Special Issue "Multienzymatic Catalysis and/or Enzyme Co-immobilization"

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

Deadline for manuscript submissions: 30 June 2021.

Special Issue Editor

Prof. Dr. Roberto Fernandez-Lafuente
grade 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
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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

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Keywords

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

Published Papers (6 papers)

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Research

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Article
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
Catalysts 2020, 10(10), 1207; https://doi.org/10.3390/catal10101207 - 19 Oct 2020
Cited by 2 | Viewed by 726
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 [...] Read more.
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)
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Communication
Combination of CTec2 and GH5 or GH26 Endo-Mannanases for Effective Lignocellulosic Biomass Degradation
Catalysts 2020, 10(10), 1193; https://doi.org/10.3390/catal10101193 - 16 Oct 2020
Cited by 2 | Viewed by 546
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 [...] Read more.
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)
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Article
Efficient Oxidation of Methyl Glycolate to Methyl Glyoxylate Using a Fusion Enzyme of Glycolate Oxidase, Catalase and Hemoglobin
Catalysts 2020, 10(8), 943; https://doi.org/10.3390/catal10080943 - 17 Aug 2020
Cited by 1 | Viewed by 773
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 [...] Read more.
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-SoGOXmut-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-SoGOXmut-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)
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Article
Co-immobilization of an Enzyme System on a Metal-Organic Framework to Produce a More Effective Biocatalyst
Catalysts 2020, 10(5), 499; https://doi.org/10.3390/catal10050499 - 02 May 2020
Cited by 3 | Viewed by 1226
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 [...] Read more.
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 (GOx) 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 GOx. Enzyme immobilization and leaching studies showed HRP/GOx@UiO-66-NH2 immobilized 6% more than HRP/GOx@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/GOx@UiO-66-NH2 displayed 189 U/mg activity and HRP/GOx@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/GOx@UiO-66-NH2 enhancing electrostatic interactions at the enzyme/MOF interface, thereby producing the most stable biocatalyst material in solution. The HRP/GOx@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)
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Review

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Review
Enzyme-Coated Micro-Crystals: An Almost Forgotten but Very Simple and Elegant Immobilization Strategy
Catalysts 2020, 10(8), 891; https://doi.org/10.3390/catal10080891 - 06 Aug 2020
Cited by 8 | Viewed by 1144
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 [...] Read more.
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)
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Review
One Pot Use of Combilipases for Full Modification of Oils and Fats: Multifunctional and Heterogeneous Substrates
Catalysts 2020, 10(6), 605; https://doi.org/10.3390/catal10060605 - 29 May 2020
Cited by 15 | Viewed by 1290
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. [...] Read more.
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)
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Planned Papers

The below list represents only planned manuscripts. Some of these manuscripts have not been received by the Editorial Office yet. Papers submitted to MDPI journals are subject to peer-review.

Title: 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 enzymes
Authors: Sara Arana-Peña, Diego Carballares , Vicente Cortes-Corberan and Roberto Fernandez-Lafuente *
Affiliation: UFRGS

Title: GH5 and GH26 endo-mannanases perform the same during lignocellulosic biomass degradation - even though GH26 endo-mannanase is more catalytically active on mannans
Authors: Samkelo Malgas
Affiliation: Enzyme Science Programme (ESP), Department of Biochemistry and Microbiology, Rhodes University, Grahamstown 6140, South Africa
Abstract: Among endo-mannanases, glycoside hydrolase (GH) family 26 enzymes have been shown to be more catalytically active on mannans than GH5 enzymes. 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 CTec2 during pre-treated lignocellulose degradation. Substrate specificity and thermo-stability studies on mannans showed that GH26 endo-mannanase was more catalytically active and stable than GH5. GH26 also exhibited higher binding affinity on mannan than GH5, while GH5 showed more affinity on 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 recorded on model substrates is a poor predictor of their activity and synergism on complex lignocelluloses.

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