Submit to Catalysts Review for Catalysts Propose a Special Issue

Journal Browser

► Journal Browser

Multienzymatic Catalysis and/or Enzyme Co-immobilization

Print Special Issue Flyer
Special Issue Editors
Special Issue Information
Keywords
Published Papers

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

Deadline for manuscript submissions: closed (20 January 2022) | Viewed by 30770

Share This Special Issue

Special Issue Editor

Prof. Dr. Roberto Fernandez-Lafuente

E-Mail Website
Guest Editor

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
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

The importance of biocatalysis is continuously growing, and many multienzymatic processes are being developed. These include cascade reactions, where the product of one enzyme is the substrate of the next enzyme, but also many other biocatalysts (e.g., use of lytic enzymes to prevent contaminations in bioprocesses). This Special Issue intends to address this diversity of multienzymatic reactions: processes where cofactor recycling is required, synergy hydrolysis of proteins and polysaccharides, a process where hydrogen peroxide is substrate or product, etc.

On the other hand, enzyme coimmobilization may produce some kinetic advantages. There is an enormous effort to develop coimmobilization technologies that can permit the specific location of each enzyme, and also to solve some of the inherent problems of coimmobilization. This way, this Special Issue also welcomes papers addressing the preparation of multienzymatic coimmobilized biocatalysts.

Prof. Dr. Roberto Fernandez-Lafuente
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. Catalysts is an international peer-reviewed open access monthly 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

multienzymatic processes
cellulose hydrolysis
juice clarification
cofactor recycling systems
hydrogen peroxide in biocatalysis
enzyme coimmobilization
enzyme location
enzyme stability
enzyme reuse

Published Papers (10 papers)

Download All Papers

Editorial

Jump to: Research, Review

3 pages, 213 KiB

Open AccessEditorial

Multienzymatic Catalysis and Enzyme Co-Immobilization

by Roberto Fernandez-Lafuente

Catalysts 2023, 13(12), 1488; https://0-doi-org.brum.beds.ac.uk/10.3390/catal13121488 - 30 Nov 2023

Cited by 1 | Viewed by 856

Abstract

The evolution of biocatalysis has undergone an unprecedented boost in response to the human demand for sustainable chemistry, which should enable researchers to make the most complex, selective and specific compounds with minimal ecological impact [...] Full article

(This article belongs to the Special Issue Multienzymatic Catalysis and/or Enzyme Co-immobilization)

Research

Jump to: Editorial, Review

15 pages, 2190 KiB

Open AccessArticle

Catalyst Replacement Policy on Multienzymatic Systems: Theoretical Study in the One-Pot Sequential Batch Production of Lactofructose Syrup

by Pablo Silva, Vanessa Arancibia, Daniela Cid, Oscar Romero, Andrés Illanes and Lorena Wilson

Catalysts 2021, 11(10), 1167; https://0-doi-org.brum.beds.ac.uk/10.3390/catal11101167 - 27 Sep 2021

Cited by 2 | Viewed by 1371

Abstract

One-pot systems are an interesting proposal to carry out multi-enzymatic reactions, though this strategy implies establishing an optimal balance between the activity and operability of the involved enzymes. This is crucial for enzymes with marked differences in their operational stability, such as one-pot production of lactofructose syrup from cheese whey permeate, which involves two enzymes—β-galactosidase (β-gal) and glucose isomerase (GI). The aim of this work was to study the behavior of one-pot sequential batch production of lactofructose syrup considering both enzymes immobilized individually, in order to evaluate and design a strategy of replacement of the catalysts according to their stabilities. To this end, the modelling and simulation of the process was carried out, considering simultaneously the kinetics of both reactions and the kinetics of inactivation of both enzymes. For the latter, it was also considered the modulating effect that sugars present in the medium may have on the stability of β-gal, which is the less stable enzyme. At the simulated reaction conditions of 40 °C, pH 7, and 0.46 (IU_GI/IU_β-gal), the results showed that considering the stability of β-gal under non-reactive conditions, meaning in absence of the effect of modulation, it is necessary to carry out four replacements of β-gal for each cycle of use of GI. On the other hand, when considering the modulation caused by the sugars on the β-gal stability, the productivity increases up to 23% in the case of the highest modulation factor studied (η = 0.8). This work shows the feasibility of conducting a one-pot operation with immobilized enzymes of quite different operational stability, and that a proper strategy of biocatalyst replacement increases the productivity of the process. Full article

(This article belongs to the Special Issue Multienzymatic Catalysis and/or Enzyme Co-immobilization)

► Show Figures

Graphical abstract

9 pages, 1250 KiB

Open AccessCommunication

Encapsulation of Combi-CLEAs of Glycosidases in Alginate Beads and Polyvinyl Alcohol for Wine Aroma Enhancement

by Luigi Tavernini, Carla Aburto, Oscar Romero, Andrés Illanes and Lorena Wilson

Catalysts 2021, 11(7), 866; https://0-doi-org.brum.beds.ac.uk/10.3390/catal11070866 - 20 Jul 2021

Cited by 3 | Viewed by 1807

Abstract

The aromatic expression of wines can be enhanced by the addition of specific glycosidases, although their poor stability remains a limitation. Coimmobilization of glycosidases as cross-linked enzyme aggregates (combi-CLEAs) offers a simple solution yielding highly stable biocatalysts. Nevertheless, the small particle size of combi-CLEAs hinders their recovery, preventing their industrial application. Encapsulation of combi-CLEAs of glycosidases in alginate beads and in polyvinyl alcohol is proposed as a solution. Combi-CLEAS of β-d-glucosidase and α-l-arabinofuranosidase were prepared and encapsulated. The effects of combi-CLEA loading and particle size on the expressed specific activity (IU/g_biocatalyst) of the biocatalysts were evaluated. Best results were obtained with 2.6 mm diameter polyvinyl alcohol particles at a loading of 60 mg_combi-CLEA/g_{polyvinyl alcohol}, exhibiting activities of 1.9 and 1.0 IU/g_biocatalyst for β-d-glucosidase and α-l-arabinofuranosidase, respectively. Afterwards, the stability of the biocatalysts was tested in white wine. All the encapsulated biocatalysts retained full activity after 140 incubation days, outperforming both free enzymes and nonencapsulated combi-CLEAs. Nevertheless, the alginate-encapsulated biocatalysts showed a brittle consistency, making recovery unfeasible. Conversely, the polyvinyl-encapsulated biocatalyst remained intact throughout the assay. The encapsulation of combi-CLEAs in polyvinyl alcohol proved to be a simple methodology that allows their recovery and reuse to harness their full catalytic potential. Full article

(This article belongs to the Special Issue Multienzymatic Catalysis and/or Enzyme Co-immobilization)

► Show Figures

Figure 1

15 pages, 2090 KiB

Open AccessFeature PaperArticle

Aqueous Extraction of Seed Oil from Mamey Sapote (Pouteria sapota) after Viscozyme L Treatment

by Veymar G. Tacias-Pascacio, Arnulfo Rosales-Quintero, Rafael C. Rodrigues, Daniel Castañeda-Valbuena, Pablo F. Díaz-Suarez, Beatriz Torrestiana-Sánchez, Erik F. Jiménez-Gómez and Roberto Fernandez-Lafuente

Catalysts 2021, 11(6), 748; https://0-doi-org.brum.beds.ac.uk/10.3390/catal11060748 - 19 Jun 2021

Cited by 10 | Viewed by 3677

Abstract

In this study, aqueous enzymatic extraction (AEE) was evaluated during the process of obtaining oil from mamey sapote seed (OMSS). Viscozyme L enzyme complex was used at pH 4 and 50 °C during the optimization of the extraction process by central composite design and response surface methodology. Optimal conditions were: 3.5% (w/w) of enzyme (regarding the seed weight), 5.5 h of incubation time, 235 rpm of agitation rate, and 1:3.5 of solid-to-liquid ratio. These conditions enabled us to obtain an OMSS yield of 66%. No statistically significant differences were found in the fatty acid profile and physicochemical properties, such as the acid and iodine values and the percentage of free fatty acids, between the oil obtained by AEE or by the conventional solvent extraction (SE). However, the oxidative stability of the oil obtained by AEE (11 h) was higher than that obtained by SE (9.33 h), therefore, AEE, in addition to being an environmentally friendly method, produces a superior quality oil in terms of oxidative stability. Finally, the high oil content in mamey sapote seed, and the high percentage of oleic acid (around 50% of the total fatty acid) found in this oil, make it a useful edible vegetable oil. Full article

(This article belongs to the Special Issue Multienzymatic Catalysis and/or Enzyme Co-immobilization)

► Show Figures

Figure 1

23 pages, 2237 KiB

Open AccessFeature PaperArticle

Multi-Combilipases: Co-Immobilizing Lipases with Very Different Stabilities Combining Immobilization via Interfacial Activation and Ion Exchange. The Reuse of the Most Stable Co-Immobilized Enzymes after Inactivation of the Least Stable Ones

by Sara Arana-Peña, Diego Carballares, Vicente Cortés Corberan and Roberto Fernandez-Lafuente

Catalysts 2020, 10(10), 1207; https://0-doi-org.brum.beds.ac.uk/10.3390/catal10101207 - 19 Oct 2020

Cited by 29 | Viewed by 2831

Abstract

The lipases A and B from Candida antarctica (CALA and CALB), Thermomyces lanuginosus (TLL) or Rhizomucor miehei (RML), and the commercial and artificial phospholipase Lecitase ultra (LEU) may be co-immobilized on octyl agarose beads. However, LEU and RML became almost fully inactivated under conditions where CALA, CALB and TLL retained full activity. This means that, to have a five components co-immobilized combi-lipase, we should discard 3 fully active and immobilized enzymes when the other two enzymes are inactivated. To solve this situation, CALA, CALB and TLL have been co-immobilized on octyl-vinyl sulfone agarose beads, coated with polyethylenimine (PEI) and the least stable enzymes, RML and LEU have been co-immobilized over these immobilized enzymes. The coating with PEI is even favorable for the activity of the immobilized enzymes. It was checked that RML and LEU could be released from the enzyme-PEI coated biocatalyst, although this also produced some release of the PEI. That way, a protocol was developed to co-immobilize the five enzymes, in a way that the most stable could be reused after the inactivation of the least stable ones. After RML and LEU inactivation, the combi-biocatalysts were incubated in 0.5 M of ammonium sulfate to release the inactivated enzymes, incubated again with PEI and a new RML and LEU batch could be immobilized, maintaining the activity of the three most stable enzymes for at least five cycles of incubation at pH 7.0 and 60 °C for 3 h, incubation on ammonium sulfate, incubation in PEI and co-immobilization of new enzymes. The effect of the order of co-immobilization of the different enzymes on the co-immobilized biocatalyst activity was also investigated using different substrates, finding that when the most active enzyme versus one substrate was immobilized first (nearer to the surface of the particle), the activity was higher than when this enzyme was co-immobilized last (nearer to the particle core). Full article

(This article belongs to the Special Issue Multienzymatic Catalysis and/or Enzyme Co-immobilization)

► Show Figures

Graphical abstract

8 pages, 586 KiB

Open AccessCommunication

Combination of CTec2 and GH5 or GH26 Endo-Mannanases for Effective Lignocellulosic Biomass Degradation

by Samkelo Malgas and Brett I. Pletschke

Catalysts 2020, 10(10), 1193; https://0-doi-org.brum.beds.ac.uk/10.3390/catal10101193 - 16 Oct 2020

Cited by 8 | Viewed by 2009

Abstract

Among endo-mannanases, glycoside hydrolase (GH) family 26 enzymes have been shown to be more catalytically active than GH5 enzymes on mannans. However, only GH5 endo-mannanases have been used for the formulation of enzyme cocktails. In this study, Bacillus sp.-derived GH5 and GH26 endo-mannanases were comparatively analysed biochemically for their synergistic action with a commercial cellulase blend, CTec2, during pre-treated lignocellulose degradation. Substrate specificity and thermo-stability studies on mannan substrates showed that GH26 endo-mannanase was more catalytically active and stable than GH5. GH26 also exhibited higher binding affinity for mannan than GH5, while GH5 showed more affinity for lignocellulosic substrates than GH26. Applying the endo-mannanases in combination with CTec2 for lignocellulose degradation led to synergism with a 1.3-fold increase in reducing sugar release compared to when CTec2 was used alone. This study showed that using the activity of endo-mannanases displayed with model substrates is a poor predictor of their activity and synergism on complex lignocelluloses. Full article

(This article belongs to the Special Issue Multienzymatic Catalysis and/or Enzyme Co-immobilization)

► Show Figures

Figure 1

13 pages, 2195 KiB

Open AccessArticle

Efficient Oxidation of Methyl Glycolate to Methyl Glyoxylate Using a Fusion Enzyme of Glycolate Oxidase, Catalase and Hemoglobin

by Xiangxian Ying, Can Wang, Shuai Shao, Qizhou Wang, Xueting Zhou, Yanbing Bai, Liang Chen, Chenze Lu, Man Zhao and Zhao Wang

Catalysts 2020, 10(8), 943; https://0-doi-org.brum.beds.ac.uk/10.3390/catal10080943 - 17 Aug 2020

Cited by 9 | Viewed by 3118

Abstract

Possessing aldehyde and carboxyl groups, glyoxylic acid and its ester derivatives serve as platform chemicals for the synthesis of vanillin, (R)-pantolactone, antibiotics or agrochemicals. Methyl glycolate is one of the by-products in the coal-to-glycol industry, and we attempted its value-added use through enzymatic oxidation of methyl glycolate to methyl glyoxylate. The cascade catalysis of glycolate oxidase from Spinacia oleracea (SoGOX), catalase from Helicobacter pylori (HpCAT) and hemoglobin from Vitreoscilla stercoraria (VsHGB) was firstly constructed, despite poor catalytic performance. To enable efficient oxidation of methyl glycolate, eight fusion enzymes of SoGOX, HpCAT and VsHGB were constructed by varying the orientation and the linker length. The fusion enzyme VsHGB-GSG-SoGOX-GGGGS-HpCAT was proved to be best, which reaction yield was 2.9 times higher than that of separated enzymes. The enzyme SoGOX was further subjected to directed evolution and site-saturation mutagenesis. The reaction yield of the resulting variant M267T/S362G was 1.9 times higher than that of the wild type. Then, the double substitution M267T/S362G was integrated with fusion expression to give the fusion enzyme VsHGB-GSG-SoGOX^mut-GGGGS-HpCAT, which crude enzyme was used as biocatalyst. The use of crude enzyme virtually eliminated side reactions and simplified the preparation of biocatalysts. Under the optimized conditions, the crude enzyme VsHGB-GSG-SoGOX^mut-GGGGS-HpCAT catalyzed the oxidation of 200 mM methyl glycolate for 6 h, giving a yield of 95.3%. The development of efficient fusion enzyme and the use of its crude enzyme paved the way for preparative scale application on enzymatic oxidation of methyl glycolate to methyl glyoxylate. Full article

(This article belongs to the Special Issue Multienzymatic Catalysis and/or Enzyme Co-immobilization)

► Show Figures

Figure 1

14 pages, 2534 KiB

Open AccessArticle

Co-immobilization of an Enzyme System on a Metal-Organic Framework to Produce a More Effective Biocatalyst

by Raneem Ahmad, Jordan Shanahan, Sydnie Rizaldo, Daniel S. Kissel and Kari L. Stone

Catalysts 2020, 10(5), 499; https://0-doi-org.brum.beds.ac.uk/10.3390/catal10050499 - 02 May 2020

Cited by 29 | Viewed by 4410

Abstract

In many respects, enzymes offer advantages over traditional chemical processes due to their decreased energy requirements for function and inherent greener processing. However, significant barriers exist for the utilization of enzymes in industrial processes due to their limited stabilities and inability to operate over larger temperature and pH ranges. Immobilization of enzymes onto solid supports has gained attention as an alternative to traditional chemical processes due to enhanced enzymatic performance and stability. This study demonstrates the co-immobilization of glucose oxidase (GO_x) and horseradish peroxidase (HRP) as an enzyme system on Metal-Organic Frameworks (MOFs), UiO-66 and UiO-66-NH₂, that produces a more effective biocatalyst as shown by the oxidation of pyrogallol. The two MOFs utilized as solid supports for immobilization were chosen to investigate how modifications of the MOF linker affect stability at the enzyme/MOF interface and subsequent activity of the enzyme system. The enzymes work in concert with activation of HRP through the addition of glucose as a substrate for GO_x. Enzyme immobilization and leaching studies showed HRP/GO_x@UiO-66-NH₂ immobilized 6% more than HRP/GO_x@UiO-66, and leached only 36% of the immobilized enzymes over three days in the solution. The enzyme/MOF composites also showed increased enzyme activity in comparison with the free enzyme system: the composite HRP/GO_x@UiO-66-NH₂ displayed 189 U/mg activity and HRP/GO_x@UiO-66 showed 143 U/mg while the free enzyme showed 100 U/mg enzyme activity. This increase in stability and activity is due to the amine group of the MOF linker in HRP/GO_x@UiO-66-NH₂ enhancing electrostatic interactions at the enzyme/MOF interface, thereby producing the most stable biocatalyst material in solution. The HRP/GO_x@UiO-66-NH₂ also showed long-term stability in the solid state for over a month at room temperature. Full article

(This article belongs to the Special Issue Multienzymatic Catalysis and/or Enzyme Co-immobilization)

► Show Figures

Graphical abstract

Review

Jump to: Editorial, Research

27 pages, 5286 KiB

Open AccessFeature PaperEditor’s ChoiceReview

Enzyme-Coated Micro-Crystals: An Almost Forgotten but Very Simple and Elegant Immobilization Strategy

by Rodolpho R. C. Monteiro, José C. S. dos Santos, Andrés R. Alcántara and Roberto Fernandez-Lafuente

Catalysts 2020, 10(8), 891; https://0-doi-org.brum.beds.ac.uk/10.3390/catal10080891 - 06 Aug 2020

Cited by 36 | Viewed by 4370

Abstract

The immobilization of enzymes using protein coated micro-crystals (PCMCs) was reported for the first time in 2001 by Kreiner and coworkers. The strategy is very simple. First, an enzyme solution must be prepared in a concentrated solution of one compound (salt, sugar, amino acid) very soluble in water and poorly soluble in a water-soluble solvent. Then, the enzyme solution is added dropwise to the water soluble solvent under rapid stirring. The components accompanying the enzyme are called the crystal growing agents, the solvent being the dehydrating agent. This strategy permits the rapid dehydration of the enzyme solution drops, resulting in a crystallization of the crystal formation agent, and the enzyme is deposited on this crystal surface. The reaction medium where these biocatalysts can be used is marked by the solubility of the PCMC components, and usually these biocatalysts may be employed in water soluble organic solvents with a maximum of 20% water. The evolution of these PCMC was to chemically crosslink them and further improve their stabilities. Moreover, the PCMC strategy has been used to coimmobilize enzymes or enzymes and cofactors. The immobilization may permit the use of buffers as crystal growth agents, enabling control of the reaction pH in the enzyme environments. Usually, the PCMC biocatalysts are very stable and more active than other biocatalysts of the same enzyme. However, this simple (at least at laboratory scale) immobilization strategy is underutilized even when the publications using it systematically presented a better performance of them in organic solvents than that of many other immobilized biocatalysts. In fact, many possibilities and studies using this technique are lacking. This review tried to outline the possibilities of this useful immobilization strategy. Full article

(This article belongs to the Special Issue Multienzymatic Catalysis and/or Enzyme Co-immobilization)

► Show Figures

Graphical abstract

58 pages, 11423 KiB

Open AccessFeature PaperEditor’s ChoiceReview

One Pot Use of Combilipases for Full Modification of Oils and Fats: Multifunctional and Heterogeneous Substrates

by Sara Arana-Peña, Diego Carballares, Ángel Berenguer-Murcia, Andrés R. Alcántara, Rafael C. Rodrigues and Roberto Fernandez-Lafuente

Catalysts 2020, 10(6), 605; https://0-doi-org.brum.beds.ac.uk/10.3390/catal10060605 - 29 May 2020

Cited by 57 | Viewed by 5284

Abstract

Lipases are among the most utilized enzymes in biocatalysis. In many instances, the main reason for their use is their high specificity or selectivity. However, when full modification of a multifunctional and heterogeneous substrate is pursued, enzyme selectivity and specificity become a problem. This is the case of hydrolysis of oils and fats to produce free fatty acids or their alcoholysis to produce biodiesel, which can be considered cascade reactions. In these cases, to the original heterogeneity of the substrate, the presence of intermediate products, such as diglycerides or monoglycerides, can be an additional drawback. Using these heterogeneous substrates, enzyme specificity can promote that some substrates (initial substrates or intermediate products) may not be recognized as such (in the worst case scenario they may be acting as inhibitors) by the enzyme, causing yields and reaction rates to drop. To solve this situation, a mixture of lipases with different specificity, selectivity and differently affected by the reaction conditions can offer much better results than the use of a single lipase exhibiting a very high initial activity or even the best global reaction course. This mixture of lipases from different sources has been called “combilipases” and is becoming increasingly popular. They include the use of liquid lipase formulations or immobilized lipases. In some instances, the lipases have been coimmobilized. Some discussion is offered regarding the problems that this coimmobilization may give rise to, and some strategies to solve some of these problems are proposed. The use of combilipases in the future may be extended to other processes and enzymes. Full article

(This article belongs to the Special Issue Multienzymatic Catalysis and/or Enzyme Co-immobilization)

► Show Figures

Graphical abstract

Journal Menu

Journal Browser

Multienzymatic Catalysis and/or Enzyme Co-immobilization

Share This Special Issue

Special Issue Editor

Special Issue Information

Keywords

Published Papers (10 papers)

Editorial

Research

Review

Further Information

Guidelines

MDPI Initiatives

Follow MDPI