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17 pages, 5373 KiB

Open AccessFeature PaperArticle

Efficient One-Step Immobilization of CaLB Lipase over MOF Support NH₂-MIL-53(Al)

by Victoria Gascón-Pérez, Mayra Belen Jiménez, Asunción Molina, Rosa María Blanco and Manuel Sánchez-Sánchez

Catalysts 2020, 10(8), 918; https://doi.org/10.3390/catal10080918 - 10 Aug 2020

Cited by 17 | Viewed by 5474

Abstract

Metal-organic framework (MOF) materials possess the widest versatility in structure, composition, and synthesis procedures amongst the known families of materials. On the other hand, the extraordinary affinity between MOFs and enzymes has led to widely investigating these materials as platforms to support these [...] Read more.

Metal-organic framework (MOF) materials possess the widest versatility in structure, composition, and synthesis procedures amongst the known families of materials. On the other hand, the extraordinary affinity between MOFs and enzymes has led to widely investigating these materials as platforms to support these catalytic proteins in recent years. In this work, the MOF material NH₂-MIL-53(Al) has been tested as a support to immobilize by one-step methodology (in situ) the enzyme lipase CaLB from Candida antarctica by employing conditions that are compatible with its enzymatic activity (room temperature, aqueous solution, and moderate pH values). Once the nature of the linker deprotonating agent or the synthesis time were optimized, the MOF material resulted in quite efficient entrapping of the lipase CaLB through this in situ approach (>85% of the present enzyme in the synthesis media) while the supported enzyme retained acceptable activity (29% compared to the free enzyme) and had scarce enzyme leaching. The equivalent post-synthetic method led to biocatalysts with lower enzyme loading values. These results make clear that the formation of MOF support in the presence of the enzyme to be immobilized substantially improves the efficiency of the biocatalysts support for retaining the enzyme and limits their leaching. Full article

(This article belongs to the Special Issue Advances in the Design and Characterization of Heterogeneous Biocatalysts)

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22 pages, 2332 KiB

Open AccessFeature PaperArticle

Composites of Crosslinked Aggregates of Eversa^® Transform and Magnetic Nanoparticles. Performance in the Ethanolysis of Soybean Oil

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

Catalysts 2020, 10(8), 817; https://doi.org/10.3390/catal10080817 - 22 Jul 2020

Cited by 31 | Viewed by 3027

Abstract

Eversa^® Transform 2.0 has been launched to be used in free form, but its immobilization may improve its performance. This work aimed to optimize the immobilization of Eversa^® Transform 2.0 by the crosslinked enzyme aggregates (CLEAs) technique, using almost all the [...] Read more.

Eversa^® Transform 2.0 has been launched to be used in free form, but its immobilization may improve its performance. This work aimed to optimize the immobilization of Eversa^® Transform 2.0 by the crosslinked enzyme aggregates (CLEAs) technique, using almost all the available tools to improve its performance. Several variables in the CLEA preparation were optimized to improve the recovered activity, such as precipitant nature and crosslinker concentration. Moreover, some feeders were co-precipitated to improve the crosslinking step, such as bovine serum albumin, soy protein, or polyethyleneimine. Starch (later enzymatically degraded) was utilized as a porogenic agent to decrease the substrate diffusion limitations. Silica magnetic nanoparticles were also utilized to simplify the CLEA handling, but it was found that a large percentage of the Eversa activity could be immobilized on these nanoparticles before aggregation. The best CLEA protocol gave a 98.9% immobilization yield and 30.1% recovered activity, exhibited a porous structure, and an excellent performance in the transesterification of soybean oil with ethanol: 89.8 wt% of fatty acid ethyl esters (FAEEs) yield after 12 h of reaction, while the free enzyme required a 48 h reaction to give the same yield. A caustic polishing step of the product yielded a biodiesel containing 98.9 wt% of FAEEs and a free fatty acids content lower than 0.25%, thus the final product met the international standards for biodiesel. The immobilized biocatalyst could be reused for at least five 12 h-batches maintaining 89.6% of the first-batch yield, showing the efficient catalyst recovery by applying an external magnetic field. Full article

(This article belongs to the Special Issue Advances in the Design and Characterization of Heterogeneous Biocatalysts)

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10 pages, 826 KiB

Open AccessFeature PaperArticle

High Stabilization of Enzymes Immobilized on Rigid Hydrophobic Glyoxyl-Supports: Generation of Hydrophilic Environments on Support Surfaces

by Alejandro H. Orrego, María Romero-Fernández, María del Carmen Millán-Linares, Justo Pedroche, José M. Guisán and Javier Rocha-Martin

Catalysts 2020, 10(6), 676; https://doi.org/10.3390/catal10060676 - 16 Jun 2020

Cited by 15 | Viewed by 3635

Abstract

Very rigid supports are useful for enzyme immobilization to design continuous flow reactors and/or to work in non-conventional media. Among them, epoxy-methacrylic supports are easily functionalized with glyoxyl groups, which makes them ideal candidates for enzyme stabilization via multipoint covalent immobilization. However, these [...] Read more.

Very rigid supports are useful for enzyme immobilization to design continuous flow reactors and/or to work in non-conventional media. Among them, epoxy-methacrylic supports are easily functionalized with glyoxyl groups, which makes them ideal candidates for enzyme stabilization via multipoint covalent immobilization. However, these supports present highly hydrophobic surfaces, which might promote very undesirable effects on enzyme activity and/or stability. The hydrophilization of the support surface after multipoint enzyme immobilization is proposed here as an alternative to reduce these undesirable effects. The remaining aldehyde groups on the support are modified with aminated hydrophilic small molecules (glycine, lysine or aspartic acid) in the presence of 2-picoline borane. The penicillin G acylase from Escherichia coli (PGA) and alcohol dehydrogenase from Thermus thermophilus HB27 (ADH2) were immobilized on glyoxyl-functionalized agarose, Relizyme and Relisorb. Despite the similar density of aldehyde groups displayed by functionalized supports, their stabilization effects on immobilized enzymes were quite different: up to 300-fold lower by hydrophobic supports than by highly hydrophilic glyoxyl-agarose. A dramatic increase in the protein stabilities was shown when a hydrophilization treatment of the hydrophobic support surface was done. The PGA immobilized on the glyoxyl-Relisorb hydrophilized with aspartic acid becomes 280-fold more stable than without any treatment, and it is even more stable than the PGA immobilized on the glyoxyl agarose. Full article

(This article belongs to the Special Issue Advances in the Design and Characterization of Heterogeneous Biocatalysts)

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15 pages, 2207 KiB

Open AccessFeature PaperArticle

Stabilization of b-Glucuronidase by Immobilization in Magnetic-Silica Hybrid Supports

by Sonali Correa, Magdalena Ripoll, Erienne Jackson, Valeria Grazú and Lorena Betancor

Catalysts 2020, 10(6), 669; https://doi.org/10.3390/catal10060669 - 13 Jun 2020

Cited by 12 | Viewed by 4129

Abstract

β-Glucuronidases are a class of enzymes that catalyze the breakdown of complex carbohydrates. They have well documented biocatalytic applications in synthesis, therapeutics, and analytics that could benefit from enzyme immobilization and stabilization. In this work, we have explored a number of immobilization strategies [...] Read more.

β-Glucuronidases are a class of enzymes that catalyze the breakdown of complex carbohydrates. They have well documented biocatalytic applications in synthesis, therapeutics, and analytics that could benefit from enzyme immobilization and stabilization. In this work, we have explored a number of immobilization strategies for Patella vulgata β-Glucuronidase that comprised a tailored combination of biomimetic silica (Si) and magnetic nanoparticles (MNPs). The individual effect of each material on the enzyme upon immobilization was first tested. Three different immobilization strategies for covalent attachment on MNPs and different three catalysts for the deposition of Si particles were tested. We produced nine different immobilized preparations and only two of them presented negligible activity. All the preparations were in the micro-sized range (from 1299 ± 52 nm to 2101 ± 67 nm of hydrodynamic diameter). Their values for polydispersity index varied around 0.3, indicating homogeneous populations of particles with low probability of agglomeration. Storage, thermal, and operational stability were superior for the enzyme immobilized in the composite material. At 80 °C different preparations with Si and MNPs retained 40% of their initial activity after 6 h of incubation whereas the soluble enzyme lost 90% of its initial activity within 11 min. Integration of MNPs provided the advantage of reusing the biocatalyst via magnetic separation up to six times with residual activity. The hybrid material produced herein demonstrated its versatility and robustness as a support for β-Glucuronidases immobilization. Full article

(This article belongs to the Special Issue Advances in the Design and Characterization of Heterogeneous Biocatalysts)

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16 pages, 3566 KiB

Open AccessFeature PaperArticle

Glyoxyl-Activated Agarose as Support for Covalently Link Novo-Pro D: Biocatalysts Performance in the Hydrolysis of Casein

by Laiane Antunes Lopes, Paula Kern Novelli, Roberto Fernandez-Lafuente, Paulo Waldir Tardioli and Raquel Lima Camargo Giordano

Catalysts 2020, 10(5), 466; https://doi.org/10.3390/catal10050466 - 25 Apr 2020

Cited by 15 | Viewed by 3977

Abstract

This study aimed to evaluate the performance of a commercial protease (Novo-Pro D (NPD)), both in soluble and immobilized forms, in the hydrolysis of proteins (using casein as model protein). Immobilization of the protease NPD on 6% agarose activated with glyoxyl groups for [...] Read more.

This study aimed to evaluate the performance of a commercial protease (Novo-Pro D (NPD)), both in soluble and immobilized forms, in the hydrolysis of proteins (using casein as model protein). Immobilization of the protease NPD on 6% agarose activated with glyoxyl groups for 24 h at 20 °C and pH 10.0 allowed preparing immobilized biocatalyst with around 90% immobilization yield, 92% recovered activity versus small substrate, and a thermal stability 5.3-fold higher than the dialyzed soluble enzyme at 50 °C and pH 8.0. Immobilization times longer than 24 h lead to a decrease in the recovered activity and did not improve the biocatalyst stability. At 50 °C and pH 6.5, the immobilized NPD was around 20-fold more stable than the dialyzed soluble protease. Versus casein, the immobilized NDP presented a 10% level of activity, but it allowed hydrolyzing casein (26 g/L) at 50 °C and pH 6.5 up to a 40% degree of hydrolysis (DH) after 2 h reaction, while under the same conditions, only a 34% DH was achieved with soluble NPD. In addition, the immobilized NPD showed good reusability, maintaining the DH of casein for at least ten 2h-reaction batches. Full article

(This article belongs to the Special Issue Advances in the Design and Characterization of Heterogeneous Biocatalysts)

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15 pages, 5025 KiB

Open AccessArticle

Laccase Immobilized Fe₃O₄-Graphene Oxide Nanobiocatalyst Improves Stability and Immobilization Efficiency in the Green Preparation of Sulfa Drugs

by Shamila Rouhani, Shohreh Azizi, Rose W. Kibechu, Bhekie B Mamba and Titus A. M. Msagati

Catalysts 2020, 10(4), 459; https://doi.org/10.3390/catal10040459 - 23 Apr 2020

Cited by 26 | Viewed by 3727

Abstract

This paper, reports on the novel and green synthesis procedure for sulfonamides that involved the immobilization of Trametes Versicolor laccase onto the Fe₃O₄–graphene nanocomposite via glutaraldehyde (GA) crosslinking (Lac/Fe₃O₄/GO). Various parameters, mainly, activation time, GA, [...] Read more.

This paper, reports on the novel and green synthesis procedure for sulfonamides that involved the immobilization of Trametes Versicolor laccase onto the Fe₃O₄–graphene nanocomposite via glutaraldehyde (GA) crosslinking (Lac/Fe₃O₄/GO). Various parameters, mainly, activation time, GA, and laccase concentration were investigated and optimized. The results showed that the optimal contact time was 4 h, GA concentration was 5% while laccase concentration was 5 mg·mL⁻¹, at which a high enzyme activity recovery was achieved (86%). In terms of the stability of immobilized laccase to temperature and storage conditions, the performance of the nanobiocatalyst was found to significantly exceed that of free laccase. The results have indicated that nearly 70% of relative activity for immobilized laccase remained after the incubation period of 2 h at 55 °C, but only 48% of free laccase remained within the same time period. Moreover, the immobilized laccase retained 88% of its initial activity after storage for 20 days. In case of the free laccase, the activity retained within the same time period was 32%. In addition, the nanobiocatalyst possessed better recycling performance as evidenced from the observation that after eight cycles of repeated use, it retained 85% of its original activity. Full article

(This article belongs to the Special Issue Advances in the Design and Characterization of Heterogeneous Biocatalysts)

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19 pages, 1737 KiB

Open AccessFeature PaperArticle

Sustainable Enzymatic Synthesis of a Solketal Ester—Process Optimization and Evaluation of Its Antimicrobial Activity

by Patricia A. Mendoza-Ortiz, Rafaela S. Gama, Omar C. Gómez, Jaine H. H. Luiz, Roberto Fernandez-Lafuente, Erika C. Cren and Adriano A. Mendes

Catalysts 2020, 10(2), 218; https://doi.org/10.3390/catal10020218 - 11 Feb 2020

Cited by 29 | Viewed by 4424

Abstract

The present study aims the enzymatic synthesis of solketal palmitate by esterification between solketal and palmitic acid using heptane as solvent. Lipases from Thermomyces lanuginosus (TLL), Candida rugosa type VII (CRL), and Pseudomonas fluorescens (PFL) were immobilized via interfacial activation on rice husk [...] Read more.

The present study aims the enzymatic synthesis of solketal palmitate by esterification between solketal and palmitic acid using heptane as solvent. Lipases from Thermomyces lanuginosus (TLL), Candida rugosa type VII (CRL), and Pseudomonas fluorescens (PFL) were immobilized via interfacial activation on rice husk silica functionalized with triethoxy(octyl)silane (Octyl–SiO₂) and used as biocatalysts. A loading of 20–22 mg of lipase/g of support was immobilized independently of the studied enzyme. TLL–Octyl–SiO₂ was the most active biocatalyst in oil hydrolysis (656.0 ± 23.9 U/g) and ester synthesis (productivity of 6.8 mmol/min.g_biocat), and it has been chosen for further ester synthesis optimization. The effect of some important parameters such as biocatalyst concentration, reaction temperature and acid:alcohol molar ratio on the reaction has been evaluated using a central composite rotatable design at fixed mechanical stirring (240 rpm) and reaction time (15 min). Subsequently, the effect of reactants concentration and molecular sieve concentration has also been examined. Under optimal conditions (56 °C, acid:alcohol molar ratio of 1:3 with a palmitic acid concentration of 1 M, and 20% wt. of TLL–Octyl–SiO₂ per volume of reaction mixture), 83% acid conversion was obtained after 150 min of reaction. The biocatalyst retained 87% of its initial activity after seven successive reaction batches. The product was identified by nuclear magnetic resonance analysis. Antimicrobial activity studies showed that the synthesized ester demonstrated antifungal activity against Candida albicans and Candida parapsilosis, with minimum inhibitory concentration (MIC) between 200 and 400 µg/mL, and bacteriostatic/fungistatic action—minimum microbicial concentration (MMC) > 400 µg/mL. Full article

(This article belongs to the Special Issue Advances in the Design and Characterization of Heterogeneous Biocatalysts)

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14 pages, 970 KiB

Open AccessArticle

Estimation of the Effectiveness Factor for Immobilized Enzyme Catalysts through a Simple Conversion Assay

by Pedro Valencia and Francisco Ibañez

Catalysts 2019, 9(11), 930; https://doi.org/10.3390/catal9110930 - 7 Nov 2019

Cited by 8 | Viewed by 7177

Abstract

A novel methodology to estimate the effectiveness factor (EF) of an immobilized enzyme catalyst is proposed here. The methodology consists of the determination of the productivity of both the immobilized enzyme catalyst and its corresponding soluble enzyme, plotted as a function of the [...] Read more.

A novel methodology to estimate the effectiveness factor (EF) of an immobilized enzyme catalyst is proposed here. The methodology consists of the determination of the productivity of both the immobilized enzyme catalyst and its corresponding soluble enzyme, plotted as a function of the reaction conversion. The ratio of these productivities corresponds to the EF estimator of the catalyst. Conversion curves were simulated in a batch reactor with immobilized enzyme and soluble enzyme for different values of the S₀/K_M ratio and Thiele modulus (Φ) to demonstrate this hypothesis. Two different reaction orders were tested: first-order kinetic and Michaelis–Menten-based kinetic with product inhibition. The results showed that the ratio of productivities between the immobilized and soluble enzymes followed the behavior profile presented by the EF with satisfactory agreement. This simple methodology to estimate the EF is based on routine conversion experiments, thus avoiding the exhaustive kinetic and mass transfer characterization of the immobilized enzyme catalyst. Full article

(This article belongs to the Special Issue Advances in the Design and Characterization of Heterogeneous Biocatalysts)

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16 pages, 2380 KiB

Open AccessArticle

Deciphering the Effect of Microbead Size Distribution on the Kinetics of Heterogeneous Biocatalysts through Single-Particle Analysis Based on Fluorescence Microscopy

by Emilio Muñoz-Morales, Susana Velasco-Lozano, Ana I. Benítez-Mateos, María J. Marín, Pedro Ramos-Cabrer and Fernando López-Gallego

Catalysts 2019, 9(11), 896; https://doi.org/10.3390/catal9110896 - 28 Oct 2019

Cited by 10 | Viewed by 4111

Abstract

Understanding the functionality of immobilized enzymes with spatiotemporal resolution and under operando conditions is an unmet need in applied biocatalysis, as well as priceless information to guide the optimization of heterogeneous biocatalysts for industrial purposes. Unfortunately, enzyme immobilization still relies on trial-and-error approximations [...] Read more.

Understanding the functionality of immobilized enzymes with spatiotemporal resolution and under operando conditions is an unmet need in applied biocatalysis, as well as priceless information to guide the optimization of heterogeneous biocatalysts for industrial purposes. Unfortunately, enzyme immobilization still relies on trial-and-error approximations that prevail over rational designs. Hence, a modern fabrication process to achieve efficient and robust heterogeneous biocatalysts demands comprehensive characterization techniques to track and understand the immobilization process at the protein–material interface. Recently, our group has developed a new generation of self-sufficient heterogeneous biocatalysts based on alcohol dehydrogenases co-immobilized with nicotinamide cofactors on agarose porous microbeads. Harnessing the autofluorescence of NAD⁺(P)H and using time-lapse fluorescence microscopy, enzyme activity toward the redox cofactors can be monitored inside the beads. To analyze these data, herein we present an image analytical tool to quantify the apparent Michaelis–Menten parameters of alcohol dehydrogenases co-immobilized with NAD(P)⁺/H at the single-particle level. Using this tool, we found a strong negative correlation between the apparent catalytic performance of the immobilized enzymes and the bead radius when using exogenous bulky substrates in reduction reactions. Therefore, applying image analytics routines to microscopy studies, we can directly unravel the functional heterogeneity of different heterogeneous biocatalyst samples tested under different reaction conditions. Full article

(This article belongs to the Special Issue Advances in the Design and Characterization of Heterogeneous Biocatalysts)

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16 pages, 1237 KiB

Open AccessArticle

Identification of a Specific Inhibitor of Human Scp1 Phosphatase Using the Phosphorylation Mimic Phage Display Method

by Takuya Yoshida, Kazuki Yamazaki, Shunta Imai, Akinori Banno, Atsushi Kaneko, Kazuhiro Furukawa and Yoshiro Chuman

Catalysts 2019, 9(10), 842; https://doi.org/10.3390/catal9100842 - 11 Oct 2019

Cited by 4 | Viewed by 3555

Abstract

Protein phosphatases are divided into tyrosine (Tyr) phosphatases and serine/threonine (Ser/Thr) phosphatases. While substrate trapping mutants are frequently used to identify substrates of Tyr phosphatases, a rapid and simple method to identify Ser/Thr phosphatase substrates is yet to be developed. The TFIIF-associating component [...] Read more.

Protein phosphatases are divided into tyrosine (Tyr) phosphatases and serine/threonine (Ser/Thr) phosphatases. While substrate trapping mutants are frequently used to identify substrates of Tyr phosphatases, a rapid and simple method to identify Ser/Thr phosphatase substrates is yet to be developed. The TFIIF-associating component of RNA polymerase II C-terminal domain (CTD) phosphatase/small CTD phosphatase (FCP/SCP) phosphatase family is one of the three types of Ser/Thr protein phosphatases. Defects in these phosphatases are correlated with the occurrence of various diseases such as cancer and neuropathy. Recently, we developed phosphorylation mimic phage display (PMPD) method with AlF₄⁻, a methodology to identify substrates for FCP/SCP type Ser/Thr phosphatase Scp1. Here, we report a PMPD method using BeF₃⁻ to identify novel substrate peptides bound to Scp1. After screening peptide phages, we identified peptides that bound to Scp1 in a BeF₃⁻-dependent manner. Synthetic phosphopeptide BeM12-1, the sequence of which was isolated at the highest frequency, directly bound to Scp1. The binding was inhibited by adding BeF₃⁻, indicating that the peptide binds to the active center of catalytic site in Scp1. The phosphorylated BeM12-1 worked as a competitive inhibitor of Scp1. Thus, PMPD method may be applicable for the identification of novel substrates and inhibitors of the FCP/SCP phosphatase family. Full article

(This article belongs to the Special Issue Advances in the Design and Characterization of Heterogeneous Biocatalysts)

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16 pages, 2615 KiB

Open AccessEditor’s ChoiceArticle

Combi-CLEAs of Glucose Oxidase and Catalase for Conversion of Glucose to Gluconic Acid Eliminating the Hydrogen Peroxide to Maintain Enzyme Activity in a Bubble Column Reactor

by Agnes Cristina Oliveira Mafra, Letícia Gazzotto Ulrich, Jakub F. Kornecki, Roberto Fernandez-Lafuente, Paulo Waldir Tardioli and Marcelo Perencin de Arruda Ribeiro

Catalysts 2019, 9(8), 657; https://doi.org/10.3390/catal9080657 - 31 Jul 2019

Cited by 35 | Viewed by 7675

Abstract

In this study combined cross-linked aggregates of catalase from bovine liver and glucose-oxidase from Aspergillus niger were prepared, and the effects of the precipitant and crosslinking agents, as well as the use of bovine serum albumin (BSA) as a feeder protein, on enzyme [...] Read more.

In this study combined cross-linked aggregates of catalase from bovine liver and glucose-oxidase from Aspergillus niger were prepared, and the effects of the precipitant and crosslinking agents, as well as the use of bovine serum albumin (BSA) as a feeder protein, on enzyme immobilization yield and thermal stability of both enzymes, were evaluated. Combi- crosslinking of enzyme aggregates (CLEAs) prepared using dimethoxyethane as precipitant, 25 mM glutaraldehyde and BSA/enzymes mass ratio of 5.45 (w/w), exhibited the highest enzyme activities and stabilities at 40 °C, pH 6.0, and 250 rpm for 5 h. The stability of both immobilized enzymes was fairly similar, eliminating one of the problems of enzyme coimmobilization. Combi-CLEAs were used in gluconic acid (GA) production in a bubble column reactor operated at 40 °C, pH 6.0 and 10 vvm of aeration, using 26 g L⁻¹ glucose as the substrate. Results showed conversion of around 96% and a reaction course very similar to the same process using free enzymes. The operational half-life was 34 h, determined from kinetic profiles and the first order inactivation model. Combi-CLEAs of glucose-oxidase and catalase were shown to be a robust biocatalyst for applications in the production of gluconic acid from glucose. Full article

(This article belongs to the Special Issue Advances in the Design and Characterization of Heterogeneous Biocatalysts)

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13 pages, 2147 KiB

Open AccessArticle

Increasing the Enzyme Loading Capacity of Porous Supports by a Layer-by-Layer Immobilization Strategy Using PEI as Glue

by Nathalia S. Rios, Sara Arana-Peña, Carmen Mendez-Sanchez, Yuliya Lokha, Vicente Cortes-Corberan, Luciana R. B. Gonçalves and Roberto Fernandez-Lafuente

Catalysts 2019, 9(7), 576; https://doi.org/10.3390/catal9070576 - 29 Jun 2019

Cited by 42 | Viewed by 4814

Abstract

A new strategy to increase the enzyme-loading capacity of porous supports was investigated. Lipase from Pseudomonas fluorescens (PFL) was immobilized on octyl-agarose (OA) beads and treated with polyethyleneimine (PEI). Then, PFL was immobilized on the previous PFL layer. Next, the biocatalyst was coated [...] Read more.

A new strategy to increase the enzyme-loading capacity of porous supports was investigated. Lipase from Pseudomonas fluorescens (PFL) was immobilized on octyl-agarose (OA) beads and treated with polyethyleneimine (PEI). Then, PFL was immobilized on the previous PFL layer. Next, the biocatalyst was coated with PEI and a third layer of PFL was added. Sodium dodecyl sulfate polyacrylamide electrophoresis showed that the amount of PFL proportionally increased with each enzyme layer; however, the effects on biocatalyst activity were not as clear. Hydrolyzing 50 mM of triacetin at 25 °C, the activity of the three-layer biocatalyst was even lower than that of the bi-layer one; on the contrary its activity was higher when the activity was measured at 4 °C in the presence of 30% acetonitrile (that reduced the activity and thus the relevance of the substrate diffusion limitations). That is, the advantage of the multilayer formation depends on the specific activity of the enzyme and on the diffusion limitations of the substrate. When octyl agarose (OA)-PFL-PEI-PFL preparation was treated with glutaraldehyde, the activity was reduced, although the enzyme stability increased and the immobilization of the last PFL layer offered results similar to the one obtained using the three-layer preparation without glutaraldehyde modification (90%). Full article

(This article belongs to the Special Issue Advances in the Design and Characterization of Heterogeneous Biocatalysts)

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14 pages, 2454 KiB

Open AccessEditor’s ChoiceArticle

Reuse of Lipase from Pseudomonas fluorescens via Its Step-by-Step Coimmobilization on Glyoxyl-Octyl Agarose Beads with Least Stable Lipases

by Nathalia S. Rios, Sara Arana-Peña, Carmen Mendez-Sanchez, Claudia Ortiz, Luciana R. B. Gonçalves and Roberto Fernandez-Lafuente

Catalysts 2019, 9(5), 487; https://doi.org/10.3390/catal9050487 - 27 May 2019

Cited by 39 | Viewed by 5139

Abstract

Coimmobilization of lipases may be interesting in many uses, but this means that the stability of the least stable enzyme determines the stability of the full combilipase. Here, we propose a strategy that permits the reuse the most stable enzyme. Lecitase Ultra (LU) [...] Read more.

Coimmobilization of lipases may be interesting in many uses, but this means that the stability of the least stable enzyme determines the stability of the full combilipase. Here, we propose a strategy that permits the reuse the most stable enzyme. Lecitase Ultra (LU) (a phospholipase) and the lipases from Rhizomucor miehei (RML) and from Pseudomonas fluorescens (PFL) were immobilized on octyl agarose, and their stabilities were studied under a broad range of conditions. Immobilized PFL was found to be the most stable enzyme under all condition ranges studied. Furthermore, in many cases it maintained full activity, while the other enzymes lost more than 50% of their initial activity. To coimmobilize these enzymes without discarding fully active PFL when LU or RML had been inactivated, PFL was covalently immobilized on glyoxyl-agarose beads. After biocatalysts reduction, the other enzyme was coimmobilized just by interfacial activation. After checking that glyoxyl-octyl-PFL was stable in 4% Triton X-100, the biocatalysts of PFL coimmobilized with LU or RML were submitted to inactivation under different conditions. Then, the inactivated least stable coimmobilized enzyme was desorbed (using 4% detergent) and a new enzyme reloading (using in some instances RML and in some others employing LU) was performed. The initial activity of immobilized PFL was maintained intact for several of these cycles. This shows the great potential of this lipase coimmobilization strategy. Full article

(This article belongs to the Special Issue Advances in the Design and Characterization of Heterogeneous Biocatalysts)

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14 pages, 5525 KiB

Open AccessArticle

Site-Specific Addressing of Particles and Coatings via Enzyme-Mediated Destabilization

by David Wedegärtner and Oliver I. Strube

Catalysts 2019, 9(4), 354; https://doi.org/10.3390/catal9040354 - 12 Apr 2019

Cited by 2 | Viewed by 3374

Abstract

Enzyme mediated addressing (EMA) is a highly specific and easy-to-apply technology for direction and deposition of particles and coatings on surfaces. Key feature of this process is an enzymatic reaction in direct proximity to the surface, which induces the deposition. The technique has [...] Read more.

Enzyme mediated addressing (EMA) is a highly specific and easy-to-apply technology for direction and deposition of particles and coatings on surfaces. Key feature of this process is an enzymatic reaction in direct proximity to the surface, which induces the deposition. The technique has previously shown great success in the handling of biological particles. In this study, addressing of non-biological nanoparticles, in particular plastics and metals, is presented. The respective particles are stabilized by an amphiphilic, enzyme-degradable block copolymer, consisting of poly(ethylene glycol) and poly(caprolactone). After contact with the enzyme pseudomonas lipase, the particles are destabilized, due to the loss of the hydrophilic part of the block copolymer. The lipase is therefore immobilized on glass supports. Immobilization is performed via adsorption or covalent bonding to epoxide groups. All deposition experiments show that addressing of individual particles occurs precisely within the predefined areas of enzyme activity. Depending on the material and reaction conditions, intact nanoparticles or coatings from such can be gained. The quintessence of the study is the indifference of the EMA regarding particle materials. From this rationale, the technique offers near unlimited materials compatibility within a precise, easy-to-apply, and upscalable process. Full article

(This article belongs to the Special Issue Advances in the Design and Characterization of Heterogeneous Biocatalysts)

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