Immobilization of Enzyme in Polymers

A special issue of Polymers (ISSN 2073-4360). This special issue belongs to the section "Biomacromolecules, Biobased and Biodegradable Polymers".

Deadline for manuscript submissions: 1 October 2024 | Viewed by 15440

Special Issue Editor


E-Mail Website
Guest Editor
Department of Biophysics and Biotechnology, Voronezh State University, 394018 Voronezh, Russia
Interests: biophysics; biotechnology; chemical enzymology; enzyme immobilization; enzyme stabilization; protein-polysaccharide complexes; hydrolases; proteases; chitosan
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Enzyme immobilization is one of the widespread modern industrial biotechnologies. Since the commercial use of the first immobilized enzymes in the 1960s, enzyme immobilization technologies and theories as well as immobilization materials and chemistry have rapid development. The enzyme immobilization allows to achieve more stable, active, and reusable biocatalysts. However, the choice of immobilization matrices is often empirical, and it is needed to be improved and standardized by the application of modern methods such as in silico design, protein and polymer engineering. Also, the rationalization of the immobilization procedure, i.g. pH and temperature, has a significant impact on the activity of the creating catalysts. For many enzymes and polymers, these problems remain unresolved, and this Special Issue is intended to enhance theoretical and practical knowledge in the field of enzyme immobilization on various polymers.

This Special Issue welcomes full papers and short communications highlighting the aspects of the current trends in the area of the enzymes immobilized on polymers.

Dr. Marina G. Holyavka
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

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

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • enzyme immobilization
  • enzyme conjugation
  • enzyme adsorption immobilization
  • enzyme covalent immobilization
  • enzyme immobilization in gels
  • matrix

Published Papers (10 papers)

Order results
Result details
Select all
Export citation of selected articles as:

Research

Jump to: Review

12 pages, 2282 KiB  
Article
Immobilization of BoPAL3 Phenylalanine Ammonia-Lyase on Electrospun Nanofibrous Membranes of Polyvinyl Alcohol/Nylon 6/Chitosan Crosslinked with Dextran Polyaldehyde
by Chun-Yen Hsieh, Pei-Yu Hong and Lu-Sheng Hsieh
Polymers 2023, 15(18), 3699; https://doi.org/10.3390/polym15183699 - 08 Sep 2023
Viewed by 761
Abstract
Phenylalanine ammonia-lyase (PAL, EC 4.3.1.24) is common in plants and catalyzes the formation of trans-cinnamic acid and ammonia via phenylalanine deamination. Recombinant Bambusa oldhamii BoPAL3 protein expressed in Escherichia coli was immobilized on an electrospun nanofibrous membrane using dextran polyaldehyde as a [...] Read more.
Phenylalanine ammonia-lyase (PAL, EC 4.3.1.24) is common in plants and catalyzes the formation of trans-cinnamic acid and ammonia via phenylalanine deamination. Recombinant Bambusa oldhamii BoPAL3 protein expressed in Escherichia coli was immobilized on an electrospun nanofibrous membrane using dextran polyaldehyde as a crosslinker. The immobilized BoPAL3 protein exhibited comparable kinetic properties with the free BoPAL3 protein and could be recycled for six consecutive cycles compared with the free BoPAL3 protein. The residual activity of the immobilized BoPAL3 protein was 84% after 30 days of storage at 4 °C, whereas the free BoPAL3 protein retained 56% residual activity in the same storage conditions. Furthermore, the resistance of the immobilized BoPAL3 protein to chemical denaturants was greatly increased. Therefore, the BoPAL3 protein can be immobilized using the natural dextran polyaldehyde crosslinker in place of the conventional chemical crosslinker. Nanofibrous membranes made from polyvinyl alcohol (PVA), nylon 6, and chitosan (CS) are incredibly stable and useful for future industrial applications. Full article
(This article belongs to the Special Issue Immobilization of Enzyme in Polymers)
Show Figures

Figure 1

18 pages, 2312 KiB  
Article
Efficiency Assessment between Entrapment and Covalent Bond Immobilization of Mutant β-Xylosidase onto Chitosan Support
by Gabriela Romero, Lellys M. Contreras, Carolina Aguirre Céspedes, Jeff Wilkesman, Josefa María Clemente-Jiménez, Felipe Rodríguez-Vico and Francisco Javier Las Heras-Vázquez
Polymers 2023, 15(15), 3170; https://doi.org/10.3390/polym15153170 - 26 Jul 2023
Cited by 2 | Viewed by 822
Abstract
The Y509E mutant of β-xylosidase from Geobacillus stearothermophilus (XynB2Y509E) (which also bears xylanase activity) has been immobilized in chitosan spheres through either entrapment or covalent bond formation methods. The maximum immobilization yield by entrapment was achieved by chitosan beads developed using [...] Read more.
The Y509E mutant of β-xylosidase from Geobacillus stearothermophilus (XynB2Y509E) (which also bears xylanase activity) has been immobilized in chitosan spheres through either entrapment or covalent bond formation methods. The maximum immobilization yield by entrapment was achieved by chitosan beads developed using a 2% chitosan solution after 1 h of maturation time in CFG buffer with ethanol. On the other hand, the highest value in covalent bond immobilization was observed when employing chitosan beads that were prepared from a 2% chitosan solution after 4 h of activation in 1% glutaraldehyde solution at pH 8. The activity expressed after immobilization by covalent bonding was 23% higher compared to the activity expressed following entrapment immobilization, with values of 122.3 and 99.4 IU.g−1, respectively. Kinetic data revealed that catalytic turnover values were decreased as compared to a free counterpart. Both biocatalysts showed increased thermal and pH stability, along with an improved storage capacity, as they retained 88% and 40% of their activity after being stored at 4 °C for two months. Moreover, XynB2Y509E immobilized by covalent binding also exhibited outstanding reusability, retaining 92% of activity after 10 cycles of reuse. In conclusion, our results suggest that the covalent bond method appears to be the best choice for XynB2Y509E immobilization. Full article
(This article belongs to the Special Issue Immobilization of Enzyme in Polymers)
Show Figures

Figure 1

18 pages, 3459 KiB  
Article
Immobilization of Hyperthermostable Carboxylesterase EstD9 from Anoxybacillus geothermalis D9 onto Polymer Material and Its Physicochemical Properties
by Ummie Umaiera Mohd. Johan, Raja Noor Zaliha Raja Abd. Rahman, Nor Hafizah Ahmad Kamarudin, Wahhida Latip and Mohd Shukuri Mohamad Ali
Polymers 2023, 15(6), 1361; https://doi.org/10.3390/polym15061361 - 09 Mar 2023
Cited by 1 | Viewed by 1354
Abstract
Carboxylesterase has much to offer in the context of environmentally friendly and sustainable alternatives. However, due to the unstable properties of the enzyme in its free state, its application is severely limited. The present study aimed to immobilize hyperthermostable carboxylesterase from Anoxybacillus geothermalis [...] Read more.
Carboxylesterase has much to offer in the context of environmentally friendly and sustainable alternatives. However, due to the unstable properties of the enzyme in its free state, its application is severely limited. The present study aimed to immobilize hyperthermostable carboxylesterase from Anoxybacillus geothermalis D9 with improved stability and reusability. In this study, Seplite LX120 was chosen as the matrix for immobilizing EstD9 by adsorption. Fourier-transform infrared (FT-IR) spectroscopy verified the binding of EstD9 to the support. According to SEM imaging, the support surface was densely covered with the enzyme, indicating successful enzyme immobilization. BET analysis of the adsorption isotherm revealed reduction of the total surface area and pore volume of the Seplite LX120 after immobilization. The immobilized EstD9 showed broad thermal stability (10–100 °C) and pH tolerance (pH 6–9), with optimal temperature and pH of 80 °C and pH 7, respectively. Additionally, the immobilized EstD9 demonstrated improved stability towards a variety of 25% (v/v) organic solvents, with acetonitrile exhibiting the highest relative activity (281.04%). The bound enzyme exhibited better storage stability than the free enzyme, with more than 70% of residual activity being maintained over 11 weeks. Through immobilization, EstD9 can be reused for up to seven cycles. This study demonstrates the improvement of the operational stability and properties of the immobilized enzyme for better practical applications. Full article
(This article belongs to the Special Issue Immobilization of Enzyme in Polymers)
Show Figures

Figure 1

13 pages, 3610 KiB  
Article
Regulation of Intersubunit Interactions in Homotetramer of Glyceraldehyde-3-Phosphate Dehydrogenases upon Its Immobilization in Protein—Kappa-Carrageenan Gels
by Olga Makshakova, Maria Antonova, Liliya Bogdanova, Dzhigangir Faizullin and Yuriy Zuev
Polymers 2023, 15(3), 676; https://doi.org/10.3390/polym15030676 - 29 Jan 2023
Cited by 2 | Viewed by 1414
Abstract
Polysaccharides, being biocompatible and biodegradable polymers, are highly attractive as materials for protein delivery systems. However, protein–polysaccharide interactions may lead to protein structural transformation. In the current study, we analyze the structural adjustment of a homotetrameric protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), upon its interactions [...] Read more.
Polysaccharides, being biocompatible and biodegradable polymers, are highly attractive as materials for protein delivery systems. However, protein–polysaccharide interactions may lead to protein structural transformation. In the current study, we analyze the structural adjustment of a homotetrameric protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), upon its interactions with both flexible coil chain and the rigid helix of κ-carrageenan. FTIR spectroscopy was used to probe the secondary structures of both protein and polysaccharide. Electrostatically driven protein–polysaccharide interactions in dilute solutions resulted in an insoluble complex formation with a constant κ-carrageenan/GAPDH ratio of 0.2, which amounts to 75 disaccharide units per mole of protein tetramer. Upon interactions with both coiled and helical polysaccharides, a weakening of the intersubunit interactions was revealed and attributed to a partial GAPDH tetramer dissociation. In turn, protein distorted the helical conformation of κ-carrageenan when co-gelled. Molecular modeling showed the energy favorable interactions between κ-carrageenan and GAPDH at different levels of oligomerization. κ-Carrageenan binds in the region of the NAD-binding groove and the S-loop in OR contact, which may stabilize the OP dimers. The obtained results highlight the mutual conformational adjustment of oligomeric GAPDH and κ-carrageenan upon interaction and the stabilization of GAPDH’s dissociated forms upon immobilization in polysaccharide gels. Full article
(This article belongs to the Special Issue Immobilization of Enzyme in Polymers)
Show Figures

Figure 1

13 pages, 4608 KiB  
Article
The Influence of Isoenzyme Composition and Chemical Modification on Horseradish Peroxidase@ZIF-8 Biocomposite Performance
by Marija D. Stanišić, Nikolina Popović Kokar, Predrag Ristić, Ana Marija Balaž, Miloš Ognjanović, Veljko R. Đokić, Radivoje Prodanović and Tamara R. Todorović
Polymers 2022, 14(22), 4834; https://doi.org/10.3390/polym14224834 - 10 Nov 2022
Cited by 3 | Viewed by 1418
Abstract
Many articles in the literature deal with horseradish peroxidase (HRP) biomineralization, but none pay attention to the isoenzyme composition of commercial HRP or the influence of the carbohydrate component of the protein molecule on the biomineralization process. To study the impact of these [...] Read more.
Many articles in the literature deal with horseradish peroxidase (HRP) biomineralization, but none pay attention to the isoenzyme composition of commercial HRP or the influence of the carbohydrate component of the protein molecule on the biomineralization process. To study the impact of these factors, we performed periodate oxidation of commercial HRP and a purified HRP-C isoform for biomineralization within ZIF-8. With purified HRP, enzyme@ZIF-8 biocomposites with higher activity were obtained, while periodate oxidation of the carbohydrate component of both commercial HRP and purified HRP-C yields biocomposites with very high activity in acetate buffer that does not degrade the ZIF-8 structure. Using acetate instead of phosphate buffer can prevent the false high activity of HRP@ZIF-8 biocomposites caused by the degradation of ZIF-8 coating. At the same time, purification and especially oxidation of the carbohydrate component of enzymes prior to biomineralization lead to significantly improved activity of the biocomposites. Full article
(This article belongs to the Special Issue Immobilization of Enzyme in Polymers)
Show Figures

Figure 1

19 pages, 3442 KiB  
Article
Characterization of Carboxylic Acid Reductase from Mycobacterium phlei Immobilized onto Seplite LX120
by Rose Syuhada Basri, Raja Noor Zaliha Raja Abd. Rahman, Nor Hafizah Ahmad Kamarudin, Wahhida Latip and Mohd Shukuri Mohamad Ali
Polymers 2022, 14(20), 4375; https://doi.org/10.3390/polym14204375 - 17 Oct 2022
Cited by 4 | Viewed by 1864
Abstract
A multi-domain oxidoreductase, carboxylic acid reductase (CAR), can catalyze the one-step reduction of carboxylic acid to aldehyde. This study aimed to immobilize bacterial CAR from a moderate thermophile Mycobacterium phlei (MpCAR). It was the first work reported on immobilizing bacterial CAR [...] Read more.
A multi-domain oxidoreductase, carboxylic acid reductase (CAR), can catalyze the one-step reduction of carboxylic acid to aldehyde. This study aimed to immobilize bacterial CAR from a moderate thermophile Mycobacterium phlei (MpCAR). It was the first work reported on immobilizing bacterial CAR onto a polymeric support, Seplite LX120, via simple adsorption. Immobilization time and protein load were optimized for MpCAR immobilization. The immobilized MpCAR showed optimal activity at 60 °C and pH 9. It was stable over a wide range of temperatures (10 to 100 °C) and pHs (4–11), retaining more than 50% of its activity. The immobilized MpCAR also showed stability in polar solvents. The adsorption of MpCAR onto the support was confirmed by Scanning Electron Microscopy (SEM), Fourier-Transform Infrared (FTIR) spectroscopy, and Brunauer–Emmett–Teller (BET) analysis. The immobilized MpCAR could be stored for up to 6 weeks at 4 °C and 3 weeks at 25 °C. Immobilized MpCAR showed great operational stability, as 59.68% of its activity was preserved after 10 assay cycles. The immobilized MpCAR could also convert approximately 2.6 mM of benzoic acid to benzaldehyde at 60 °C. The successfully immobilized MpCAR on Seplite LX120 exhibited improved properties that benefit green industrial processes. Full article
(This article belongs to the Special Issue Immobilization of Enzyme in Polymers)
Show Figures

Figure 1

9 pages, 1487 KiB  
Article
κ-Carrageenan Hydrogel as a Matrix for Therapeutic Enzyme Immobilization
by Olga N. Makshakova, Liliya R. Bogdanova, Anastasiya O. Makarova, Aleksandra M. Kusova, Elena A. Ermakova, Mariia A. Kazantseva and Yuriy F. Zuev
Polymers 2022, 14(19), 4071; https://doi.org/10.3390/polym14194071 - 28 Sep 2022
Cited by 5 | Viewed by 1401
Abstract
During the last few decades, polysaccharide hydrogels attract more and more attention as therapeutic protein delivery systems due to their biocompatibility and the simplicity of the biodegradation of natural polymers. The protein retention by and release from the polysaccharide gel network is regulated [...] Read more.
During the last few decades, polysaccharide hydrogels attract more and more attention as therapeutic protein delivery systems due to their biocompatibility and the simplicity of the biodegradation of natural polymers. The protein retention by and release from the polysaccharide gel network is regulated by geometry and physical interactions of protein with the matrix. In the present work, we studied the molecular details of interactions between κ-carrageenan and three lipases, namely the lipases from Candida rugosa, Mucor javanicus, and Rhizomucor miehei—which differ in their size and net charge—upon protein immobilization in microparticles of polysaccharide gel. The kinetics of protein release revealed the different capability of κ-carrageenan to retain lipases, which are generally negatively charged; that was shown to be in line with the energy of interactions between polysaccharides and positively charged epitopes on the protein surface. These data create a platform for the novel design of nanocarriers for biomedical probes of enzymatic origin. Full article
(This article belongs to the Special Issue Immobilization of Enzyme in Polymers)
Show Figures

Figure 1

12 pages, 2370 KiB  
Article
Novel Immobilized Biocatalysts Based on Cysteine Proteases Bound to 2-(4-Acetamido-2-sulfanilamide) Chitosan and Research on Their Structural Features
by Svetlana S. Olshannikova, Nataliya V. Malykhina, Maria S. Lavlinskaya, Andrey V. Sorokin, Nikolay E. Yudin, Yulia M. Vyshkvorkina, Anatoliy N. Lukin, Marina G. Holyavka and Valeriy G. Artyukhov
Polymers 2022, 14(15), 3223; https://doi.org/10.3390/polym14153223 - 08 Aug 2022
Cited by 9 | Viewed by 1614
Abstract
Briefly, 2-(4-Acetamido-2-sulfanilamide) chitosan, which is a chitosan water-soluble derivative, with molecular weights of 200, 350, and 600 kDa, was successfully synthesized. The immobilization of ficin, papain, and bromelain was carried out by complexation with these polymers. The interaction mechanism of 2-(4-acetamido-2-sulfanilamide) chitosan with [...] Read more.
Briefly, 2-(4-Acetamido-2-sulfanilamide) chitosan, which is a chitosan water-soluble derivative, with molecular weights of 200, 350, and 600 kDa, was successfully synthesized. The immobilization of ficin, papain, and bromelain was carried out by complexation with these polymers. The interaction mechanism of 2-(4-acetamido-2-sulfanilamide) chitosan with bromelain, ficin, and papain was studied using FTIR spectroscopy. It was found that the hydroxy, thionyl, and amino groups of 2-(4-acetamido-2-sulfanilamide) chitosan were involved in the complexation process. Molecular docking research showed that all amino acid residues of the active site of papain formed hydrogen bonds with the immobilization matrix, while only two catalytically valuable amino acid residues took part in the H-bond formation for bromelain and ficin. The spectral and in silico data were in good agreement with the catalytic activity evaluation data. Immobilized papain was more active compared to the other immobilized proteases. Moreover, the total and specific proteolytic activity of papain immobilized on the carrier with a molecular weight of 350 kDa were higher compared to the native one due to the hyperactivation. The optimal ratio of protein content (mg × g −1 of carrier), total activity (U × mL−1 of solution), and specific activity (U × mg−1 of protein) was determined for the enzymes immobilized on 2-(4-acetamido-2-sulfanilamide) chitosan with a molecular weight of 350 kDa. Full article
(This article belongs to the Special Issue Immobilization of Enzyme in Polymers)
Show Figures

Figure 1

Review

Jump to: Research

19 pages, 1761 KiB  
Review
Carrier Variety Used in Immobilization of His6-OPH Extends Its Application Areas
by Elena Efremenko, Ilya Lyagin, Aysel Aslanli, Nikolay Stepanov, Olga Maslova and Olga Senko
Polymers 2023, 15(3), 591; https://doi.org/10.3390/polym15030591 - 24 Jan 2023
Cited by 2 | Viewed by 1627
Abstract
Organophosphorus hydrolase, containing a genetically introduced hexahistidine sequence (His6-OPH), attracts the attention of researchers by its promiscuous activity in hydrolytic reactions with various substrates, such as organophosphorus pesticides and chemical warfare agents, mycotoxins, and N-acyl homoserine lactones. The application of [...] Read more.
Organophosphorus hydrolase, containing a genetically introduced hexahistidine sequence (His6-OPH), attracts the attention of researchers by its promiscuous activity in hydrolytic reactions with various substrates, such as organophosphorus pesticides and chemical warfare agents, mycotoxins, and N-acyl homoserine lactones. The application of various carrier materials (metal-organic frameworks, polypeptides, bacterial cellulose, polyhydroxybutyrate, succinylated gelatin, etc.) for the immobilization and stabilization of His6-OPH by various methods, enables creation of biocatalysts with various properties and potential uses, in particular, as antidotes, recognition elements of biosensors, in fibers with chemical and biological protection, dressings with antimicrobial properties, highly porous sorbents for the degradation of toxicants, including in flow systems, etc. The use of computer modeling methods in the development of immobilized His6-OPH samples provides in silico prediction of emerging interactions between the enzyme and immobilizing polymer, which may have negative effects on the catalytic properties of the enzyme, and selection of the best options for experiments in vitro and in vivo. This review is aimed at analysis of known developments with immobilized His6-OPH, which allows to recognize existing recent trends in this field of research, as well as to identify the reasons limiting the use of a number of polymer molecules for the immobilization of this enzyme. Full article
(This article belongs to the Special Issue Immobilization of Enzyme in Polymers)
Show Figures

Graphical abstract

14 pages, 964 KiB  
Review
Polyelectrolytes for Enzyme Immobilization and the Regulation of Their Properties
by Vladimir I. Muronetz, Denis V. Pozdyshev and Pavel I. Semenyuk
Polymers 2022, 14(19), 4204; https://doi.org/10.3390/polym14194204 - 07 Oct 2022
Cited by 6 | Viewed by 1689
Abstract
In this review, we considered aspects related to the application of polyelectrolytes, primarily synthetic polyanions and polycations, to immobilize enzymes and regulate their properties. We mainly focused on the description of works in which polyelectrolytes were used to create complex and unusual systems [...] Read more.
In this review, we considered aspects related to the application of polyelectrolytes, primarily synthetic polyanions and polycations, to immobilize enzymes and regulate their properties. We mainly focused on the description of works in which polyelectrolytes were used to create complex and unusual systems (self-regulated enzyme–polyelectrolyte complexes, artificial chaperones, polyelectrolyte brushes, layer-by-layer immobilization and others). These works represent the field of “smart polymers”, whilst the trivial use of charged polymers as carriers for adsorption or covalent immobilization of proteins is beyond the scope of this short review. In addition, we have included a section on the molecular modeling of interactions between proteins and polyelectrolytes, as modeling the binding of proteins with a strictly defined, and already known, spatial structure, to disordered polymeric molecules has its own unique characteristics. Full article
(This article belongs to the Special Issue Immobilization of Enzyme in Polymers)
Show Figures

Figure 1

Back to TopTop