Matrix For Enzyme Immobilization Research Articles

Multivariate mesoporous MOFs with regulatable hydrophilic/hydrophobic surfaces as a versatile platform for enzyme immobilization

Zeolitic imidazole frameworks materials-8 (ZIF-8), a member of the metal-organic framework (MOFs) series, have been extensively used as a host matrix for enzyme immobilization. However, the microporous structure and hydrophobicity, as well as the protonation of the precursor 2-methylimidazole (2-MeIm) of ZIF-8, remain considerable challenges in maintaining the activity of immobilized enzymes. Here, novel multivariate mesoporous MOFs (mMOFs) with regulatable hydrophilic/hydrophobic surfaces were designed by the multivariate competitive strategy and pore modification engineering. 3-Methyl-1H-1,2,4-triazole (3-MTZ) and 5-methyltetrazole (5-MTA) were employed to partially replace 2-MeIm. These were then combined with zinc sulfate heptahydrate (soft templates) to yield mMOFs in a methanol solution. As a proof-of-concept application, we used mMOFs as carriers for enzyme immobilization and investigated the properties of the immobilized enzymes. Benefiting from their mesoporous structure, hydrophilic surface, and improved microenvironment, multivariate mMOFs exhibit a strong ability to stabilize enzyme conformation and increase enzyme activity compared with traditional ZIF-8. Our study offers an avenue for the controllable preparation of well-designed MOF structures, which will further broaden the application opportunities of MOF materials for enzyme immobilization.

Development of chia gum/alginate-polymer support for horseradish peroxidase immobilization and its application in phenolic removal

Chia gum’s molecular structure with distinctive properties as well as the alginate-based hydrogel’s three-dimensionally cross-linked structure can provide a potent matrix for immobilization of enzyme. Herein, chia gum (CG)/alginate (A)-polymeric complex was synthesized and employed as a support material for the immobilization of horseradish peroxidase (HRP). HRP was successfully immobilized on the developed ACG-polymeric support, and the highest immobilization recovery (75%) was observed at 1.0% CG and 2% A, pH 7.0, and 50 units of the enzyme. The structure, morphology, and thermal properties of the prepared ACG-HRP were demonstrated using Fourier Transform Infrared (FTIR), Scanning Electron Microscope, and Thermogravimetric (TGA) analyses. ACG-HRP showed a good reusability (60%) over ten reuses. The immobilized ACG-HRP displayed an acidic pH optimum (6.0), a higher temperature optimum (50 °C), and improved thermal stability (30–50 °C) compared to the soluble HRP at pH 7.0, 40 °C and (30–40 °C), respectively. ACG-HRP has a lower affinity for hydrogen peroxide (H2O2) and guaiacol and a higher oxidizing affinity for a number of phenolic substrates. The ACG-HRP demonstrated greater resistance to heavy metals, isopropanol, urea, Triton X-100, and urea, as well as improved efficiency for eliminating phenol and p-chlorophenol. The developed ACG-polymeric support provided improved enzyme properties, allowed the reuse of the immobilized HRP in 10 cycles, and made it promising for several biotechnological applications.

Enhanced Bioactivity of Enzyme/MOF Biocomposite via Host Framework Engineering.

This study reports the successful development of a sustainable synthesis protocol for a phase-pure metal azolate framework (MAF-6) and its application in enzyme immobilization. An esterase@MAF-6 biocomposite was synthesized, and its catalytic performance was compared with that of esterase@ZIF-8 and esterase@ZIF-90 in transesterification reactions. Esterase@MAF-6, with its large pore aperture, showed superior enzymatic performance compared to esterase@ZIF-8 and esterase@ZIF-90 in catalyzing transesterification reactions using both n-propanol and benzyl alcohol as reactants. The hydrophobic nature of the MAF-6 platform was shown to activate the immobilized esterase into its open-lid conformation, which exhibited a 1.5- and 4-times enzymatic activity as compared to free esterase in catalyzing transesterification reaction using n-propanol and benzyl alcohol, respectively. The present work offers insights into the potential of MAF-6 as a promising matrix for enzyme immobilization and highlights the need to explore MOF matrices with expanded pore apertures to broaden their practical applications in biocatalysis.

Correction: Electrochemical biosensor for monitoring fish spoilage based on nanocellulose as enzyme immobilization matrix

Correction: Electrochemical biosensor for monitoring fish spoilage based on nanocellulose as enzyme immobilization matrix

Immobilization of Tyrosinase and Its Application

Immobilized enzymes are more robust and resistant to environmental changes than free enzymes in solution. More crucially, the immobilized enzyme systems' heterogeneity enables facile recovery of enzymes and products, multiple reuses, continuous enzymatic processes, quick reaction termination, and a more comprehensive range of bioreactor designs. This paper examines recent findings on enzyme immobilization using diverse approaches for various uses. The information gathered from the reactions catalyzed by the encapsulated tyrosinase provided a good view of hetero-biocatalysts in the phenol biosensor industries. This review proposes an effective method for immobilizing tyrosinase biomolecules into a silica aerogel matrix. Silica matrix has been utilized to encapsulate a wide range of biomolecules, mainly in sol-gel composites. We also discovered that silica aerogel synthesized from sol-gel method retains all the immobilized enzyme activity. The use of a silica matrix for enzyme immobilization, in conjunction with a moderate immobilization method, results in the successful retention of enzyme activity. Future studies should explore practical encapsulating approaches and inventively modified supports to enhance the commercialization of immobilized enzymes and offer fresh perspectives to the industrial sector.

Electrochemical biosensor for monitoring fish spoilage based on nanocellulose as enzyme immobilization matrix

Electrochemical biosensor for monitoring fish spoilage based on nanocellulose as enzyme immobilization matrix

Microcontact printing of choline oxidase using a polycation-functionalized zwitterionic polymer as enzyme immobilization matrix.

Highly sensitive and selective choline microbiosensors were constructed by microcontact printing (μCP) of choline oxidase (ChOx) in a crosslinked, polyamine-functionalized zwitterionic polymer matrix on microelectrode arrays (MEAs). μCP has emerged as a potential means to create implantable, multiplexed sensor microprobes, which requires the targeted deposition of different sensor materials to specific microelectrode sites on a MEA. However, the less than sufficient enzyme loading and inadequate spatial resolution achieved with current μCP approaches has limited adoption of the method for electroenzymatic microsensors. A novel polymer, poly(2-methacryloyloxyethyl phosphorylcholine)-g-poly(allylamine hydrochloride) (PMPC-g-PAH), has been developed to address this challenge. PMPC-g-PAH contributes to a higher viscosity "ink" that enables thicker immobilized ChOx deposits of high spatial resolution while also providing a hydrophilic, biocompatible microenvironment for the enzyme. Electroenzymatic choline microbiosensors with sensitivity of 639 ± 96 nA μM-1 cm-2 (pH 7.4; n = 4) were constructed that also are selective against both ascorbic acid and dopamine, which are potential electroactive interfering compounds in the mammalian brain. The high sensitivities achieved can lead to smaller MEA microprobes that minimize tissue damage and make possible the monitoring of multiple neurochemicals simultaneously in vivo with high spatial resolution.

Recent Approaches on the Application of Agro Waste Derived Biocomposites as Green Support Matrix for Enzyme Immobilization

Recent Approaches on the Application of Agro Waste Derived Biocomposites as Green Support Matrix for Enzyme Immobilization

Conducting Matrices of the Poly(-O-Toluidine, PTOL) Film for Immobilizing Uricase

Polyaniline, PANI as conducting polymer has been used as enzyme immobilization matrix in biosensors due to its excellent stability and electrochromic effects, conductivity, and redox reversibility. However, at pH above 4, the electron transfer ability and redox activity decrease, henceit is necessary to modify the PANI structure to increase the PANI pH range. In this study, the PANI derivative, the poly(-o-toluidine, PTOL) film was produced for the uricase immobilization matrix. This study aimed to develop a supporting material that maintains catalytic activity uricase with a facile and inexpensive procedure. The PTOL was prepared by using the chemical oxidative polymerization technique with ammonium persulfate (dissolved in 1.0 M HCl) as an oxidizing agent. The FTIR spectra confirmed the success of PTOL formation by the appearance of its characteristic peaks. The UV-Vis spectra indicated that PTOL was in an emeraldine state with a semi-crystalline structure that was shown by its XRD pattern. While, SEM analysis described that the PTOL film was uniform with some granular aggregation in its surface morphology, but there is no holes or porosity were found. The use of PTOL film as a uricase matrix could maintain the uricase activity against the substrate (uric acid, UA) with excellent analytical characteristics. Therefore, PTOL film could be used as conducting matrices for immobilizing enzymes (e.g. biosensors).

Enhancement of Glucose Oxidase-Based Bioanode Performance by Comprising Spirulina platensis Microalgae Lysate

In this study, Spirulina platensis-based lysate was used as a biological redox mediator to design glucose oxidase (GOx) based biofuel cell bioanode. Chemically oxidized multi-walled carbon nanotubes (CNT) were deposited on carbon-electrode and were covered with eco-friendly algae cell-based lysate that facilitated the electron transfer and served as a biocompatible matrix for enzyme immobilization, which reduced the inactivation of GOx by CNT. The designed GC/PEI/CNT/S.p./GOx bioanode exhibited an open circuit potential (OCP) of −262 mV vs Ag/AgCl(3MKCl) in the presence of 12.5 mM of glucose. The maximum power output of the proposed bioanode was 21.8 times higher and reached 3.2 μW cm−2 at −51 mV vs Ag/AgCl(3MKCl) if the S. platensis cell lysate was used for bioanode design. The amperometric responses of GC/PEI/CNT/S.p./GOx bioelectrode towards the addition of glucose were linear at glucose concentrations ranging between 250 μM and 5 mM. These characteristics enable applying this bioanode as a part of biofuel cell and the electrode of an amperometric glucose biosensor, which response within 15 s, with a detection limit of 118 μM and a sensitivity of 15.09 μA mM−1 cm−2.

The Effect of Zeolite/Chitosan Hybrid Matrix for Thermal-stabilization Enhancement on the Immobilization of Aspergillus fumigatus α-Amylase

In this paper, the A. fumigatus α-amylase had been immobilized onto zeolite/chitosan hybrid to improve its thermal-stabilization for industrial needs. The methods applied enzyme production, isolation, partial purification, immobilization, and characterization. The optimum temperatures of the native and immobilized enzymes were 50 and 55˚C, respectively. The native enzyme has KM of 3.478 ± 0.271 mg mL-1 substrate and Vmax of 2.211± 0.096 µmole mL-1 min-1, while the immobilized enzyme has KM value of 12.051 ± 4.949 mg mL-1 substrate and Vmax of 1.602 ± 0.576 µmole mL-1 min-1. The residual activity of the immobilized enzyme retained up 10.97% after fifth reuse cycles. The native enzyme has ΔGi of 104.35 ± 1.09 kJ mole-1 and t½ of 38.75 ± 1.53 min, while the immobilized enzyme has ΔGi of 108.03 ± 0.05 kJ mole-1 and t½ of 180.03 ± 3.31 min. According to the increase in half-life (t½), stability improvement of the A. fumigatusα-amylase was 4.65 times greater than the native enzyme. Thus, the zeolite/chitosan hybrid is used as a new supporting matrix for further enzyme immobilization to stabilize the enzymes. Doi: 10.28991/ESJ-2022-06-03-06 Full Text: PDF

Thermo‐responsive macroporous p(NIPAM) cryogel affords enhanced thermal stability and activity for ɑ‐glucosidase enzyme by entrapping in situ

AbstractThe concept of using a macroporous thermo‐responsive poly(N‐isopropylacrylamide) (p(NIPAM)) polymer matrix for enzyme immobilization having lower critical solution temperature (LCST) is rationalized by the availability of the many compartments (pores) to entrap enzymes to operate within pores of three‐dimensional matrix providing special environmental conditions. Therefore, the ɑ‐glucosidase (ɑ‐G) immobilization within p(NIPAM) cryogel (ɑ‐G@p(NIPAM)) was carried out under the storage conditions of enzymes, generally ~ −20°C to afford the unnecessary loss of enzyme functionality in comparison to the other enzyme entrapment methods. The LCST value for the prepared p(NIPAM)‐based cryogels was determined as 34.8 ± 1.4°C. The immobilization yield, immobilization efficiency, and activity recovery% values were calculated as 89.4 ± 3.1, 66.2 ± 3.3, and 74.0% ± 3.3%, respectively, at pH 6.8 and 37°C for ɑ‐G@p(NIPAM) cryogel system. Interestingly, the optimum working conditions were attained as 25°C and pH 6.8 with higher activity, 98.4% ± 0.2% for the prepared ɑ‐G@p(NIPAM) cryogel system. The reuse and storage stability studies revealed that the prepared ɑ‐G@p(NIPAM) cryogel system is more effective than the native ɑ‐G enzyme; for example, it showed at least 80% activity after the fifth usage and provided higher activity up to a 10‐day room temperature storage time. Moreover, the kinetic parameters such as Km and Vmax of native ɑ‐G enzyme and ɑ‐G@p(NIPAM) cryogel system were calculated by non‐linear Lineweaver–Burk plot equations.

Preparation of immobilized lipase on Co2+-chelated carboxymethyl cellulose based MnFe2O4 magnetic nanocomposite particles

Lipase immobilization was performed via physical adsorption on a novel metal-chelating nanostructure consisted of (S)-N-(5-Amino-1-carboxypentyl) iminodiacetic acid (NTA), as a tetra-dentate chelating agent, grafted onto MnFe2O4 nanoparticles coated with carboxymethyl cellulose (CMC) for Co2+-chelated affinity lipase adsorption. The influential immobilization parameters for enzyme adsorption were investigated. The maximum amount of immobilized lipase (8.5 mg/g adsorbent) was obtained at modified conditions with initial enzyme concentration of 2 mg/mL and adsorbent dosage of 1 mg/mL after 90 min incubation at 50 mM buffer citrate at pH 5. The Fourier transform infrared spectroscopy (FTIR), energy dispersive spectroscopy (EDS), and elemental mapping provided evidence of successful immobilization of lipase. The results of kinetic study revealed that the adsorption of lipase followed pseudo second order kinetic model. Investigation on the enzymatic properties demonstrated that the immobilized lipase possesses remarkable thermal stability at 60 °C for 120 min, as the catalytic activity of immobilized lipase was 75% higher than that of free lipase at the same temperature. Beside, the immobilized lipase still retained 70% of its initial activity after 7 successive recycles, suggesting the new designed Co2+-chelated magnetic nanostructure as a great matrix for the non-covalent enzyme immobilization.

Electrochemical Analysis of Metabolites as a Method for Cytochromes P450 Activity Determination

The review deals with the electrochemical methods for determination of metabolites of cytochromes P450 catalyzed reactions. We have focused on the electrochemical determination of metabolites of drugs and some endogenous compounds. We have reviewed bielectrode systems for determination of cytochrome P450 activity, where one electrode serves as a matrix for enzyme immobilization and a source of electrons for heme iron ion reduction and initialization of the catalytic reaction towards a substrate and the second one is being used for quantification of the products formed by their electrochemical oxidation. Such systems allow one to elude additional steps of separation of reaction substrates and products. The review also includes discussion of the ways to increase the analytical sensitivity and decrease the limit of detection of the investigated metabolites by chemical modification of electrodes. We demonstrate the possibilities of these systems for cytochrome P450 kinetics analysis and the perspectives of their further improvement, such as increasing the sensitivity of metabolite electrochemical determination by modern electrode modificators, including carbon-based, and construction of devices for automatic monitoring of the products.

Determination of Glucose with Cellulose Acetate/Glucose Oxidase Modified Carbon Paste Electrodes

An amperometric biosensor for glucose determination was developed using a carbon paste electrode (CPE) modified with cellulose acetate (CA)/glucose oxidase (GOx) bilayer membrane through the electrostatic interaction between them. The CA membrane was used as matrix for enzyme immobilization via microencapsulation technique, is enzyme placed between two membranes. CA/GOx membrane was attached to CPE surface containing ferrocene (Fc). By using proposed amperometric biosensor, glucose concentration was determined as well as its characteristic. The modified Fc–graphite electrode with CA/GOx bilayer membrane for glucose had optimum measurement conditions at work emf of 874 mV, CA concentration of 10% and amount Fc of 0.021 mg. The biosensor showed good performance at glucose concentration range of 0.05–3 mM and limit of detection was 0.024 mM. Proposed biosensor has good reproducibility with relative standard deviation (RSD) was less than 5% up to 7 times use (in the defined condition 4 ˚C). Glucose measurement result in human serum of diabetes mellitus patients showed conformity with result of reference method, MediSense Optium glucose test kit.

Passive fuel delivery and efficient anoxic condition in anode improve performance of methanol biofuel cell

The operational stability, power efficiency, and technical simplicity of enzymatic biofuel cells are the focus area of research for practical applications of this green energy-generating device. Here, we report some critical findings on these issues on a methanol-fuelled pure biofuel cell fabricated by using alcohol oxidase and bilirubin oxidase as anodic and cathodic catalysts, respectively. The cell was fabricated with a new design strategy comprising efficient anoxic condition in the anodic chamber, adequate airflow to the cathode for enhancing oxygen reduction reactions, and a passive fuel pumping facility to the anode. A magnetic nanoparticle-based bio-nanocomposite matrix on the carbon-cloth electrode offered as a biocompatible enzyme immobilization matrix for harvesting electrons in the cell through the direct electron transfer mechanism as validated by cyclic voltammetry. Six units of the cells, when connected in a series, the device's potential increased to 4.3-fold (3.1 V) and rested at a stable state under a load with a half-life of ∼ 372 days and a coulombic efficiency of 60%. This high operational stability has been attributed to the efficient anoxic setup in the anodic chamber that supported the stability of alcohol oxidase, the activity of which was intact even after 49 days of the operation. This work also demonstrated that the prolonged interaction of molecular oxygen with the oxidase drastically inactivates it without affecting the structural integrity of the enzyme protein. This enzymatic fuel cell with improved design and functions is a step forward for achieving practical application as a standalone power supply to small-scale devices.

Dual Valorization of Lignin as a Versatile and Renewable Matrix for Enzyme Immobilization and (Flow) Bioprocess Engineering.

Lignin has emerged as an attractive alternative in the search for more eco‐friendly and less costly materials for enzyme immobilization. In this work, the terephthalic aldehyde‐stabilization of lignin is carried out during its extraction to develop a series of functionalized lignins with a range of reactive groups (epoxy, amine, aldehyde, metal chelates). This expands the immobilization to a pool of enzymes (carboxylase, dehydrogenase, transaminase) by different binding chemistries, affording immobilization yields of 64–100 %. As a proof of concept, a ω‐transaminase reversibly immobilized on polyethyleneimine‐lignin is integrated in a packed‐bed reactor. The stability of the immobilized biocatalyst is tested in continuous‐flow deamination reactions and maintains the same conversion for 100 cycles. These results outperform previous stability tests carried out with the enzyme covalently immobilized on methacrylic resins, with the advantage that the reversibility of the immobilized enzyme allows recycling and reuse of lignin beyond the enzyme inactivation. Additionally, an in‐line system also based on lignin is added into the downstream process to separate the reaction products by catch‐and‐release. These results demonstrate a fully closed‐loop sustainable flow‐biocatalytic system based exclusively on lignin.

Enzymatic Cascade Reactions Mediated by Highly Efficient Biomimetic Quasi Metal-Organic Frameworks.

The integration of chemo- and enzymatic catalysis for effective multistep cascades has presented critical challenges for decades. In this work, the biomimetic quasi NH2-MIL-101 (qNM) with highly efficient peroxidase-like activity was synthesized via a palmitic acid-induced strategy followed by pyrolysis. The effects of the amount of palmitic acid and calcination temperature on the synthesis of qNM were optimized. It was found that qNM was an excellent catalyst for oxidations of various peroxidase substrates, and a possible mechanism was proposed, i.e., the presence of FeII species in qNM was responsible for its excellent activity, which facilitated the transition between FeII and FeIII species to produce more hydroxyl radicals by H2O2 decomposition. The qNM served as the potential matrix for enzyme immobilization through a cross-linking method, and kinetic studies revealed that the catalytic efficiency (kcat/Km) for the immobilized GOx (23.7 mM-1 s-1) is comparable to that of free GOx (26.9 mM-1 s-1). The immobilized GOx also showed improved stability against high temperatures and organic solvents compared to free GOx, and analysis of the secondary structure of GOx indicated that the improved stability resulted from enzyme rigidity by the intense covalent linkage with qNM. Furthermore, qNM contributed its biomimetic activity to cooperate with a single enzyme (GOx) or two enzymes (β-Gal and GOx) for the enzymatic cascade reactions. Compared with the mixture of each component in the solution, the combination of the single-enzyme system (GOx) or the two-enzyme system (β-Gal and GOx) in qNM achieved 2.67-fold and 1.83-fold enhancements in the activity of catalytic cascades, respectively. This study provides new insights into the construction of effective and synergistic cascade reactions by integrating biomimetic MOF with natural enzyme, which holds potential for applications in biotechnology and ecofriendly and biomimetic catalysis.

Environmentally Friendly Preparation of Magnetic Composites Featuring Proteolytic Properties

The enzyme-containing magnetic composites are presented. The magnetic matrix for enzyme immobilization is obtained by sequential application of an amine-containing polysaccharide—chitosan and a synthetic polymer—poly(ethylene-alt-maleic acid) to the magnetite microparticles to form the interpolyelectrolyte complex shell. Then, the enzyme (trypsin) is immobilized by covalent or noncovalent binding. Thus, the suggested composites can be readily obtained in the environmentally friendly manner. The enzyme capacity of the resulting composites reaches 28.0–32.6 mg/g. The maximum hydrolysis rates of the H-Val-Leu-Lys-pNA substrate provided by these composites range within 0.60·10–7–0.77·10–7 M/min.

Reed Membrane as a Novel Immobilization Matrix for the Development of an Optical Phenol Biosensor

Background: Biocompatible and easily available immobilization matrix is vital for the construction of enzyme-based biosensor. Methods: Reed membrane was selected as a novel immobilization matrix to construct an optical phenol biosensor. Tyrosinase from mushroom was immobilized in a reed membrane using glutaraldehyde as a cross-linker. The detection scheme was based on the measurement of the color intensity of the adduct resulting from the reaction of 3-methyl-2-benzothiazolinone hydrazone (MBTH) with the quinone produced from the oxidation of phenol by tyrosinase. The performance of such method including specificity, sensitivity, repeatability and practical use were validated. Results: The prepared biosensor demonstrated optimum performance at pH 6-7, temperature of 40°C and a linear response in the phenol concentration range of 5-100 μM. It also showed good operation stability for repeated measurements (over 200 times) and good storage stability after it had been kept at 4°C for 2 months. Conclusion: Reed membrane is a novel matrix for immobilization of enzyme. The prepared biosensor permits good sensitivity, reproducibility and stability. It is anticipated that reed membrane is a promising solid support for fabricating other enzyme-based biosensors.