after functionalizing Basotect® with hydrophilic copolymer poly(ethylene-alt-maleic anhydride) (PEMA). Prior to laccase immobilization, the PEMA coating of Basotect® was verified by ATR-FTIR analysis. Subsequent quantification of available high-reactive PEMA anhydride moieties revealed an amount of 1028 ± 73 nmol per mg Basotect®. The surface-bound enzyme amounts were quantified as 4.1–5.8 μg per mg Basotect®. A theoretical surface-covered enzyme mass for the ideal case that an enzyme monolayer was immobilized onto the Basotect® surface was calculated and compared to the amount of adsorptive and covalently bound enzymes before and after treatment with SDS. Furthermore, the enzyme activities were determined for the different immobilization approaches, and the stability during storage over time and against sodium dodecyl sulfate treatment was monitored. Additionally, PEMA-BT-bound laccase was tested for the elimination of anthropogenic micropollutant bisphenol A from contaminated water in a cost-effective and environmentally-friendly way and resulted in a degradation rate higher than 80%. Full article
(This article belongs to the Special Issue Immobilized Biocatalysts)
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Open AccessArticle Polyelectrolyte Complex Beads by Novel Two-Step Process for Improved Performance of Viable Whole-Cell Baeyer-Villiger Monoxygenase by Immobilization
Catalysts 2017, 7(11), 353; doi:10.3390/catal7110353
Received: 18 October 2017 / Revised: 10 November 2017 / Accepted: 13 November 2017 / Published: 21 November 2017
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Abstract
A novel immobilization matrix for the entrapment of viable whole-cell Baeyer–Villiger monooxygenase was developed. Viable recombinant Escherichia coli cells overexpressing cyclohexanone monooxygenase were entrapped in polyelectrolyte complex beads prepared by a two-step reaction of oppositely-charged polymers including highly defined cellulose sulphate. Immobilized cells
[...] Read more.
A novel immobilization matrix for the entrapment of viable whole-cell Baeyer–Villiger monooxygenase was developed. Viable recombinant Escherichia coli cells overexpressing cyclohexanone monooxygenase were entrapped in polyelectrolyte complex beads prepared by a two-step reaction of oppositely-charged polymers including highly defined cellulose sulphate. Immobilized cells exhibited higher operational stability than free cells during 10 repeated cycles of Baeyer–Villiger biooxidations of rac-bicyclo[3.2.0]hept-2-en-6-one to the corresponding lactones (1R,5S)-3-oxabicyclo-[3.3.0]oct-6-en-3-one and (1S,5R)-2-oxabicyclo-[3.3.0]oct-6-en-3-one. The morphology of polyelectrolyte complex beads was characterised by environmental scanning electron microscopy; the spatial distribution of polymers in the beads and cell viability were examined using confocal laser scanning microscopy, and the texture was characterised by the mechanical resistance measurements. Full article
(This article belongs to the Special Issue Immobilized Biocatalysts)
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Open AccessCommunication In-Situ Self-Assembly of Zinc/Adenine Hybrid Nanomaterials for Enzyme Immobilization
Catalysts 2017, 7(11), 327; doi:10.3390/catal7110327
Received: 16 October 2017 / Revised: 27 October 2017 / Accepted: 27 October 2017 / Published: 3 November 2017
PDF Full-text (3181 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
In this study, a one-step and facile immobilization of enzymes by self-assembly of zinc ions and adenine in aqueous solution with mild conditions was reported. Enzymes, such as glucose oxidase (GOx) and horseradish peroxidase (HRP), could be efficiently encapsulated in Zn/adenine coordination polymers
[...] Read more.
In this study, a one-step and facile immobilization of enzymes by self-assembly of zinc ions and adenine in aqueous solution with mild conditions was reported. Enzymes, such as glucose oxidase (GOx) and horseradish peroxidase (HRP), could be efficiently encapsulated in Zn/adenine coordination polymers (CPs) with high loading capacity over 90%. When the enzyme was immobilized by CPs, it displayed high catalytic efficiency, high selectivity and enhanced stability due to the protecting effect of the rigid framework. As a result, the relative activity of Zn/adenine nano-CP-immobilized GOx increased by 1.5-fold at pH 3 and 4-fold at 70 to 90 °C, compared to free GOx. The immobilized GOx had excellent reusability (more than 90% relative activity after being reused eight times). Furthermore, the use of this system as a glucose biosensor was also demonstrated by co-immobilization of two enzymes, detecting glucose down to 1.84 µM with excellent selectivity. The above work indicated that in-situ self-assembly of Zn/adenine CPs could be a simple and efficient method for biocatalyst immobilization. Full article
(This article belongs to the Special Issue Immobilized Biocatalysts)
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