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Immobilized Biocatalysts II

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

Deadline for manuscript submissions: closed (31 March 2023) | Viewed by 16247

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Prof. Dr. Luciana R. B. Gonçalves

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Departamento de Engenharia Química, Universidade Federal do Ceará, Campus do Pici, Bloco 709, CEP 60455760, Fortaleza 60000-000, CE, Brazil
Interests: biocatalysis; enzyme immobilization; bioprocess engineering and biochemical reaction engineering
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Dear Colleagues,

Although enzymes as catalysts are environmentally beneficial, their applications require a certain degree of profitability to justify their cost-competitiveness with conventional chemical catalysts. To make enzymatic biocatalysts increasingly economically viable, advances in modern biotechnology and protein engineering have contributed to improving the various production processes. However, because the implementation of bioprocesses is a reality in the industry, some critical obstacles still need to be overcome. For example, the efficiency of enzymatic processes can be increased by using immobilized catalysts (heterogeneous biocatalysts). In this way, it is possible to protect the enzymes from the various interactions with the reaction medium, increase their stability, allow reuse, or prolong the bioreactor’s operation time. Immobilization also increases the shelf life of an enzyme, thus facilitating its large-scale use and the economic formulation of biotechnological industries. There are several ways to immobilize enzymes, and three of the most common methods are physical adsorption, cross-linking or covalent bonding, and encapsulation. However, despite the great diversity of methods developed to date, no general approach applies to all enzymes. Therefore, for each case (process), it is necessary to choose the most straightforward and cheapest protocol that will result in a biocatalyst with high operational activity and stability. In this context, this Special Issue aims to bring together contributions for improving immobilized enzyme performance. The idea is not limited to biocatalyst preparation; the study of kinetics and reactor design are also welcome, including mass transfer limitations and scale-up.

Prof. Dr. Luciana R. B. Gonçalves
Guest Editor

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Keywords

enzyme immobilization
heterogeneous biocatalysts
enzyme stabilization
novel immobilization platforms
bioreactor design

Related Special Issue

Immobilized Biocatalysts, 3rd Edition in Catalysts (1 article)

Published Papers (9 papers)

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Research

18 pages, 4812 KiB

Open AccessArticle

Polymer-Grafted 3D-Printed Material for Enzyme Immobilization—Designing a Smart Enzyme Carrier

by Daniela Eixenberger, Aditya Kumar, Saskia Klinger, Nico Scharnagl, Ayad W. H. Dawood and Andreas Liese

Catalysts 2023, 13(7), 1130; https://doi.org/10.3390/catal13071130 - 20 Jul 2023

Cited by 3 | Viewed by 1160

Abstract

One way to enhance the flow properties of packed bed reactors, including efficient mass transfer and high catalyst conversion rates, is the use of 3D printing. By creating optimized structures that prevent channeling and high pressure drops, it is possible to achieve the desired target. Nevertheless, additively manufactured structures most often possess a limited surface-area-to-volume-ratio, especially as porous printed structures are not standardized yet. One way to achieve surface-enhanced 3D-printed structures is surface modification to introduce surface-initiated polymers. In addition, when stimuli-sensitive polymers are chosen, autonomous process control is prospective. The current publication deals with the application of surface-induced polymerization on 3D-printed structures with the subsequent application as an enzyme carrier. Surface-induced polymerization can easily increase the number of enzymes by a factor of six compared to the non-modified 3D-printed structure. In addition, the swelling behavior of polyacrylic acid is proven, even with immobilized enzymes, enabling smart reaction control. The maximum activity of Esterase 2 (Est2) from Alicyclobacillus acidocaldarius per g carrier, determined after 2 h of polymer synthesis, is 0.61 U/g_support. Furthermore, universal applicability is shown in aqueous and organic systems, applying an Est2 and Candida antarctica lipase B (CalB) catalyzed reaction and leaving space for improvement due to compatibility of the functionalization process and the here chosen organic solvent. Overall, no enzyme leaching is detectable, and process stability for at least five subsequent batches is ensured. Full article

(This article belongs to the Special Issue Immobilized Biocatalysts II)

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13 pages, 2461 KiB

Open AccessFeature PaperArticle

Immobilization of Alpha Acetolactate Decarboxylase in Hybrid Gelatin/Alginate Support for Application to Reduce Diacetyl Off-Flavor in Beer

by Gustavo P. Costa, Leonardo B. Queiroz, Vitor Manfroi, Rafael C. Rodrigues and Plinho F. Hertz

Catalysts 2023, 13(3), 601; https://doi.org/10.3390/catal13030601 - 16 Mar 2023

Cited by 1 | Viewed by 1399

Abstract

Beer production is the largest among alcoholic beverages. Its production process is complex and demands several steps. Lager beers commonly present an off-flavor of butter that is due to the presence of diacetyl, and to avoid such a problem, a long period of maturation (3–5 weeks) is required. Another way is the application of (α-acetolactate decarboxylase) ALDC to accelerate the process. The objectives of the present work were to develop a low-cost support using gelatin, a residue from capsules from the nutraceutical industry, to immobilize the ALDC enzyme. For this, the yield, efficiency and activity recovered, and the stability of free and immobilized enzymes at different temperatures and pH were evaluated. To evaluate the capacity of immobilized enzymes when applied directly to beer and their operational stability, three concentrations of glutaraldehyde (1%, 2.5% and 5%) were tested in distilled water as a cross-linking agent. The best results obtained were 95.6%, 27.0% and 23.6%, respectively, for yield, efficiency and activity recovery. Immobilization provided a high activity over a wide pH range. The immobilized enzyme showed greater stability at temperatures of 50 and 60 °C. The immobilized derivative showed adequate reuse capacity, and its dehydrated form had excellent activity after long periods of storage. Full article

(This article belongs to the Special Issue Immobilized Biocatalysts II)

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19 pages, 3610 KiB

Open AccessArticle

Performance of Eversa Transform 2.0 Lipase in Ester Production Using Babassu Oil (Orbignya sp.) and Tucuman Oil (Astrocaryum vulgar): A Comparative Study between Liquid and Immobilized Forms in Fe₃O₄ Nanoparticles

by João Brandão Júnior, Jean Gleison Andrade do Nascimento, Michael Pablo França Silva, Eliane de Aquino Lima Brandão, Viviane de Castro Bizerra, Kaiany Moreira dos Santos, Juliana de França Serpa, José Cleiton Sousa dos Santos, Aluísio Marques da Fonseca, Diego Lomonaco Vasconcelos de Oliveira and Maria Cristiane Martins de Souza

Catalysts 2023, 13(3), 571; https://doi.org/10.3390/catal13030571 - 11 Mar 2023

Cited by 16 | Viewed by 2567

Abstract

In this study, biodiesel was produced through the enzymatic esterification of vegetable oils from two common Brazilian palm trees: babassu and tucuman. The oils were hydrolyzed by a chemical route and their free fatty acids esterified with ethanol and methanol using the lipase enzyme Eversa^® Transform 2.0 in free forms and supported in iron magnetic nanoparticles (Fe₃O₄) (enzymatic load: 80 UpNPBg⁻¹). These enzymatic reactions were performed at an oil–alcohol molar ratio of 1:1, reaction temperature of 37 °C, agitation at 150 rpm, and reaction times of 2, 4, 6 and 8 h for the reactions catalyzed by the soluble enzyme and 8 h for the reactions using the biocatalyst. The conversions of fatty acids in ethyl and methyl esters obtained were monitored by gas chromatography (CG). The results obtained from ester synthesis using enzyme catalysts in free form were better: babassu 52.6% (methanol) and 57.5% (ethanol), and for tucuman 96.7% (methanol) and 93.4% (ethanol). In the case of immobilized enzymes, the results obtained ranged from 68.7% to 82.2% for babassu and from 32.5% to 86.0% for tucuman, with three cycles of reuse and without significant catalyst loss. Molecular coupling studies revealed the structures of lipase and that linoleic acid bonded near the active site of the enzyme with the best free energy of −6.5 Kcal/mol. Full article

(This article belongs to the Special Issue Immobilized Biocatalysts II)

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18 pages, 3721 KiB

Open AccessFeature PaperArticle

Magnetic CLEAs of β-Galactosidase from Aspergillus oryzae as a Potential Biocatalyst to Produce Tagatose from Lactose

by Lucas A. de Freitas, Marylane de Sousa, Laiza B. Ribeiro, Ítalo W. L. de França and Luciana R. B. Gonçalves

Catalysts 2023, 13(2), 306; https://doi.org/10.3390/catal13020306 - 30 Jan 2023

Cited by 4 | Viewed by 2118

Abstract

β-galactosidase is an enzyme capable of hydrolysing lactose, used in various branches of industry, mainly the food industry. As the efficient industrial use of enzymes depends on their reuse, it is necessary to find an effective method for immobilisation, maintaining high activity and stability. The present work proposes cross-linked magnetic cross-linked enzyme aggregates (mCLEAs) to prepare heterogeneous biocatalysts of β-galactosidase. Different concentrations of glutaraldehyde (0.6%, 1.0%, 1.5%), used as a cross-linking agent, were studied. The use of dextran-aldehyde as an alternative cross-linking agent was also evaluated. The mCLEAs presented increased recovered activity directly related to the concentration of glutaraldehyde. Modifications to the protocol to prepare mCLEAs with glutaraldehyde, adding a competitive inhibitor or polymer coating, have not been effective in increasing the recovered activity of the heterogeneous biocatalysts or its thermal stability. The biocatalyst prepared using dextran-aldehyde presented 73.6% recovered activity, aside from substrate affinity equivalent to the free enzyme. The thermal stability at 60 °C was higher for the biocatalyst prepared with glutaraldehyde (mCLEA-GLU-1.5) than the one produced with dextran-aldehyde (mCLEA-DEX), and the opposite happened at 50 °C. Results obtained for lactose hydrolysis, the use of its product to produce a rare sugar (D-tagatose) and operational and storage stability indicate that heterogeneous biocatalysts have adequate characteristics for industrial use. Full article

(This article belongs to the Special Issue Immobilized Biocatalysts II)

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14 pages, 3854 KiB

Open AccessArticle

Use of Bioprinted Lipases in Microwave-Assisted Esterification Reactions

by Jéssica Jéssi Carvalho de Melo, Gardenia Laís Passos da Silva, Danyelle Andrade Mota, Luma Mirely de Souza Brandão, Ranyere Lucena de Souza, Matheus M. Pereira, Álvaro Silva Lima and Cleide Mara Faria Soares

Catalysts 2023, 13(2), 299; https://doi.org/10.3390/catal13020299 - 28 Jan 2023

Cited by 4 | Viewed by 1731

Abstract

In this study, a comparative evaluation was performed in batch esterification reactions under conventional heating (CH) and assisted by microwave irradiation (MW) using bioprinted lipases. Microwave-irradiation-assisted reactions generally provide higher productivities and improve synthesis performance in terms of increased rate and reduced reaction times, resulting in higher interest yields in less time. Productivity was calculated with the enzymes: Burkholderia cepacia lipase (BCL), Candida rugosa lipase (CRL), and porcine pancreas lipase (PPL) using different fatty acids (lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), stearic acid (18:0), and oleic acid (18:1)) and alcohols at a molar ratio of 1:8. The microwave reactor was operated at a temperature of 45 °C, and power varied between 50 W and 200 W. Bioprinted BCL (bBCL) showed the highest productivity among the tested lipases. In the reaction with the best result, bBCL with lauric acid under MW, the reaction time decreased from 24 h (CH) to 25 min (MW) and the productivity increased 33 times compared with the reactions under CH. The increase in productivity demonstrates its activation that occurred as a result of conformational changes of the enzyme in the bioprinting process, confirmed by Fourier transform infrared (FTIR) spectrometric analysis, which reduces the content of bBCL α-helix with lauric acid. The biocatalyst showed high operational stability over eight cycles, while losing only 19% of its initial activity with half-life times of 12.8 batches. The storage time was five weeks, maintaining ≈80% activity. The results demonstrate the prospect of a new enzymatic route to obtain hyperactive catalysts, with the use of bioprinted lipases in esterification reactions under microwave irradiation, for the synthesis of esters with a view to large-scale industrial application. Full article

(This article belongs to the Special Issue Immobilized Biocatalysts II)

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13 pages, 1627 KiB

Open AccessArticle

Heterofunctional Methacrylate Beads Bearing Octadecyl and Vinyl Sulfone Groups: Tricks to Obtain an Interfacially Activated Lipase from Thermomyces lanuginosus and Covalently Attached to the Support

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

Catalysts 2023, 13(1), 108; https://doi.org/10.3390/catal13010108 - 3 Jan 2023

Cited by 7 | Viewed by 1413

Abstract

Lipase from Thermomyces lanuginosus (TLL) has been immobilized on a methacrylate macroporous resin coated with octadecyl groups (Purolite Lifetech^®® ECR8806F). This immobilization protocol gave a biocatalyst with significantly higher stability than that obtained using octyl agarose. To further improve the biocatalyst features, we tried to covalently immobilize the enzyme using this support. For this purpose, the support was activated with divinyl sulfone. The results showed that at least 1/3 of the immobilized enzyme molecules were not covalently immobilized. To solve the problem, we produced an aminated support and then activated it with divinyl sulfone. This permitted the full covalent immobilization of the previously immobilized TLL. The use of different blocking agents as the reaction endpoint (using ethylenediamine, Asp, Gly, and Cys) greatly altered the biocatalyst functional features (activity, specificity, or stability). For example, the blocking with ethylenediamine increased the ratio of the activity versus R- and S-methyl mandelate by a three-fold factor. The blocking with Cys produced the most stable biocatalyst, maintaining close to 90% of the activity under conditions where the just adsorbed enzyme maintained less than 55%. That way, this strategy to modify the support has permitted obtaining an enzyme interfacially activated versus the octadecyl layer and, later, covalently immobilized by reaction with the vinyl sulfone groups. Full article

(This article belongs to the Special Issue Immobilized Biocatalysts II)

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22 pages, 7498 KiB

Open AccessArticle

Co-Enzymes with Dissimilar Stabilities: A Discussion of the Likely Biocatalyst Performance Problems and Some Potential Solutions

by Amalie Vang Høst, Roberto Morellon-Sterling, Diego Carballares, John M. Woodley and Roberto Fernandez-Lafuente

Catalysts 2022, 12(12), 1570; https://doi.org/10.3390/catal12121570 - 3 Dec 2022

Cited by 3 | Viewed by 1377

Abstract

Enzymes have several excellent catalytic features, and the last few years have seen a revolution in biocatalysis, which has grown from using one enzyme to using multiple enzymes in cascade reactions, where the product of one enzyme reaction is the substrate for the subsequent one. However, enzyme stability remains an issue despite the many benefits of using enzymes in a catalytic system. When enzymes are exposed to harsh process conditions, deactivation occurs, which changes the activity of the enzyme, leading to an increase in reaction time to achieve a given conversion. Immobilization is a well-known strategy to improve many enzyme properties, if the immobilization is properly designed and controlled. Enzyme co-immobilization is a further step in the complexity of preparing a biocatalyst, whereby two or more enzymes are immobilized on the same particle or support. One crucial problem when designing and using co-immobilized enzymes is the possibility of using enzymes with very different stabilities. This paper discusses different scenarios using two co-immobilized enzymes of the same or differing stability. The effect on operational performance is shown via simple simulations using Michaelis–Menten equations to describe kinetics integrated with a deactivation term. Finally, some strategies for overcoming some of these problems are discussed. Full article

(This article belongs to the Special Issue Immobilized Biocatalysts II)

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19 pages, 3960 KiB

Open AccessArticle

Improved Catalytic Performance of Lipase Eversa^® Transform 2.0 via Immobilization for the Sustainable Production of Flavor Esters—Adsorption Process and Environmental Assessment Studies

by José Miguel Júnior, Fernanda R. Mattos, Guilherme R. Costa, Ana B. R. Zurlo, Roberto Fernandez-Lafuente and Adriano A. Mendes

Catalysts 2022, 12(11), 1412; https://doi.org/10.3390/catal12111412 - 11 Nov 2022

Cited by 6 | Viewed by 1988

Abstract

The aim of this study was to produce several flavor esters via esterification of octanoic acid with different commercial short-chain alcohols (methanol, propanol, isoamyl alcohol, hexanol and benzyl alcohol) and fusel oil in solvent-free systems. Lipase Eversa^® Transform 2.0 immobilized via mechanism of interfacial activation on poly(styrenene-divinylbenzene) (PSty-DVB) beads was used as heterogeneous biocatalyst and its catalytic performance was compared with that of the soluble lipase. The heterogeneous biocatalyst was prepared by employing 5 mmol·L⁻¹ buffer sodium acetate at pH 5.0 and 25 °C using an initial protein loading of 40 mg·g⁻¹. The maximum amount of immobilized protein reached was 31 mg·g⁻¹, corresponding to an immobilization yield of 80%. Mass transfer studies demonstrated that the lipase was preferentially adsorbed inside the pores of the support, which was confirmed by scanning electron microscopy analysis. Lipase immobilization can be described by a pseudo-first-order kinetic model via a physisorption process. When used as biocatalysts of the target reactions, the highest conversion percentage (between 65% and 85% of acid conversion after 60–90 min of reaction) values were achieved for esterification reactions catalyzed by immobilized lipase. Reusability tests revealed high retention of the original activity of the immobilized lipase after six successive batch reactions using isoamyl alcohol (47%) and fusel oil (72%). The proposed reaction systems can be considered green processes (EcoScale score above 80), with exception of methanol medium, classified as an acceptable green process (EcoScale score of 68). These results show that the heterogeneous biocatalyst prepared can be an economic and sustainable option for flavor esters production on an industrial scale. Full article

(This article belongs to the Special Issue Immobilized Biocatalysts II)

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18 pages, 2831 KiB

Open AccessArticle

Site-Specific Covalent Immobilization of Methylobacterium extorquens Non-Blue Laccse Melac13220 on Fe₃O₄ Nanoparticles by Aldehyde Tag

by Abidan Ainiwaer, Ao Li, Xingwang Zhao, Yujiao Xu, Siping Han and Renjun Gao

Catalysts 2022, 12(11), 1379; https://doi.org/10.3390/catal12111379 - 7 Nov 2022

Cited by 1 | Viewed by 1341

Abstract

In the present study, the non-blue laccase Melac13220 from Methylobacterium extorquens was immobilized using three methods to overcome problems related to the stability and reusability of the free enzyme: entrapment of the enzyme with sodium alginate, crosslinking of the enzyme with glutaraldehyde and chitosan-, and site-specific covalent immobilization of the enzyme on Fe₃O₄ nanoparticles by an aldehyde tag. The site-specific covalent immobilization method showed the highest immobilization efficiency and vitality recovery. The optimum temperature of Melac13220 was increased from 65 °C to 80 °C. Immobilized Melac13220 showed significant tolerance to some organic solvents and maintained approximately 80% activity after 10 cycles of use. Differential scanning calorimetry (DSC) indicated that the melting temperature of the enzyme was increased (from 57 °C to 79 °C). Immobilization of Melac13220 also led to improvement in dye decolorization such that Congo Red was completely decolorized within 10 h. The immobilized enzyme can be easily prepared without purification, demonstrating the advantages of using the aldehyde tag strategy and providing a reference for the practical application of different immobilized laccase methods in the industrial field. Full article

(This article belongs to the Special Issue Immobilized Biocatalysts II)

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