Covalent Immobilization Of Enzymes Research Articles

Enhancing Enzyme Stability and Functionality: Covalent Immobilization of Trypsin on Magnetic Gum Arabic Modified Fe3O4 Nanoparticles.

This study aimed to fabricate gum Arabic (GA)-coated Fe3O4 nanoparticles bearing numerous active aldehyde groups on their surface, followed by an assessment of their capability as a magnetic support for the covalent immobilization of the trypsin enzyme for the first time. FT-IR, XRD, TGA, and SEM results demonstrated the successful synthesis of GA-coated Fe3O4 nanoparticles, along with the covalent immobilization of the enzyme onto the support. Immobilization enhanced the relative enzymatic activity across a range of aqueous solution pH levels (ranging from 4 to 11) and temperatures (ranging from 20 to 80 °C) without altering the optimum pH and temperature for trypsin activity. Kinetic studies using Michaelis-Menten plots revealed changes in kinetic parameters, including a lower Vmax and higher Km for immobilized trypsin compared to the free enzyme. The immobilization onto magnetic gum Arabic nanoparticles resulted in an improved stability of trypsin in the presence of various solvents, maintaining a stability order comparable to that of the free enzyme due to the stabilizing effect of the support. The reusability results showed that the immobilized enzyme can retain over 93% of its activity for up to 15 cycles.

Upgrading Epoxy Supports for Enzyme Immobilization by Affinity Function Doping—A Case Study with Phenylalanine Ammonia-Lyase from Petroselinum crispum

This article provides a method to upgrade epoxy-functionalized carriers for covalent enzyme immobilization to selective carriers suitable for covalent immobilization of metal affinity-tagged enzymes without the need of preliminary enzyme purification. Affinity function doping of the epoxy-functionalized surface introduces an advanced possibility to avoid the costly and time-consuming downstream processes required for efficient immobilization on non-selective epoxy carriers. Our approach is based on the partial functionalization of surface epoxides via a proper diamine-derived linker and an ethylenediaminetetraacetic dianhydride-based chelator charged with cobalt ions. The solid macroporous carriers, doped with metal affinity functions, have both coordinative binding ability (rapid anchoring the metal affinity-tagged enzymes to the surface) and subsequent covalent bond-forming ability (preferred binding of the tagged enzyme to the surface after proper washing by the residual epoxide functions), enabling a single operation for the enrichment and immobilization of a recombinant phenylalanine ammonia-lyase from parsley fused to a polyhistidine affinity tag. The immobilized PcPAL was applied in the ammonia elimination of racemic phenylalanine, 4-chlorophenylalanine, and 4-bromophenylalanine to produce the corresponding d-phenylalanines, in addition to the formation of (E)-cinnamates, as well as in ammonia addition reactions to (E)-cinnamates, yielding the corresponding enantiopure l-phenylalanines.

Urease immobilized poly(AAm-AGE) cryogel for depletion of urea from human serum

Polyacrylamide-allyl glycidyl ether [poly(AAm-AGE)] cryogel monolith was prepared for covalent immobilization of urease enzyme and aimed for usage of urea depletion from human serum by immobilized urease. Immobilization was succeeded via epoxy groups of allyl glycidyl ether with an immobilization yield of 71.9 ± 2.65%. Characterization was performed by Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscopy (SEM), Energy Dispersive X-Ray (EDX) analysis and swelling analysis to reveal functionalities and morphological, elemental and macroporous structure of the cryogel. Effects of pH, temperature and urea concentration on catalytic activity profiles of free and immobilized urease were investigated. Thermal and storage stabilities of free urease were enhanced upon immobilization. Additionally, urea removal from artificial human serum was achieved by 92% and immobilized urease was reusable up to ten reuses.

Magnetic macroporous chitin microsphere as a support for covalent enzyme immobilization

In this study, a novel magnetic macroporous chitin microsphere (MMCM) was developed for enzyme immobilization. Chitin nanofibers were prepared and subsequently subjected to self-assembly with magnetic nanoparticles and PMMA (polymethyl methacrylate). Following this, microspheres were formed through spray drying, achieving a porous structure through etching. The MMCM serves as an effective support for immobilizing enzymes, allowing for their covalent immobilization both on the microsphere's surface and within its pores. The substantial surface area resulting from the porous structure leads to a 2.1-fold increase in enzyme loading capacity compared to non-porous microspheres. The MMCM enhances stability of the immobilized enzymes under various pH and temperature conditions. Furthermore, after 20 days of storage at 4 °C, the residual activity of the immobilized enzyme was 2.93 times that of the free enzyme. Even after being recycled 10 times, the immobilized enzyme retained 56.7 % of its initial activity. It's noteworthy that the active sites of the enzymes remained unchanged after immobilization using the MMCM, and kinetic analysis revealed that the affinity of the immobilized enzymes rivals that of the free enzymes.

Rational design of biocatalysts based on covalent immobilization of acylase enzymes

Acylases catalyze the hydrolysis of amide bonds. Penicillin G acylase (PGA) is used for the semi-synthesis of penicillins and cephalosporins. Although protein immobilization increases enzyme stability, the design of immobilized systems is difficult and usually it is empirically performed. We describe a novel application of our strategy for the Rational Design of Immobilized Derivatives (RDID) to produce optimized acylase-based immobilized biocatalysts for enzymatic bioconversion. We studied the covalent immobilization of the porcine kidney aminoacylase-1 onto aldehyde-based supports. Predictions of the RDID1.0 software and the experimental results led to the selection of glyoxyl-Sepharose CL 4B support and pH 10.0. One of the predicted clusters of reactive amino groups generates an enzyme-support configuration with highly accessible active sites, contributing with 82% of the biocatalyst’s total activity. For Escherichia coli PGA, the predictions and experimental results show similar maximal amounts of immobilized protein and activity at pH 8.0 and 10.0 on glyoxyl-Sepharose CL 10B. However, thermal stability of the immobilized derivative is higher at pH 10.0 due to an elevated probability of multipoint covalent attachment. In this case, two clusters of amino groups are predicted to be relevant for PGA immobilization in catalytically competent configurations at pH 10.0, showing accessible active sites and contributing with 36% and 44% of the total activity, respectively. Our results support the usefulness of the RDID strategy to model different protein engineering approaches (site-directed mutagenesis or obtainment of fusion proteins) and select the most promising ones, saving time and laboratory work, since the in silico-designed modified proteins could have higher probabilities of success on bioconversion processes.

Continuous fixed-bed column studies to remove polycyclic aromatic hydrocarbons by degrading enzymes immobilized on polyimide aerogels

The main sources of polycyclic aromatic hydrocarbons (PAHs), entering aquatic environments are industrial discharges, petroleum spills, combustion of fossil fuels, urban runoff, and atmospheric deposition. For the biodegradation of PAHs, the use of biocatalysts, such as enzymes, is an environmentally friendly method. Despite this, it is necessary to immobilize the enzymes to facilitate their recovery and reusability, as well as to prevent their loss. Using covalent bonding, (PAHs) degrading enzymes including naphthalene and catechol 2,3 dioxygenase enzymes were immobilized on modified polyimide aerogels. Covalent immobilization of enzymes on surface-modified polyimide aerogels resulted in around 9- and 6-fold lower enzyme leaching for naphthalene and catechol 2,3 dioxygenase enzymes compared to enzyme immobilization using adsorption. The Fourier Transform Infrared Spectrum (FT-IR) confirmed the enzyme immobilization and aerogel modification. SEM images indicated completely different textures for surface-modified aerogel attributed to the presence of functional groups after surface modification that enhanced enzyme loading. The removal capacity (∼20mganthracenegaerogel) of enzyme loaded aerogel was determined to select an appropriate range for column tests. Finally, the effects of flow rate, size of aerogels and inlet concentration of anthracene on the removal efficiency of pollutants were examined. Using the derived model as a basis for prediction, in terms of removal efficiency, the highest result was achieved to be 84.01% at a flow rate of 22 mL min−1, initial concentration of 34 mg L−1and aerogel size of 2 cm while under these conditions, the removal efficiency was experimentally measured to be 87.14 %. A fixed-bed column packed with enzyme-loaded aerogel for the removal of PAHs provides novel insight into the application of aerogel base materials for water treatment and PAH removal with possibilities of scaling up for larger applications.

Isocyanide-based multi-component reactions for carrier-free and carrier-bound covalent immobilization of enzymes.

Strategies for the covalent immobilization of enzymes depend on the type of functional group selected to perform the coupling reaction, and on the relative importance of selectivity, loading capacity, immobilization time and activity/stability of the resulting immobilized preparation. However, no single strategy is applicable for all covalent immobilization methods or can meet all these criteria, exemplifying the challenge of introducing a versatile process broadly compatible with the residues on the surface of proteins and the functional groups of common linkers. Here, we describe the use of isocyanide-based multi-component reactions for the carrier-bound and carrier-free covalent immobilization of enzymes. Isocyanide-based multi-component reactions can accept a wide variety of functional groups such as epoxy, acid, amine and aldehyde, as well as many commercially available bi-functional linkers, and are therefore suitable for either covalent coupling of enzymes on a solid support or intermolecular cross-linking of enzymes. In this strategy, an isocyanide is directly added to the reaction medium, the enzyme supplies either the exposed amine or carboxylic acid groups, and the support (in carrier-bound immobilization) or the bi-functional cross-linking agent (in carrier-free immobilization) provides another reactive functional group. The protocol offers operational simplicity, high efficiency and a notable reduction in time over alternative strategies, and can be performed by users with expertise in chemistry or biology. The immobilization reactions typically require 1-24 h.

Polydopamine-assisted one-step immobilization of lipase on α-alumina membrane for fouling control in the treatment of oily wastewater

Covalent enzyme immobilization is generally a time-consuming and multistep procedure that uses toxic solvents and requires more than one chemical, making industrial upscaling unattractive. Using an aqueous polydopamine (PDA) solution for enzyme immobilization is a greener alternative. Usually, enzyme immobilization using PDA is performed in two steps: dopamine polymerization on the material surface followed by enzyme immobilization. A few recent studies applied a one-step strategy by mixing dopamine and enzyme in the coating solution, reducing the immobilization time, chemical consumption, and wastewater generation. This study compares the two-step and one-step approaches to immobilizing the lipase Eversa Transform 2.0 (ET2) on an α-alumina membrane. The one-step immobilization method achieved similar enzyme loading, membrane hydrolytic activity, and enzyme-specific activity to those of the two-step method. The ET2 immobilized using both strategies showed excellent fouling resistance and self-cleaning performance in oily wastewater filtration. The membrane modified by the one-step approach exhibited a lower reduction in pure water permeance after oil fouling (35%) and a higher permeance recovery (90%) than the one modified by the two-step method (40% and 74%, respectively). This better performance can be due to the higher hydrophilicity of the modified membrane and higher stability over reaction time shown by the enzyme immobilized by the one-step strategy. The higher stability can be attributed to more attachment points between the enzyme and PDA, increasing the enzyme rigidity and preventing conformational changes.

Plasma polymerized functional supermagnetic Fe3O4 nanostructured templates for laccase immobilization: A robust catalytic system for bio-inspired dye degradation.

Fe3O4 magnetic nanoparticles, synthesized using co-precipitation method, were epoxy functionalized via plasma polymerization of 2,3-epoxypropylmethacrylate (EPMA) precursor. The EPMA-functionalized Fe3O4 nanoparticles (EPMA-f-MN) were employed as templates for facile, one-step covalent immobilization of laccase enzyme at room temperature. Samples were rigorously characterized by FTIR, TGA, SEM, TEM, XRD techniques, while Mössbauer spectroscopy (MöS) and vibrating sample magnetometry (VSM) confirmed the supermagnetic nature of Fe3O4 nanoparticles. Activities of free and immobilized laccase (ImLac) were assayed by spectrophotometrically monitoring the enzymatic reduction of substrate 2,2-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS) at 420 nm, corresponding to the λmax of ABTS.+. In addition to possessing higher thermal stability and a broader pH tolerance window compared to free laccase, the supermagnetic property of the Fe3O4 renders the ImLac system conveniently recoverable and recyclable. Practical applicability of ImLac towards catalytic degradation of industrial dyes was also ably demonstrated using Acid Blue 193 (AB 193) as a commercially used model textile dye, which belongs to the family of azo dyes. Over 95% degradation of the dye was achieved within a period of 4 hours. ImLac could be used for more than 10 dye degradation cycles with >90 % of retention in enzyme activity.

Assessing the predictive capabilities of design heuristics and coarse-grain simulation toward understanding and optimizing site-specific covalent immobilization of β-lactamase.

For industrial applications, covalent immobilization of enzymes provides minimum leakage, recoverability, reusability, and high stability. Yet, the suitability of a given site on the enzyme for immobilization remains a trial-and-error procedure. Here, we investigate the reliability of design heuristics and a coarse-grain molecular simulation in predicting the optimum sites for covalent immobilization of TEM-1 β-lactamase. We utilized Escherichia coli-lysate-based cell-free protein synthesis (CFPS) to produce variants containing a site-specific incorporated unnatural amino acid with a unique moiety to facilitate site directed covalent immobilization. To constrain the number of potential immobilization sites, we investigated the predictive capability of several design heuristics. The suitability of immobilization sites was determined by analyzing expression yields, specific activity, immobilization efficiency, and stability of variants. These experimental findings are compared with coarse-grain simulation of TEM-1 domain stability and thermal stability and analyzed for a priori predictive capabilities. This work demonstrates that the design heuristics successfully identify a subset of locations for experimental validation. Specifically, the nucleotide following amber stop codon and domain stability correlate well with the expression yield and specific activity of the variants, respectively. Our approach highlights the advantages of combining coarse-grain simulation and high-throughput experimentation using CFPS to identify optimal enzyme immobilization sites.

2,5-Furandicarboxaldehyde as a bio-based crosslinking agent replacing glutaraldehyde for covalent enzyme immobilization.

In the quest for a bio-based and safer substitute for glutaraldehyde, we have investigated 2,5 diformylfuran (DFF) as bifunctional crosslinking agent for the covalent immobilization of glucoamylase on amino-functionalized methacrylic resins. Immobilization experiments and systematic comparison with glutaraldehyde at four different concentrations for the activation step showed that DFF leads to comparable enzymatic activities at all tested concentrations. Continuous flow experiment confirms a similar long term stability of the immobilized formulations obtained with the two crosslinkers. The NMR study of DFF in aqueous solution evidenced a much simpler behaviour as compared to glutaraldehyde, since no enolic forms can form and only a mono-hydrated form was observed. Unlike in the case of glutaraldehyde, DFF reacts covalently with the primary amino groups via imine bond formation only. Nevertheless, the stability of the covalent immobilization was confirmed also at acidic pH (4.5), most probably because of the higher stability of the imine bonds formed with the aromatic aldehydes. In terms of toxicity DFF has the advantage of being poorly soluble in water and, more importantly, poorly volatile as compared to glutaraldehyde, which displays severe respiratory toxicity. We have performed preliminary ecotoxicity assays using Aliivibrio fischeri, a marine bacterium, evidencing comparable behaviour (below the toxicity threshold) for both dialdehydes at the tested concentrations.

Synthesis of luminescent core–shell polymer particles carrying amino groups for covalent immobilization of enzymes

Synthesis of luminescent core–shell polymer particles carrying amino groups for covalent immobilization of enzymes

Assessment of Enzymatic Bioremediation for Catechol in Water by the Role of Immobilized Catechol 1,2-dioxygenase on Biosilica of Diatoms Frustule and other Polymers

The highest specific activity for (C12O) enzyme purification was within the second stage of the purificationsteps represented by precipitation with ammonium sulfate AS 2% (weight / volume), reaching about 14.56U. mg-1 after achieving the purification by 6.6 folds with a recovery rate of 78.3%. The optimum catalyticactivity of the enzyme (C12O) was within the pH of 6.5-8.5 and the temperature of 25 ° C. Immobilization of(C12O) enzyme by biosilica of diatom frustules DFS/G GA gave a double shifting of 20 ° C from the optimumtemperature for enzyme activity, within all the purification stages, as the percentage of catechol degradationthrough it at the temperature of 45 ° C reached about 93% and 87.2% and 85% were in the precipitationstage with ammonium sulfate AS and the precipitation with streptomycin sulfate SS and the enzymatic crudeextract CEE respectively. While the shifting was only 10 ° C in the case of immobilization on polymericsupports types: PAN / G and PAA / G. In contrast, the soluble (C12O) enzyme which did not show anyshifting by its optimum temperature , and its highest activity values were within the optimum temperatureof 25 ° C. While the method of immobilization for (C12O) enzyme by biosilica of diatom frustules DFS/Ggave a 1.5-degree shifting for the optimum pH of the enzyme activity, within all the purification stages, thehighest rates of catechol degradation were reached through it at the base pH 9-10 about 91.8% and 86.5. %And 85.0% in the AS, CEE and SS stages, respectively. Whereas, the enzyme immobilization methods of(C12O) by PAN / G and PAA / G did not show such displacement during the enzymatic purification stagewith streptomycin sulfate SS but were limited to the primarily stages of enzymatic purification. , As it wasable to shift the optimal pH of the (C12O) enzyme by 1.5 degrees also within the purification stages of ASand CEE. The rates of catechol biodegradation through it at the base pH 9-10 were about 79.0% and 71.8%,respectively, in the case of immobilization by PAN /G and PAA / G with percentages of 71.8% and 62.0%,respectively. In contrast, soluble (C12O) enzyme did not show any shifting by its optimum pH, and itshighest activity values were within the optimum pH of 6.5-8.5. The reason for these results may be attributedto the role of the large surface area ready for covalent immobilization of (C12O) enzyme by biosilica ofdiatom frustules compared with the surface area of pores of the two types of polymeric membranes used incurrent study.

Peroxidase driven micromotors for dynamic bioremediation

Phenolics are size products present in tons concentrations in industrial wastewater that can cause adverse health effects when released in the environment. As such, there is a growing interest in the development of efficient strategies for the removal of phenolic compounds from polluted water. Herein we describe the use of poly(3,4-ethylenedioxythiophene) (PEDOT)-Au/peroxidase micromotors as dynamic biocatalytic platforms for the removal of model phenolics (phenol, bisphenol A, guaiacol, pyrogallol and catechol). Micromotors are synthetized by using a simplified template electrodeposition protocol followed by covalent enzyme immobilization in the inner Au layer. Kinetic parameters revealed that enzyme immobilization in the inner micromotor layer increased over 2-fold the enzymatic activity, along with increasing operational pH and thermal stabilities. The micromotors can propel at speed of up to 60 µm/s, generating an enhanced fluid mixing that results in removal efficiencies of up to 60% as compared with the 27% removal when using free peroxidase under the same conditions. In addition, excellent activities of almost 100% were obtained within ten cycles of removal using the micromotors. This newly developed bioremediation strategy holds considerable promise in for its application in large scale water treatment systems and many relevant environmental processes.

Microporous regenerated cellulose-based macrogels for covalent immobilization of enzymes

In this study, regenerated cellulose-based macrogels with abundant carboxyl groups and interconnected microporous structures were synthesized from cellulose fibers (CFs)/cellulose nanofibers (CNFs) and utilized as stable, efficient, and recyclable platforms for enzyme immobilization. CNFs with different carboxyl contents were prepared by a TEMPO (2, 2, 6, 6 -tetramethylpiperidine-1-oxyl radical)-medium oxidation system. The cellulose-based macrogels were prepared by dissolving CFs and CNFs in a NaOH/urea aqueous solution and a followed extrusion process. The carboxyl groups within the macrogels were then activated by an N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC·HCl)/Nhydroxysuccinimide (NHS) system, which provides these macrogels with efficient binding sites for enzyme immobilization. The three-dimensional network structures of the macrogels facilitate the enzymes to infiltrate into the core of the macrogels and be fixed on the abundant pore walls. The immobilized enzymes maintain activity at a higher pH than free enzymes. Moreover, the immobilized enzymes have higher temperature tolerance, and their optimal reaction temperature is about 10 °C higher than that of free enzymes. The immobilized enzymes retain 24% of the initial activity after repeated use for 7 times. The microporous regenerated cellulose-based macrogels show potential in the fields of enzyme immobilization and biocatalysis.

Effect of amine length in the interference of the multipoint covalent immobilization of enzymes on glyoxyl agarose beads

Trypsin, chymotrypsin, penicillin G acylase and ficin extract have been stabilized by immobilization on glyoxyl agarose, adding different aliphatic compounds bearing a primary amine group during the immobilization: ethyl amine, butyl amine, hexyl amine (at concentrations ranging from 0 to 20 mM) and octyl amine (from 0 to 10 mM) to analyze their effects on the immobilized enzyme stability. As expected, the presence of amines reduced the intensity of the enzyme-support multipoint covalent attachment, and therefore the enzyme stability. However, it is clear that this effect is higher using octyl amine for all enzymes (in some cases the enzyme immobilized in the presence of 10 mM octyl amine was almost inactivated while the reference kept over 50 % of the initial activity). This way, it seems that the most important effect of the presence of aminated compounds came from the generation of steric hindrances to the enzyme/support multi-reaction promoted by the ammines that are interacting with the aldehyde groups. In some instances, just 1 mM of aminated compounds is enough to greatly decrease enzyme stability. The results suggested that, if the composition of the enzyme extract is unknown, to eliminate small aminated compounds may be necessary to maximize the enzyme-support reaction.

Immobilization of enzyme cocktails on dopamine functionalized magnetic cellulose nanocrystals to enhance sugar bioconversion: A biomass reusing loop

A combined enzymatic treatment/acid hydrolysis technique was utilized to synthesize cellulose nanocrystals (CNCs) from sugar beet pulp. CNCs were functionalized with magnetite nanoparticles and dopamine making a versatile nano-carrier (DA/Fe3O4NPs@CNCs) for covalent enzyme immobilization. Oxygene/amine functionalities, high magnetization value, and specific surface area of DA/Fe3O4NPs@CNCs made it a reusable and green candidate for conjugation to hydrolytic enzyme cocktails (three cellulases, two hemicellulases, and their combinations) to prepare an innovative and practical nano-biocatalyst for biomass conversion. The conjugated enzymes showed an enhanced optimum temperature (∼ 10 °C), improved thermal stability, and shifted optimum pH toward alkaline pHs. Covalent attachment could successfully suppress the enzyme leaching and provide easy recovery/reuse of the nano-biocatalyst up to 10 cycles, with > 50% of initial activity. Application of the nano-biocatalyst in hydrolysis of rice straw and sugar beet pulp showed an increase (20-76%) in the yield of fermentable sugars compared to the free enzyme cocktails.

Paper-Based Lactate Biofuel Cell Using Poly(glycidyl methacrylate)-Grafted MgO-Templated Carbon

Enzymatic lactate/O2 biofuel cells (BFCs) are energy conversion systems in which enzymes oxidize lactate at a bioanode and reduce oxygen at a biocathode. These enzymes are highly safe toward humans, and highly selective for substrates. In addition, lactate is contained abundant in a human sweat. Thus, the lactate/O2 BFCs have been attracting attention as a next-generation wearable power source1).We have produced a paper-based lactate biofuel cell in which lactate oxidase (LOx) and a mediator are physically absorbed on MgO-templated carbon (MgOC) having mesopores suitable for enzyme2).By using MgOC for electrode, long-term stability may be improved because the elution of the enzyme is suppressed by entrapping the enzyme in the mesopore3). On the other hand, covalent immobilization of mediators and enzymes seems to be effective for further improvement of stability of the electrodes. We recently reported that poly(glycidyl methacrylate) (poly(GMA)) bearing pendant glycidyl groups, grafted on the surface of MgO-templated carbon (GMgOC), was useful for forming strong multipoint covalent bonds with amino functional groups on the surface of flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) molecules4). The stability of FAD-GDH-immobilized GMgOC electrode showed good stability with a simple immobilization scheme by suppressing elution of FAD-GDH from the electrode surface.In this study, we newly fabricated a paper-based lactate biofuel cell using a GMgOC electrode formed by screen printing. A lactate biofuel cell anode, in which Azure A, as a mediator, was covalently immobilized on the GMgOC. LOx was also immobilized in the GMgOC. We evaluated the output current and the stability of the electrode. The synthesis reaction of the GMgOC is shown in Fig. 1(a). MgOC was irradiated with an electron beem to generate radicals, and then glycidyl methacrylate was introduced to form GMgOC on the electrode surface. Polyvinylidene difluoride(PVdF) as a binder and N-methyl-2-pyrrolidinone(NMP) as a solvent were added to GMgOC as a carbon ink.The paper-based carbon electrodes were fabricated by screen printing. A carbon leads and MgOC ink was printed on a water-repellent paper substrate, successively. The AzureA as a mediator and LOx as an enzyme were dropped on the porous carbon electrode surface. Chronoamperometry was performed using the prepared bioanode. The measurement was performed by the three-electrode method using a platinum wire as the counter electrode and a saturated KCl/Ag/AgCl electrode as the reference electrode, respectively. The chlonoamperometry was performed in 1 mol dm-3 phosphate buffer containing 0.1 mol dm-3 lactate. The potential was set at 0.1 V vs. Ag/AgCl.Figure 1(b) shows the cyclic voltammetry of the GMgOC electode. A catalytic oxidation current was observed at a nobler potential than -0.1 V. The peak current value of the present enzyme electrode was larger than that of an enzyme electrode in which MgOC was used as the electrode material. The results indicated that the use of GMgOC will obtain a larger peak value because of immobilizing mediator and enzymes makes electrons to transport more efficiently to the electrode.A chronoamperograms using GMgOC and MgOC electrodes were measured at 0.1 V. Figure 1(c) shows that the current value of MgOC was 0.09 mA cm-2 after 30000 s, and that of GMgOC was 1.7 mA cm-2. It was found to exhibit that the current density was about twice as high as MgOC. It’s because, mediators and enzymes are immobilized by physical adsorption on the carbon surface when using MgOC, while elution of LOx could be suppressed by using GMgOC because the amino group of the enzyme shell and the epoxy group on the carbon surface are bonded successfully.Reference1) E. Katz et al., J. Electroanal. Chem., 479 (1999) 64.2) I. Shitanda et al., J. Electrochem. Soc, 166(12) (2019) B1063-B1068.3) S. Tsujimura et al., Electrochemistry 83 (2015) 372.4) I. Shitanda, T. Kato et al., Bull. Chem. Soc. Jpn., 93 (2020) 32.AcknowledgmentsThis work was partially supported by JST-ASTEP Grant Number JPMJTS1513, JSPS Grant Number 17H02162 and Private University Research Branding Project (2017-2019) from Ministry of Education, Culture, Sports, Science and Technology, and Tokyo University of Science Grant for President's Research Promotion． Figure 1

Silk Fibroin-Based Materials for Catalyst Immobilization.

Silk fibroin is a widely and commercially available natural protein derived from silkworm cocoons. Thanks to its unique amino acid composition and structure, which lead to localized nanoscale pockets with limited but sufficient hydration for protein interaction and stabilization, silk fibroin has been studied in the field of enzyme immobilization. Results of these studies have demonstrated that silk fibroin offers an important platform for covalent and noncovalent immobilization of enzymes through serving as a stabilization matrix/support with high retention of the biological activity of the enzymes of interest. In the hope of providing suggestions for potential future research directions, this review has been written to briefly introduce and summarize key advances in silk fibroin-based materials for immobilization of both enzymes/biocatalysts (including alkaline phosphatase, β-glucosidase, glucose oxidase, lipase, urease, uricase, horseradish peroxidase, catalase, xanthine oxidase, tyrosinase, acetylcholinesterase, neutral protease, α-chymotrypsin, amylase, organophosphorus hydrolase, β-galactosidase, carbonic anhydrase, laccase, zymolyase, phenylalanine ammonia-lyase, thymidine kinase, and several others) and non-enzymatic catalysts (such as Au, Pd, Fe, α-Fe2O3, Fe3O4, TiO2, Pt, ZnO, CuO, Cu2O, Mn3O4, and MnO2).

Stabilization and optimization of purified diamine oxidase by immobilization onto activated PVC membrane

ABSTRACT The covalent immobilization of enzymes is required for most biocatalytic processes as it can broaden their applicability in various workflows. Diamine oxidase (DAO) has found important utility in food and allied industries for protection of food freshness and safety. In this study, an activated PVC strip was developed, covalently immobilized to purified DAO from pea seedlings. A comparative investigation was done on the parameters affecting catalytic activity of the free and immobilized enzyme, such as pH, temperature, and enzyme concentration. The immobilization preserved 81% of the initial enzyme activity against the substrate, putrescine dihydrochloride. The optimal pH levels of free and immobilized DAO were 7.0 and 6.5, respectively. The highest activity of the immobilized DAO was observed at 40°C while 34°C for the free enzyme. Moreover, the immobilized DAO retained about 52% of its initial activity after 10 repetitive uses and the activity was maintained after 30 days at 4°C. Therefore, this strategy may provide an excellent support for enzyme immobilization having better catalytic ability and operational stability than its free counterpart. Eventually, the results observed can be used further for various applications such as in food industry, to evaluate the freshness of real samples, and in biotechnological field to fabricate specific biosensors to detect biogenic amines content for evaluating food hygienic quality. Thus, the prepared enzyme catalyst presents a new approach for successful industrial applications.