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Enzyme Immobilization Ⅳ

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

Deadline for manuscript submissions: closed (1 May 2022) | Viewed by 24892

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Prof. Dr. Roberto Fernandez-Lafuente

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Institute of Catalysis and Petrochemsitry-CSIC, Campus UAM-CSIC, C/ Marie Curie 2, Cantoblanco, 28049 Madrid, Spain
Interests: biocatalysis; enzyme immobilization; enzyme stabilization; enzyme chemical modification; bioprocess optimization
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Dear Colleagues,

After the success of the previous Special Issues edited by Molecules (2014, 2016, and 2019), I am very happy to announce the launch of a fourth Special Issue on “Enzyme Immobilization”. At present, enzyme immobilization has shown to be a solution to many enzyme limitations—for example, improving enzyme stability, activity, selectivity, or specificity; reducing inhibition problems; and even coupling immobilization and purification.

Although the coupling of genetic and chemical tools produces improved immobilized enzymes, the control of the orientation of the enzyme on the support surface and of the intensity of the support–enzyme interactions is still an open question.

Thus, the main topics of the submitted papers should be the same as in the previous Issues. For example, 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. The use of the immobilized enzymes to take advantage of the catalytically improved properties will be also suitable for this Special Issue. Papers related to the modification of immobilized enzymes, or to the modification of the support surface after enzyme immobilization, are also very welcome.

Prof. Dr. Roberto Fernandez-Lafuente
Guest Editor

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Keywords

enzyme stabilization by immobilization
modulation of enzyme properties by immobilization
multipoint covalent attachment
multisubunit immobilization
immobilized enzyme characterization
cascade reactions
nanoparticles
enzymes co-immobilization

Published Papers (11 papers)

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Editorial

Jump to: Research

3 pages, 200 KiB

Open AccessEditorial

Enzyme Immobilization

by Roberto Fernandez-Lafuente

Molecules 2023, 28(3), 1373; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules28031373 - 01 Feb 2023

Cited by 2 | Viewed by 1850

Abstract

The development of enzyme immobilization started in the middle of the previous century as a potential answer to the problem of the enzyme recovery and reuse [...] Full article

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

Research

Jump to: Editorial

12 pages, 1178 KiB

Open AccessArticle

Enzymatic Synthesis of Ascorbyl Palmitate in a Rotating Bed Reactor

by Jessica Holtheuer, Luigi Tavernini, Claudia Bernal, Oscar Romero, Carminna Ottone and Lorena Wilson

Molecules 2023, 28(2), 644; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules28020644 - 08 Jan 2023

Cited by 6 | Viewed by 1609

Abstract

Ascorbyl palmitate, an ascorbic acid ester, is an important amphipathic antioxidant that has several applications in foods, pharmaceuticals, and cosmetics. The enzymatic synthesis of ascorbyl palmitate is very attractive, but few efforts have been made to address its process scale-up and implementation. This study aimed at evaluating the enzymatic synthesis of ascorbyl palmitate in a rotating basket reactor operated in sequential batches. Different commercial immobilized lipases were tested, and the most suitable reaction conditions were established. Among those lipases studied were Amano Lipase PS, Lipozyme^® TL IM, Lipozyme^® Novo 40086, Lipozyme^® RM IM and Lipozyme^® 435. Initially, the enzymes were screened based on previously defined synthesis conditions, showing clear differences in behavior. Lipozyme^® 435 proved to be the best catalyst, reaching the highest values of initial reaction rate and yield. Therefore, it was selected for the following studies. Among the solvents assayed, 2-methyl-2-butanol and acetone showed the highest yields, but the operational stability of the catalyst was better in 2-methyl-2-butanol. The tests in a basket reactor showed great potential for large-scale application. Yields remained over 80% after four sequential batches, and the basket allowed for easy catalyst recycling. The results obtained in basket reactor are certainly a contribution to the enzymatic synthesis of ascorbyl palmitate as a competitive alternative to chemical synthesis. This may inspire future cost-effectiveness studies of the process to assess its potential as a viable alternative to be implemented. Full article

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

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19 pages, 2510 KiB

Open AccessArticle

Immobilization of Penicillin G Acylase on Vinyl Sulfone-Agarose: An Unexpected Effect of the Ionic Strength on the Performance of the Immobilization Process

by Thays N. da Rocha, Roberto Morellon-Sterlling, Javier Rocha-Martin, Juan M. Bolivar, Luciana R. B. Gonçalves and Roberto Fernandez-Lafuente

Molecules 2022, 27(21), 7587; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules27217587 - 05 Nov 2022

Cited by 5 | Viewed by 1490

Abstract

Penicillin G acylase (PGA) from Escherichia coli was immobilized on vinyl sulfone (VS) agarose. The immobilization of the enzyme failed at all pH values using 50 mM of buffer, while the progressive increase of ionic strength permitted its rapid immobilization under all studied pH values. This suggests that the moderate hydrophobicity of VS groups is enough to transform the VS-agarose in a heterofunctional support, that is, a support bearing hydrophobic features (able to adsorb the proteins) and chemical reactivity (able to give covalent bonds). Once PGA was immobilized on this support, the PGA immobilization on VS-agarose was optimized with the purpose of obtaining a stable and active biocatalyst, optimizing the immobilization, incubation and blocking steps characteristics of this immobilization protocol. Optimal conditions were immobilization in 1 M of sodium sulfate at pH 7.0, incubation at pH 10.0 for 3 h in the presence of glycerol and phenyl acetic acid, and final blocking with glycine or ethanolamine. This produced biocatalysts with stabilities similar to that of the glyoxyl-PGA (the most stable biocatalyst of this enzyme described in literature), although presenting just over 55% of the initially offered enzyme activity versus the 80% that is recovered using the glyoxyl-PGA. This heterofuncionality of agarose VS beads opens new possibilities for enzyme immobilization on this support. Full article

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

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13 pages, 3939 KiB

Open AccessArticle

Tuning Immobilized Commercial Lipase Preparations Features by Simple Treatment with Metallic Phosphate Salts

by José R. Guimarães, Diego Carballares, Paulo W. Tardioli, Javier Rocha-Martin and Roberto Fernandez-Lafuente

Molecules 2022, 27(14), 4486; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules27144486 - 13 Jul 2022

Cited by 8 | Viewed by 1524

Abstract

Four commercial immobilized lipases biocatalysts have been submitted to modifications with different metal (zinc, cobalt or copper) phosphates to check the effects of this modification on enzyme features. The lipase preparations were Lipozyme^®TL (TLL-IM) (lipase from Thermomyces lanuginose), Lipozyme^®435 (L435) (lipase B from Candida antarctica), Lipozyme^®RM (RML-IM), and LipuraSelect (LS-IM) (both from lipase from Rhizomucor miehei). The modifications greatly altered enzyme specificity, increasing the activity versus some substrates (e.g., TLL-IM modified with zinc phosphate in hydrolysis of triacetin) while decreasing the activity versus other substrates (the same preparation in activity versus R- or S- methyl mandelate). Enantiospecificity was also drastically altered after these modifications, e.g., LS-IM increased the activity versus the R isomer while decreasing the activity versus the S isomer when treated with copper phosphate. Regarding the enzyme stability, it was significantly improved using octyl-agarose-lipases. Using all these commercial biocatalysts, no significant positive effects were found; in fact, a decrease in enzyme stability was usually detected. The results point towards the possibility of a battery of biocatalysts, including many different metal phosphates and immobilization protocols, being a good opportunity to tune enzyme features, increasing the possibilities of having biocatalysts that may be suitable for a specific process. Full article

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

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14 pages, 2506 KiB

Open AccessArticle

Development of a Hybrid Bioinorganic Nanobiocatalyst: Remarkable Impact of the Immobilization Conditions on Activity and Stability of β-Galactosidase

by Luigi Tavernini, Oscar Romero, Carla Aburto, Fernando López-Gallego, Andrés Illanes and Lorena Wilson

Molecules 2021, 26(14), 4152; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26144152 - 08 Jul 2021

Cited by 4 | Viewed by 1715

Abstract

Hybrid bioinorganic biocatalysts have received much attention due to their simple synthesis, high efficiency, and structural features that favor enzyme activity and stability. The present work introduces a biomineralization strategy for the formation of hybrid nanocrystals from β-galactosidase. The effects of the immobilization conditions were studied, identifying the important effect of metal ions and pH on the immobilization yield and the recovered activity. For a deeper understanding of the biomineralization process, an in silico study was carried out to identify the ion binding sites at the different conditions. The selected β-galactosidase nanocrystals showed high specific activity (35,000 IU/g biocatalyst) and remarkable thermal stability with a half-life 11 times higher than the soluble enzyme. The nanobiocatalyst was successfully tested for the synthesis of galacto-oligosaccharides, achieving an outstanding performance, showing no signs of diffusional limitations. Thus, a new, simple, biocompatible and inexpensive nanobiocatalyst was produced with high enzyme recovery (82%), exhibiting high specific activity and high stability, with promising industrial applications. Full article

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

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18 pages, 2601 KiB

Open AccessArticle

Lipozyme 435-Mediated Synthesis of Xylose Oleate in Methyl Ethyl Ketone

by Maria Carolina Pereira Gonçalves, Jéssica Cristina Amaral, Roberto Fernandez-Lafuente, Ruy de Sousa Junior and Paulo Waldir Tardioli

Molecules 2021, 26(11), 3317; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26113317 - 01 Jun 2021

Cited by 11 | Viewed by 2637

Abstract

In this paper, we have performed the Lipozyme 435-catalyzed synthesis of xylose oleate in methyl ethyl ketone (MEK) from xylose and oleic acid. The effects of substrates’ molar ratios, reaction temperature, reaction time on esterification rates, and Lipozyme 435 reuse were studied. Results showed that an excess of oleic acid (xylose: oleic acid molar ratio of 1:5) significantly favored the reaction, yielding 98% of xylose conversion and 31% oleic acid conversion after 24 h-reaction (mainly to xylose mono- and dioleate, as confirmed by mass spectrometry). The highest Lipozyme 435 activities occurred between 55 and 70 °C. The predicted Ping Pong Bi Bi kinetic model fitted very well to the experimental data and there was no evidence of inhibitions in the range assessed. The reaction product was purified and presented an emulsion capacity close to that of a commercial sugar ester detergent. Finally, the repeated use of Lipozyme 435 showed a reduction in the reaction yields (by 48 and 19% in the xylose and oleic acid conversions, respectively), after ten 12 h-cycles. Full article

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

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16 pages, 19903 KiB

Open AccessArticle

Modulation of the Biocatalytic Properties of a Novel Lipase from Psychrophilic Serratia sp. (USBA-GBX-513) by Different Immobilization Strategies

by Mónica Ruiz, Esteban Plata, John J. Castillo, Claudia C. Ortiz, Gina López, Sandra Baena, Rodrigo Torres and Roberto Fernandez-Lafuente

Molecules 2021, 26(6), 1574; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26061574 - 12 Mar 2021

Cited by 6 | Viewed by 2336

Abstract

In this work, the effect of different immobilization procedures on the properties of a lipase obtained from the extremophilic microorganism Serratia sp. USBA-GBX-513, which was isolated from Paramo soils of Los Nevados National Natural Park (Colombia), is reported. Different Shepharose beads were used: octyl-(OC), octyl-glyoxyl-(OC-GLX), cyanogen bromide (BrCN)-, and Q-Sepharose. The performance of the different immobilized extremophile lipase from Serratia (ESL) was compared with that of the lipase B from Candida antarctica (CALB). In all immobilization tests, hyperactivation of ESL was observed. The highest hyperactivation (10.3) was obtained by immobilization on the OC support. Subsequently, the thermal stability at pH 5, 7, and 9 and the stability in the presence of 50% (v/v) acetonitrile, 50% dioxane, and 50% tetrahydrofuran solvents at pH 7 and 40 °C were evaluated. ESL immobilized on octyl-Sepharose was the most stable biocatalyst at 90 °C and pH 9, while the most stable preparation at pH 5 was ESL immobilized on OC-GLX-Sepharose supports. Finally, in the presence of 50% (v/v) tetrahydrofuran (THF) or dioxane at 40 °C, ESL immobilized on OC-Sepharose was the most stable biocatalyst, while the immobilized preparation of ESL on Q-Sepharose was the most stable one in 40% (v/v) acetonitrile. Full article

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

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10 pages, 1888 KiB

Open AccessArticle

Stabilization of β-Galactosidase on Modified Gold Nanoparticles: A Preliminary Biochemical Study to Obtain Lactose-Free Dairy Products for Lactose-Intolerant Individuals

by Asim Muhammed Alshanberi, Rukhsana Satar and Shakeel Ahmed Ansari

Molecules 2021, 26(5), 1226; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26051226 - 25 Feb 2021

Cited by 9 | Viewed by 2056

Abstract

The unique chemical, optical, and electrical characteristics of nanoparticles make their utilization highly successful in every field of biological sciences as compared to their bulk counterpart. These properties arise as a result of their miniature size, which provides them an excellent surface area-to-volume ratio, inner structure, and shape, and hence increases their surface characteristics. Therefore, this study was undertaken to engineer gold nanoparticles (AuNPs) for improving their catalytic activity and stability in biotechnological processes. The characterization of AuNPs was performed by XRD, UV spectra, and TEM. The synthesized AuNPs were surface-modified by polyvinyl alcohol (PVA) for binding the enzyme in excellent yield. The developed immobilized enzyme system (PVA-AuNPs-β-galactosidase) displayed pH optima at pH 7.0 and temperature optima at 40 °C. Moreover, the stability of PVA-AuNPs-β-galactosidase was significantly enhanced at wider pH and temperature ranges and at higher galactose concentrations, in contrast to the free enzyme. β-galactosidase bound to PVA-modified AuNPs exhibited greater operational activity, even after its sixth reuse. The developed nanosystem may prove useful in producing lactose-free dairy products for lactose-intolerant patients. Full article

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

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16 pages, 2731 KiB

Open AccessArticle

Constructing an Efficient Bacillus subtilis Spore Display by Using Cohesin−Dockerin Interactions

by He Wang, Xiaomin Jiang, Yongchang Qian and Lianghong Yin

Molecules 2021, 26(4), 1186; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26041186 - 23 Feb 2021

Cited by 6 | Viewed by 2464

Abstract

Bacillus subtilis spore display has become a field of increasing interest in the past two decades. To improve the efficiency of B. subtilis spore display, its directed modification was performed based on the cellulosome architecture by introducing onto them divergent cohesin (Coh) modules that can specifically bind to the target enzyme bearing the matching dockerins (Doc). In this study, five different pairs of cohesins and dockerins, selected from four cellulolytic microbes, were examined for their capabilities in displaying a tetrameric enzyme β-galactosidase from Bacillus stearothermophilus IAM11001 on the surface of B. subtilis WB600 spores. Immunofluorescence microscopy, western blotting, dot blotting, and enzyme assay was applied to confirm its surface expression. All the resultant five Coh–Doc based spore display can hydrolyze o-nitrophenyl-β-D-galactopyranoside. Further, the optimized Coh–Doc based spore display exhibited the highest display efficiency. Overall, the results of current study may open new perspectives on the use of Coh–Doc interaction, which will find application in improving the efficiency of B. subtilis spore display. Full article

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

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25 pages, 2541 KiB

Open AccessFeature PaperArticle

Effect of Concentrated Salts Solutions on the Stability of Immobilized Enzymes: Influence of Inactivation Conditions and Immobilization Protocol

by Sabrina Ait Braham, El-Hocine Siar, Sara Arana-Peña, Diego Carballares, Roberto Morellon-Sterling, Hossein Bavandi, Diandra de Andrades, Jakub F. Kornecki and Roberto Fernandez-Lafuente

Molecules 2021, 26(4), 968; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26040968 - 12 Feb 2021

Cited by 18 | Viewed by 3191

Abstract

This paper aims to investigate the effects of some salts (NaCl, (NH₄)₂SO₄ and Na₂SO₄) at pH 5.0, 7.0 and 9.0 on the stability of 13 different immobilized enzymes: five lipases, three proteases, two glycosidases, and one laccase, penicillin G acylase and catalase. The enzymes were immobilized to prevent their aggregation. Lipases were immobilized via interfacial activation on octyl agarose or on glutaraldehyde-amino agarose beads, proteases on glyoxyl agarose or glutaraldehyde-amino agarose beads. The use of high concentrations of salts usually has some effects on enzyme stability, but the intensity and nature of these effects depends on the inactivation pH, nature and concentration of the salt, enzyme and immobilization protocol. The same salt can be a stabilizing or a destabilizing agent for a specific enzyme depending on its concentration, inactivation pH and immobilization protocol. Using lipases, (NH₄)₂SO₄ generally permits the highest stabilities (although this is not a universal rule), but using the other enzymes this salt is in many instances a destabilizing agent. At pH 9.0, it is more likely to find a salt destabilizing effect than at pH 7.0. Results confirm the difficulty of foreseeing the effect of high concentrations of salts in a specific immobilized enzyme. Full article

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

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14 pages, 1864 KiB

Open AccessArticle

Immobilization of Eversa^® Transform via CLEA Technology Converts It in a Suitable Biocatalyst for Biolubricant Production Using Waste Cooking Oil

by José Renato Guimarães, Letícia Passos Miranda, Roberto Fernandez-Lafuente and Paulo Waldir Tardioli

Molecules 2021, 26(1), 193; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules26010193 - 02 Jan 2021

Cited by 34 | Viewed by 2880

Abstract

The performance of the previously optimized magnetic cross-linked enzyme aggregate of Eversa (Eversa-mCLEA) in the enzymatic synthesis of biolubricants by transesterification of waste cooking oil (WCO) with different alcohols has been evaluated. Eversa-mCLEA showed good activities using these alcohols, reaching a transesterification activity with isoamyl alcohol around 10-fold higher than with methanol. Yields of isoamyl fatty acid ester synthesis were similar using WCO or refined oil, confirming that this biocatalyst could be utilized to transform this residue into a valuable product. The effects of WCO/isoamyl alcohol molar ratio and enzyme load on the synthesis of biolubricant were also investigated. A maximum yield of around 90 wt.% was reached after 72 h of reaction using an enzyme load of 12 esterification units/g oil and a WCO/alcohol molar ratio of 1:6 in a solvent-free system. At the same conditions, the liquid Eversa yielded a maximum ester yield of only 34%. This study demonstrated the great changes in the enzyme properties that can be derived from a proper immobilization system. Moreover, it also shows the potential of WCO as a feedstock for the production of isoamyl fatty acid esters, which are potential candidates as biolubricants. Full article

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

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