Recent Trends in Enzyme Immobilization

A special issue of Catalysts (ISSN 2073-4344). This special issue belongs to the section "Biocatalysis".

Deadline for manuscript submissions: closed (31 July 2023) | Viewed by 14684

Special Issue Editors

College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China
Interests: biocatalysis; enzyme engineering; fermentation engineering; industrial microbiology
Special Issues, Collections and Topics in MDPI journals
The Key Laboratory of Industrial Biotechnology, Ministry of Education, National Engineering Research Center of Cereal Fermentation and Food Biomanufacturing, Jiangsu Provincial Research Center for Bioactive Product Processing Technology, Jiangnan University, Wuxi 214122, China
Interests: biomanufacturing; environmental bioremediation; enzyme immobilization; metabolic engineering; fermentation process engineering

Special Issue Information

Dear Colleagues,

At present, enzymes have been widely used as biological catalysts in several fields, including foods, feedstuffs, detergents, pharmaceuticals, cosmetics, biofuels, fine chemicals, energy generation, paper manufacturing, textile, leather, environmental protection, and so on. However, the application of their free forms in industrial production may be limited by their high loadings, fragile nature, and difficult recovery and recycling. These disadvantages can be overcome by enzyme immobilization, which can promote the effective recovery and recycling of the enzymes and improve their properties, such as activity, selectivity, and long-term operational stability in industrial applications.

For this Special Issue, contributions on all aspects of enzyme immobilization are welcome, which may be related to fundamental science or practical applications and can be outlined by the keywords given below.

Prof. Dr. Yihan Liu
Prof. Dr. Liang Zhang
Guest Editors

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Keywords

  • enzyme immobilization approaches
  • novel materials for enzyme immobilization
  • enhanced enzyme characteristics through immobilization
  • co-immobilization of multiple enzymes for catalysis
  • application of immobilized enzymes

Published Papers (10 papers)

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16 pages, 4424 KiB  
Article
Immobilization of Phospholipase D for Production of Phosphatidylserine by a Pickering Emulsion Strategy
by Hui Sun, Shujing Zhang, Dianqing Liu, Zhiqi Huang, Yuxin Ge, Jiayi Hou, Fuping Lu and Yihan Liu
Catalysts 2023, 13(10), 1318; https://doi.org/10.3390/catal13101318 - 23 Sep 2023
Cited by 1 | Viewed by 740
Abstract
As a natural phospholipid, phosphatidylserine (PS) plays a key role in the food, cosmetic, and pharmaceutical industries. Recently, substantial attention has been focused on the phospholipase D (PLD)-mediated synthesis of PS. However, the application of free PLD is usually limited by high cost, [...] Read more.
As a natural phospholipid, phosphatidylserine (PS) plays a key role in the food, cosmetic, and pharmaceutical industries. Recently, substantial attention has been focused on the phospholipase D (PLD)-mediated synthesis of PS. However, the application of free PLD is usually limited by high cost, poor reusability, and low stability. In this study, PLD from Streptomyces antibiotics (saPLD) was efficiently immobilized on SiO2 through physical adsorption to develop saPLD@SiO2. The stability of the saPLD@SiO2 was higher than that of the free saPLD over an extensive range of temperature and pH conditions. Furthermore, the PS yield of saPLD@SiO2 was approximately 41% in the first cycles, and still kept 60% of its initial PS yield after 14 cycles. After a 25-day storage period, the saPLD@SiO2 retained 62.5% of its initial activity, while the free saPLD retained only 34.3%, suggesting that saPLD@SiO2 has better stability than free saPLD. A Pickering emulsion was produced by dispersing saPLD@SiO2 in solutions (ethyl propanoate and acetate/acetic acid buffer) using ultrasound. The engineered Pickering emulsion demonstrated excellent catalytic activity, with a 62% PS yield after 6 h, while free saPLD had only 18%. The results indicated that a high-performance and sustainable biocatalysis method was established for the effective synthesis of PS. Full article
(This article belongs to the Special Issue Recent Trends in Enzyme Immobilization)
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13 pages, 1772 KiB  
Article
Biocatalysts Based on Immobilized Lipases for the Production of Ethyl Esters of Fatty Acids including Bioactive Gamma-Linolenic Acid from Borage Oil
by Gonzalo Berzal, Martín García-González, Natalia Castejón, Paz García-García, Gloria Fernández-Lorente and Francisco J. Señoráns
Catalysts 2023, 13(9), 1275; https://doi.org/10.3390/catal13091275 - 05 Sep 2023
Viewed by 840
Abstract
In the present work, borage oil (Borago officinalis) was used as the main source of gamma linolenic acid (GLA) to obtain ethyl esters by enzymatic ethanolysis using immobilized enzymes for its application in the food industry. Commercial Thermomyces lanuginosus lipase (TLL) [...] Read more.
In the present work, borage oil (Borago officinalis) was used as the main source of gamma linolenic acid (GLA) to obtain ethyl esters by enzymatic ethanolysis using immobilized enzymes for its application in the food industry. Commercial Thermomyces lanuginosus lipase (TLL) was compared to chemical ethanolysis in alkaline medium. In addition, TLL was immobilized by adsorption on hydrophobic porous support (Octadecyl-Sepabeads®) to compare the results. Fatty acid ethyl ester (FAEE) yields of both reactions were compared under the same conditions (25 °C and 200 rpm) and analyzed by GC-MS. Moreover, the conversion yield for borage oil ethanolysis catalyzed by TLL immobilized on C18-Sepabeads® supports was similar to the chemical pathway (93.4% and 99.5%, respectively). When this biocatalyst was used in a solvent-free system (at 40 °C and 200 rpm), it was possible to obtain a high FAEE yield of 84.3% in the first 24 h of reaction. Furthermore, it was possible to re-use the immobilized biocatalyst for the performance of five reaction cycles maintaining 68% of its initial activity. Thus, the use of immobilized enzymes in solvent-free systems is an eco-friendly alternative to obtain GLA ethyl esters for its possible application in cosmetics and food. Full article
(This article belongs to the Special Issue Recent Trends in Enzyme Immobilization)
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17 pages, 1122 KiB  
Article
Comparison of Four Immobilization Methods for Different Transaminases
by Tobias Heinks, Nicolai Montua, Michelle Teune, Jan Liedtke, Matthias Höhne, Uwe T. Bornscheuer and Gabriele Fischer von Mollard
Catalysts 2023, 13(2), 300; https://doi.org/10.3390/catal13020300 - 28 Jan 2023
Cited by 4 | Viewed by 2250
Abstract
Biocatalytic syntheses often require unfavorable conditions, which can adversely affect enzyme stability. Consequently, improving the stability of biocatalysts is needed, and this is often achieved by immobilization. In this study, we aimed to compare the stability of soluble and immobilized transaminases from different [...] Read more.
Biocatalytic syntheses often require unfavorable conditions, which can adversely affect enzyme stability. Consequently, improving the stability of biocatalysts is needed, and this is often achieved by immobilization. In this study, we aimed to compare the stability of soluble and immobilized transaminases from different species. A cysteine in a consensus sequence was converted to a single aldehyde by the formylglycine-generating enzyme for directed single-point attachment to amine beads. This immobilization was compared to cross-linked enzyme aggregates (CLEAs) and multipoint attachments to glutaraldehyde-functionalized amine- and epoxy-beads. Subsequently, the reactivity and stability (i.e., thermal, storage, and solvent stability) of all soluble and immobilized transaminases were analyzed and compared under different conditions. The effect of immobilization was highly dependent on the type of enzyme, the immobilization strategy, and the application itself, with no superior immobilization technique identified. Immobilization of HAGA-beads often resulted in the highest activities of up to 62 U/g beads, and amine beads were best for the hexameric transaminase from Luminiphilus syltensis. Furthermore, the immobilization of transaminases enabled its reusability for at least 10 cycles, while maintaining full or high activity. Upscaled kinetic resolutions (partially performed in a SpinChemTM reactor) resulted in a high conversion, maintained enantioselectivity, and high product yields, demonstrating their applicability. Full article
(This article belongs to the Special Issue Recent Trends in Enzyme Immobilization)
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18 pages, 2586 KiB  
Article
Design and Optimization of Laccase Immobilization in Cellulose Acetate Microfiltration Membrane for Micropollutant Remediation
by Béla Varga, Mónika Meiczinger, Miklós Jakab and Viola Somogyi
Catalysts 2023, 13(2), 222; https://doi.org/10.3390/catal13020222 - 18 Jan 2023
Cited by 4 | Viewed by 1647
Abstract
The industrial and environmental applications of laccase, especially in wastewater treatment, have gained focus in recent years. Therefore, developing the proper laccase immobilization techniques, which could improve the stability of the enzymes and simplify the required downstream processes, is needed. A novel two-step [...] Read more.
The industrial and environmental applications of laccase, especially in wastewater treatment, have gained focus in recent years. Therefore, developing the proper laccase immobilization techniques, which could improve the stability of the enzymes and simplify the required downstream processes, is needed. A novel two-step immobilization process was developed, resulting in cross-linked enzyme aggregates (CLEA) in the pores of the membrane. Laccase adsorption on a biodegradable cellulose acetate microfiltration membrane along with cross-linking was investigated to maximize the enzyme load and immobilization efficiency. The optimization was done regarding the: pH, temperature, enzyme concentration, adsorption time, cross-linker concentration, and temperature. It was concluded that the highest immobilization efficiency (76%) could be achieved in acidic buffers at 29 °C with high surface activity (1174 U·m2) at the cost of partial denaturation and membrane fouling. The membrane was successfully utilized for the enzymatic treatment of diclofenac, and 58% removal efficiency was achieved. The results indicated that cellulose acetate is a suitable carrier for adsorption-based immobilization of laccase for the potential for environmental utilisation. Full article
(This article belongs to the Special Issue Recent Trends in Enzyme Immobilization)
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22 pages, 4895 KiB  
Article
Immobilization of Glucose Oxidase on Glutathione Capped CdTe Quantum Dots for Bioenergy Generation
by Daniel Lozano-López, Marisol Galván-Valencia, Ivone Rojas-de Soto, Ricardo A. Escalona-Villalpando, Janet Ledesma-García and Sergio Durón-Torres
Catalysts 2022, 12(12), 1659; https://doi.org/10.3390/catal12121659 - 17 Dec 2022
Cited by 2 | Viewed by 1619
Abstract
An efficient immobilization of Glucose oxidase (GOx) on an appropriate substrate is one of the main challenges of developing fuel cells that allow energy to be obtained from renewable substrates such as carbohydrates in physiological environments. The research importance of biofuel cells relies [...] Read more.
An efficient immobilization of Glucose oxidase (GOx) on an appropriate substrate is one of the main challenges of developing fuel cells that allow energy to be obtained from renewable substrates such as carbohydrates in physiological environments. The research importance of biofuel cells relies on their experimental robustness and high compatibility with biological organisms such as tissues or the bloodstream with the aim of obtaining electrical energy even from living systems. In this work, we report the use of 5,10,15,20 tetrakis (1-methyl-4-pyridinium) porphyrin and glutathione capped CdTe Quantum dots (GSH-CdTeQD) as a support matrix for the immobilization of GOx on carbon surfaces. Fluorescent GSH-CdTeQD particles were synthesized and their characterization by UV-Vis spectrophotometry showed a particle size between 5–7 nm, which was confirmed by DLS and TEM measurements. Graphite and Toray paper electrodes were modified by a drop coating of porphyrin, GSH-CdTeQD and GOx, and their electrochemical activity toward glucose oxidation was evaluated by cyclic voltammetry, chronoamperometry and electrochemical impedance spectroscopy. Additionally, GOx modified electrode activity was explored by scanning electrochemical microscopy, finding that near to 70% of the surface was covered with active enzyme. The modified electrodes showed a glucose sensitivity of 0.58 ± 0.01 μA/mM and an apparent Michaelis constant of 7.8 mM. The addition of BSA blocking protein maintained the current response of common interferent molecules such as ascorbic acid (AA) with less than a 5% of interference percentage. Finally, the complex electrodes were employed as anodes in a microfluidic biofuel cell (μBFC) in order to evaluate the performance in energy production. The enzymatic anodes used in the μBFC allowed us to obtain a current density of 7.53 mAcm−2 at the maximum power density of 2.30 mWcm−2; an open circuit potential of 0.57 V was observed in the biofuel cell. The results obtained suggest that the support matrix porphyrin and GSH-CdTeQD is appropriate to immobilize GOx while preserving the enzyme’s catalytic activity. The reported electrode arrangement is a viable option for bioenergy production and/or glucose quantification. Full article
(This article belongs to the Special Issue Recent Trends in Enzyme Immobilization)
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17 pages, 2173 KiB  
Article
Crystal Contact Engineering for Enhanced Cross-Linking Efficiency of HheG Crystals
by Marcel Staar, Sophie Staar and Anett Schallmey
Catalysts 2022, 12(12), 1553; https://doi.org/10.3390/catal12121553 - 01 Dec 2022
Cited by 1 | Viewed by 1216
Abstract
The generation of cross-linked enzyme crystals is a very attractive method for immobilization of enzymes displaying high crystalizability. However, the commonly used cross-linker glutaraldehyde is not always compatible with enzyme activity. Therefore, we previously reported the engineering of halohydrin dehalogenase HheG from Ilumatobacter [...] Read more.
The generation of cross-linked enzyme crystals is a very attractive method for immobilization of enzymes displaying high crystalizability. However, the commonly used cross-linker glutaraldehyde is not always compatible with enzyme activity. Therefore, we previously reported the engineering of halohydrin dehalogenase HheG from Ilumatobacter coccineus to enable thiol-specific cross-linking during CLEC generation by insertion of cysteine residues in the crystal contact. To broaden the applicability of this approach, herein crystal contact engineering of HheG has been performed to incorporate additional lysine residues as defined cross-linking sites for CLEC generation. Using the primary amine-specific cross-linker dithiobis(succinimidyl propionate) (DSP), CLECs of HheG variant V46K were obtained that displayed a high gain in thermal stability compared to wild-type HheG, while using only a low cross-linker concentration. Moreover, respective V46K CLECs exhibited a 10 K higher reaction temperature optimum as well as significantly improved activity and stability at acidic pH and in the presence of organic co-solvents. Overall, our study demonstrates that lysine-specific cross-linkers can also be used as an alternative to glutaraldehyde for stable CLEC generation of halohydrin dehalogenases, and that cross-linking efficiency is significantly improved upon crystal contact engineering. Full article
(This article belongs to the Special Issue Recent Trends in Enzyme Immobilization)
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13 pages, 2189 KiB  
Article
A Two-Step Cross-Linked Hydrogel Immobilization Strategy for Diacetylchitobiose Deacetylase
by Guoyun Sun, Ziyang Huang, Zhiqian Zhang, Yanfeng Liu, Jianghua Li, Guocheng Du, Xueqin Lv and Long Liu
Catalysts 2022, 12(9), 932; https://doi.org/10.3390/catal12090932 - 23 Aug 2022
Cited by 2 | Viewed by 1394
Abstract
Free enzymes often face economic problems due to their non-recyclability, which limits their applications for industrial manufacturing. Organic biopolymers are frequently used to fabricate hydrogel for enzyme immobilization due to their advantages of non-toxicity, biocompatibility, biodegradability, and flexibility. However, for highly thermostable enzymes, [...] Read more.
Free enzymes often face economic problems due to their non-recyclability, which limits their applications for industrial manufacturing. Organic biopolymers are frequently used to fabricate hydrogel for enzyme immobilization due to their advantages of non-toxicity, biocompatibility, biodegradability, and flexibility. However, for highly thermostable enzymes, simple cross-linking causes either low immobilizing efficiency or low thermal stability. Herein, we developed a novel enzyme immobilization strategy with two-step cross-linked gelatin hydrogel for thermostable enzymes working at high temperature. The hydrogel was firstly “soft cross-linked” to immobilize most enzyme molecules and then “hard cross-linked” to gain strong thermal stability. We selected the enzyme diacetylchitobiose deacetylase (Dac), which was firstly derived from hyperthermophilic bacteria, to demonstrate the advantages of our method. With the optimized fabrication steps, our hydrogel showed ~87% Dac immobilization efficiency and excellent stability against heating, dehydrating, long-time storing, and massive recycling. Importantly, our hydrogel showed ~85.0% relative enzyme activity at 80 °C and retained ~65.8% activity after 10 rounds of catalysis. This strategy showed high immobilizing efficiency and strong thermal stability and we believe it could improve the industrial potential for those enzymes. Full article
(This article belongs to the Special Issue Recent Trends in Enzyme Immobilization)
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23 pages, 1451 KiB  
Article
The High ‘Lipolytic Jump’ of Immobilized Amano A Lipase from Aspergillus niger in Developed ‘ESS Catalytic Triangles’ Containing Natural Origin Substrates
by Tomasz Siódmiak, Jacek Dulęba, Natalia Kocot, Dorota Wątróbska-Świetlikowska and Michał Piotr Marszałł
Catalysts 2022, 12(8), 853; https://doi.org/10.3390/catal12080853 - 03 Aug 2022
Cited by 5 | Viewed by 1970 | Correction
Abstract
Lipase Amano A from Aspergillus niger (AA-ANL) is among the most commonly applied enzymes in biocatalysis processes, making it a significant scientific subject in the pharmaceutical and medical disciplines. In this study, we investigated the lipolytic activity of AA-ANL immobilized onto polyacrylic support [...] Read more.
Lipase Amano A from Aspergillus niger (AA-ANL) is among the most commonly applied enzymes in biocatalysis processes, making it a significant scientific subject in the pharmaceutical and medical disciplines. In this study, we investigated the lipolytic activity of AA-ANL immobilized onto polyacrylic support IB-150A in 23 oils of natural origin containing various amounts of polyunsaturated fatty acids (PUFAs) and monounsaturated fatty acids (MUFAs). The created systems were expressed as an ‘ESS catalytic triangle’. A distinct ‘jump’ (up to 2400%) of lipolytic activity of immobilized AA-ANL compared to free lipase and hyperactivation in mostly tested substrates was observed. There was a ‘cutoff limit’ in a quantitative mutual ratio of ω-PUFAs/MUFAs, for which there was an increase or decrease in the activity of the immobilized AA-ANL. In addition, we observed the beneficial effect of immobilization using three polyacrylic supports (IB-150A, IB-D152, and IB-EC1) characterized by different intramolecular interactions. The developed substrate systems demonstrated considerable hyperactivation of immobilized AA-ANL. Moreover, a ‘lipolytic jump’ in the full range of tested temperature and pH was also observed. The considerable activity of AA-ANL-IB-150A after four reuse cycles was demonstrated. On the other hand, we observed an essential decrease in stability of immobilized lipase after 168 h of storage in a climate chamber. The tested kinetic profile of immobilized AA-ANL confirmed the decreased affinity to the substrate relative to lipase in the free form. Full article
(This article belongs to the Special Issue Recent Trends in Enzyme Immobilization)
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13 pages, 4901 KiB  
Article
Improvement in the Environmental Stability of Haloalkane Dehalogenase with Self-Assembly Directed Nano-Hybrid with Iron Phosphate
by Jianxiong Chen, Xiaodong Ming, Zitao Guo, Yi Shi, Moying Li, Zhongpeng Guo, Yu Xin, Zhenghua Gu, Liang Zhang and Xuan Guo
Catalysts 2022, 12(8), 825; https://doi.org/10.3390/catal12080825 - 27 Jul 2022
Cited by 1 | Viewed by 1297
Abstract
Haloalkane dehalogenase (DhaA) catalyzes the hydrolysis of halogenated compounds through the cleavage of carbon halogen bonds. However, the low activity, poor environmental stability, and difficult recycling of free DhaA greatly increases the economic cost of practical application. Inspired by the organic–inorganic hybrid system, [...] Read more.
Haloalkane dehalogenase (DhaA) catalyzes the hydrolysis of halogenated compounds through the cleavage of carbon halogen bonds. However, the low activity, poor environmental stability, and difficult recycling of free DhaA greatly increases the economic cost of practical application. Inspired by the organic–inorganic hybrid system, an iron-based hybrid nanocomposite biocatalyst FeHN@DhaA is successfully constructed to enhance its environmental tolerability. A series of characterization methods demonstrate that the synthesized enzyme–metal iron complexes exhibit granular nanostructures with good crystallinity. Under optimized conditions, the activity recovery and the effective encapsulation yield of FeHN@DhaA are 138.54% and 87.21%, respectively. Moreover, it not only exhibits excellent immobilized enzymatic properties but also reveals better tolerance to extreme acid, and is alkali compared with the free DhaA. In addition, the immobilized enzyme FeHN@DhaA can be easily recovered and has a satisfactory reusability, retaining 57.8% of relative activity after five reaction cycles. The results of this study might present an alternative immobilized DhaA-based clean biotechnology for the decontamination of organochlorine pollutants. Full article
(This article belongs to the Special Issue Recent Trends in Enzyme Immobilization)
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4 pages, 222 KiB  
Correction
Correction: Siódmiak et al. The High ‘Lipolytic Jump’ of Immobilized Amano A Lipase from Aspergillus niger in Developed ‘ESS Catalytic Triangles’ Containing Natural Origin Substrates. Catalysts 2022, 12, 853
by Tomasz Siódmiak, Jacek Dulęba, Natalia Kocot, Dorota Wątróbska-Świetlikowska and Michał Piotr Marszałł
Catalysts 2023, 13(3), 480; https://doi.org/10.3390/catal13030480 - 27 Feb 2023
Viewed by 651
Abstract
The authors wish to make the following corrections to this paper [...] Full article
(This article belongs to the Special Issue Recent Trends in Enzyme Immobilization)
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