Immobilization Of Dehydrogenase Research Articles

One‐pot immobilization of formate dehydrogenase on silica nanoparticles based on catechol chemistry with or without polyethyleneimine

AbstractConverting carbon dioxide into formic acid with formate dehydrogenase (FDH) as a catalyst is a gentle and eco‐friendly process that not only conserves energy and reduces emissions but transforms waste into valuable resource. Enzyme immobilization is a common strategy for improving the stability and recyclability of enzyme, however, the procedures are usually complicated that include surface modification and binding reactions in order to form reliable linkages. Co‐deposition of polydopamine (PDA) with polyethyleneimine (PEI) is a popular surface modification approach in versatile fields, but their co‐deposition with enzymes is rarely reported. Herein, one‐pot immobilization of FDH on SiO2 nanoparticles based on catechol chemistry was attempted for the first time with or without PEI, and the resultant immobilized enzymes, FDH/PDA/PEI‐SiO2 and FDH/PDA‐SiO2, were characterized, and the effects of the composition and deposition conditions on the catalytic performance were investigated. FDH/PDA‐SiO2 exhibited a higher apparent specific activity than FDH/PDA/PEI‐SiO2, which was separately 0.167 and 0.136 mM/h/mg. The activity recovery rate was 464% and 378% of that of free enzyme. After 5 cycles, their relative activity was 82.3% and 87.4%. This research is significant in presenting a simple and high‐efficient one‐pot immobilization method that can be extended to other kinds of enzymes.

Catalytic Synthesis of (S)-CHBE by Directional Coupling and Immobilization of Carbonyl Reductase and Glucose Dehydrogenase.

Ethyl (S)-4-chloro-3-hydroxybutyrate ((S)-CHBE) is an important chiral intermediate in the synthesis of the cholesterol-lowering drug atorvastatin. Studying the use of SpyTag/SpyCatcher and SnoopTag/SnoopCatcher systems for the asymmetric reduction reaction and directed coupling coenzyme regeneration is practical for efficiently synthesizing (S)-CHBE. In this study, Spy and Snoop systems were used to construct a double-enzyme directed fixation system of carbonyl reductase (BsCR) and glucose dehydrogenase (BsGDH) for converting 4-chloroacetoacetate (COBE) to (S)-CHBE and achieving coenzyme regeneration. We discussed the enzymatic properties of the immobilized enzyme and the optimal catalytic conditions and reusability of the double-enzyme immobilization system. Compared to the free enzyme, the immobilized enzyme showed an improved optimal pH and temperature, maintaining higher relative activity across a wider range. The double-enzyme immobilization system was applied to catalyze the asymmetric reduction reaction of COBE, and the yield of (S)-CHBE reached 60.1% at 30 °C and pH 8.0. In addition, the double-enzyme immobilization system possessed better operational stability than the free enzyme, and maintained about 50% of the initial yield after six cycles. In summary, we show a simple and effective strategy for self-assembling SpyCatcher/SnoopCatcher and SpyTag/SnoopTag fusion proteins, which inspires building more cascade systems at the interface. It provides a new method for facilitating the rapid construction of in vitro immobilized multi-enzyme complexes from crude cell lysate.

Novel Approach for the Immobilization of Cellobiose Dehydrogenase in PEDOT:PSS Conductive Layer on Planar Gold Electrodes

Third-generation biosensors use enzymes capable of direct electron transfer (DET) to the sensor surface. They are of interest for continuous glucose monitoring in blood or interstitial fluid, but they are rarely investigated. One reason is the hindered DET of the enzymes to the metallic electrodes. In this publication, a novel method for the immobilization of cellobiose dehydrogenase (CDH) DET enzymes employing conductive poly(3,4-ethylenedioxythiophene)-poly (styrene sulfonate) (PEDOT:PSS) inks and a protective polyethylene glycol dimethacrylate (PEG-DMA) hydrogel layer on gold electrodes is reported. This layer stack showed a glucose-specific current response for voltages between −0.2 and 0.4 V in physiological PBS buffer, and enabled interference-less sensing in a solution of acetaminophen, ascorbic acid, dopamine, and uric acid at 0 V. A Michaelis–Menten fit led to a maximum current density (Imax) of 257 ± 7.9 nA/mm2 and a Michaelis–Menten constant (Km) of 28.4 ± 2.2 mM, with a dynamic range of 0.1–20 mM glucose and a limit of detection of 0.1 mM. After 16 h of continuous measurement of 20 mM glucose, the signal decreased to 60% of its initial value. Storage stability was successfully verified until up to 10 days. In summary, this paper shows a simplified approach for the fabrication of third-generation biosensors using CDH-PEDOT:PSS and PEG-DMA hydrogel inks.

Immobilization of Alcohol Dehydrogenases on Silica-Based Supports and Their Application in Enantioselective Ketone Reductions

The use of immobilized alcohol dehydrogenases (ADHs) offers numerous advantages, especially in the reaction conditions required by industrial applications. Looking for more efficient and cost-effective methods of ADH immobilization, in this study we explored silica-based supports as an alternative to the use of functionalized polymeric resins. Three commercially available ADHs were immobilized by adsorption and covalent bond formation. The obtained supported biocatalysts were applied for the bioreduction of acetophenone and some derivatives with good yields and excellent enantioselectivity. The important intermediate (S)-1-[3,5-bis(trifluoromethyl)phenyl]ethanol was obtained with a high enantiomeric excess (>99%) by using the highest performing immobilized ADH sample. The reusability of this biocatalyst was investigated in a flow system for five consecutive runs; the experiments showed that the biocatalyst could be recycled without a loss of activity and enantioselectivity. Finally, cross-linking with the glutaraldehyde of the supported biocatalyst was also carried out to prevent the leaching of the enzyme during the catalytic reactions.

Photoelectrochemical Enzyme Biosensor for Malate Using Quantum Dots on Indium Tin Oxide/Plastics as a Sensing Surface.

A photoelectrochemical biosensor for malate was developed using an indium tin oxide (ITO) layer deposited on a poly(ethylene terephthalate) plastic sheet as a transparent electrode material for the immobilization of malate dehydrogenase together with CdTe quantum dots. Different approaches were compared for the construction of the bioactive layer; the highest response was achieved by depositing malate dehydrogenase together with CdTe nanoparticles and covering it with a Nafion/water (1:1) mixture. The amperometric signal of this biosensor was recorded during irradiation with a near-UV LED in the flow-through mode. The limit of detection was 0.28 mmol/L, which is adequate for analyzing malic acid levels in drinks such as white wines and fruit juices. The results confirm that the cheap ITO layer deposited on the plastic sheet after cutting into rectangular electrodes allows for the economic production of photoelectrochemical (bio)sensors. The combination of NAD+-dependent malate dehydrogenase with quantum dots was also compatible with such an ITO surface.

Development of a novel magnetic metal-organic framework for the immobilization of short-chain dehydrogenase for the asymmetric reduction of pro-chiral ketone

Short-chain dehydrogenase/reductase (SDR) acts as a biocatalyst in the synthesis of chiral alcohols with high optical purity. Herein, we achieved immobilization via crosslinking on novel magnetic metal–organic framework nanoparticles with a three-layer shell structure (Fe3O4@PDA@Cu (PABA)). The results of scanning electron microscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, X-ray photoelectron spectroscopy, and energy dispersive X-ray spectroscopy confirmed the morphology and cross-linking property of immobilized SDR, which was more durable, stable, and reusable and exhibited better kinetic performance than free enzyme. The SDR and glucose dehydrogenase (GDH) were co-immobilized and then used for the asymmetric reduction of COBE and ethyl 2-oxo-4-phenylbutanoate (OPBE). These finding suggest that enzymes immobilized on novel MOF nanoparticles can serve as promising biocatalysts for asymmetric reduction prochiral ketones into chiral alcohols.

A novel high-stability bioelectrochemical sensor based on sol-gel immobilization of lactate dehydrogenase and AuNPs-rGO signal enhancement for serum pyruvate detection

Pyruvate participates in diverse metabolic pathways in the body and is normally present in human blood at 40–120 μM, with concentrations outside this range associated with various diseases. Therefore, accurate and stable blood pyruvate level tests are necessary for effective disease detection. However, traditional analytical techniques require complicated instrumentation and are time consuming and expensive, prompting researchers to develop improved methods based on biosensors and bioassays. Here, we designed a highly stable bioelectrochemical pyruvate sensor affixed to a glassy carbon electrode (GCE). To maximize biosensor stability, 0.1 U of lactate dehydrogenase was affixed to the GCE using a sol-gel process, resulting in generation of Gel/LDH/GCE. Next, 2.0 mg/mL AuNPs-rGO was added to enhance current signal strength, resulting in generation of the bioelectrochemical sensor Gel/AuNPs-rGO/LDH/GCE. AuNPs-rGO synthesized in advance was verified as correct using transmission electron microscopy and UV–Vis, Fourier-transform infrared and X-ray photoelectron spectroscopy. Pyruvate detection conducted via differential pulse voltammetry in phosphate buffer (pH 7.4, 100 mM) at 37 °C for 1–4500 μM pyruvate provided detection sensitivity as high as 254.54 μA/mM/cm2. The reproducibility, regenerability and storage stability were analyzed with the relative standard deviation of 5 bioeletrochemical sensors detection was 4.60% and biosensor accuracy after 9 cycles was 92%, with accuracy remaining at 86% after 7 days. In the presence of D-glucose, citric acid, dopamine, uric acid and ascorbic acid, the Gel/AuNPs-rGO/LDH/GCE sensor exhibited excellent stability, high anti-interference ability and better performance than conventional spectroscopic methods for detection of pyruvate in artificial serum.

Simple and Portable Self-Powered Sensor Using Ultrasonically Dispersed Graphene as an Electron Transfer Promoter for Ultra-Trace Atrazine Monitoring

Atrazine (ATZ) is a typical environmental endocrine disruptor that is persistent, highly residual, and widely present in various environmental media. Thus, it is of great significance to establish a sensitive and portable detection method for ATZ. In this work, a simple and portable enzymatic biofuel cell-based self-powered sensor (EBFC-SPS) for ATZ is proposed with the ultrasonically dispersed graphene (US-G) serving as the electron transfer promoter for both the enzymatic bioanode and molecularly imprinted cathode. US-G acts as a molecular bridge to stabilize the glucose dehydrogenase (GDH) immobilization and accelerate the electron transfer between GDH and anode, promoting the spontaneous electricity generation by the bioanode and sustainable power supply to the EBFC-SPS while ensuring cathode recognition and electron transfer. The portable detection of ultra-trace ATZ is achieved using a digital multimeter with a wide linear range of 0.5 pM to 100 nM, a low detection limit of 0.37 pM, excellent selectivity and stability in complex actual water samples based on the separation strategy of the bioanode power supply, and molecularly imprinted cathode target recognition. A promising platform for the design of portable sensing strategies with the EBFC-SPS has therefore been provided, which can also be extended to a wider range of environmental pollutants.

Sortase E-mediated site-specific immobilization of green fluorescent protein and xylose dehydrogenase on gold nanoparticles

Sortase, a bacterial transpeptidase enzyme, is an attractive tool for protein engineering due to its ability to break a peptide bond at a specific site and then reform a new bond with an incoming nucleophile. Here, we present the immobilization of two recombinant proteins, enhanced green fluorescent protein (eGFP) and xylose dehydrogenase (XylB) over triglycine functionalized PEGylated gold nanoparticles (AuNPs) using C. glutamicum sortase E. For the first time, we used a new class of sortase from a non-pathogenic organism for sortagging. The site-specific conjugation of proteins with LAHTG-tagged sequences on AuNPs via covalent cross-linking was successfully detected by surface-enhanced Raman scattering (SERS) and UV–vis spectral analysis. The sortagging was initially validated by an eGFP model protein and later with the xylose dehydrogenase enzyme. The catalytic activity, stability, and reusability of the immobilized XylB were studied with the bioconversion of xylose to xylonic acid. When compared to the free enzyme, the immobilized XylB was able to retain 80% of its initial activity after four sequential cycles and exhibited no significant variations in instability after each cycle for about 72 h. These findings suggest that C. glutamicum sortase could be useful for immobilizing site-specific proteins/enzymes in biotransformation applications for value-added chemical production.

Ionic liquids modified Cu3(PO4)2 hybrid nanoflower for dehydrogenase immobilization by biomimetic mineralization

Ionic liquid modified organic-inorganic hybrid materials were applied for the immobilization of thermophilic and halophilic phenylalanine dehydrogenase from Natranaerobius thermophiles (NTAaDH). Biosynthesis of L-homophenylalanine by asymmetric reductive amination was studied. ILs modification induced the formation of nanoflowers with different morphologies. The activity of Cu3(PO4)2-[EMIM]BF4-NTAaDH was 1.92-fold of Cu3(PO4)2-NTAaDH, which indicated activity enhancement of ionic liquid modification. The correlation between the logP value of organic solvents and the activity of immobilized enzyme showed that high logP value increased the activity of enzyme. The effect of ionic liquid on the morphology of immobilized enzyme particles was studied by SEM. Mechanism of ionic liquids modification was investigated by multi-level interactions between the enzyme and carrier, and the cage effects enhanced enzyme stability. Ionic liquids modified organic-inorganic hybrid materials have excellent biocompatibility and conductivity, and are expected to expand the application in biocatalysis.

Covalent Immobilization of Dehydrogenases on Carbon Felt for Reusable Anodes with Effective Electrochemical Cofactor Regeneration.

This study presents the immobilization with aldehyde groups (glyoxyl carbon felt) of alcohol dehydrogenase (ADH) and formate dehydrogenase (FDH) on carbon-felt-based electrodes. The compatibility of the immobilization method with the electrochemical application was studied with the ADH bioelectrode. The electrochemical regeneration process of nicotinamide adenine dinucleotide in its oxidized form (NAD+ ), on a carbon felt surface, has been deeply studied with tests performed at different electrical potentials. By applying a potential of 0.4 V versus Ag/AgCl electrode, a good compromise between NAD+ regeneration and energy consumption was observed. The effectiveness of the regeneration of NAD+ was confirmed by electrochemical oxidation of ethanol catalyzed by ADH in the presence of NADH, which is the no active form of the cofactor for this reaction. Good reusability was observed by using ADH immobilized on glyoxyl functionalized carbon felt with a residual activity higher than 60 % after 3 batches.

Hierarchically porous metal organic framework immobilized formate dehydrogenase for enzyme electrocatalytic CO2 reduction

The utilization of CO2 has become an important issue aiming to the achievement of carbon neutrality. The enzyme electrocatalytic CO2 reduction to produce carbonaceous fuels has been increasingly appealing. While the low CO2 aqueous solubility severely limits the CO2 reduction efficiency, and the expensive cofactor restricts the application potential. In this study, microporous UiO-66-NH2 was firstly converted into hierarchically porous structure (HP-UiO-66-NH2) containing both micropores and mesopores to simultaneously enhance the enrichment of CO2 and the immobilization of formate dehydrogenase by optimizing the pore structure. The relative contents of the protein secondary structures were similar after immobilization. The mechanism of enzyme electrocatalytic CO2 reduction was proposed. The enzyme stability was highly enhanced after immobilization and the CO2 reduction was enhanced by the enzyme electrocatalytic system through the combination of CO2 enrichment and electrocatalytic NADH regeneration. The optimized catalytic system can obtain formate yield of 1.826 mM in 3 h (5.57 times higher than the free enzyme system) with a generation rate of 6086.7 μmol gcat‑1h‑1.

Bio-inspired CO2 reduction reaction catalysis using soft-oxometalates

The enzyme, Formate Dehydrogenase, is biological catalyst responsible for the hydrogenation of carbon dioxide to formic acid. The present research has discovered CO2 reduction activities and their application using certain metal containing (Mo- or W-)/ NAD + -linked Formate Dehydrogenases. However, the enzyme must be immobilized for easy separation, increased stability and reusability. The shortcomings associated with conventional immobilization method include leaching, mass transfer limitation and low activity. We here present a perspective, wherein, we assess the efficacy of soft-oxometalates and macrocycles as a promising alternative to Formate Dehydrogenase immobilization. The mechanistic pathway and stability of Formate Dehydrogenase from different sources are discussed and compared with their tailored ‘chemical counterparts’ soft-oxometalates and macrocycles based systems such as {Mo132}, {Mo154}, {MoV9}, Co and Mn based Corroles. The structure, properties and mechanism of CO2 reduction by different Soft-oxometalates and metal based macrocycles were found to be synonymous with that of metal based Formate Dehydrogenase. We comprehensively summarize different reported approaches to valorize CO2 to C1 and C2 products such as photochemical, electrochemical and systems chemistry to showcase our efforts in the ongoing pursuit of CO2 valorization, inspired by the workings of such enzymes, alongside the efforts of several other leading groups. The revelatory insights in the perspective could be used not only for developing bio-inspired CO2 Reduction Reaction but also constructing artificial cell automata for artificial life like system.

Covalent Immobilization of Aldehyde and Alcohol Dehydrogenases on Ordered Mesoporous Silicas

PurposeThis work studies the immobilization of two enzymes, the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (AldDH) both from Saccharomyces cerevisiae, which could be used to produce high value-added molecules from carboxylic acids embedded in anaerobic digestate.MethodsIn particular, three mesoporous siliceous materials, with different specific surface areas and pore sizes, (MSU-H, MSU-F and MCF0.75) were used as supports for covalent immobilization. The support materials were characterized by complementary techniques. Then, after a functionalization, creating a covalent bond between the enzyme and the support was performed. The specific activity and immobilization yield of the biocatalysts were then evaluated.ResultsThe best results were obtained with MSU-H and MSU-F, resulting in an immobilization yield greater than 50% in all cases, a specific activity of 0.13 IU/gsupp with the AldDH/MSU-H, 0.10 IU/gsupp with AldDH/MSU-F, 48.6 IU/gsupp with ADH/MSU-H and 12.6 IU/gsupp with ADH/MSU-H. These biocatalysts were then characterized by optimal pH and temperature and the stability factor was evaluated. With ADH/MSU-F no decrease in activity was observed after 120 h incubated at 50 °C. Finally, the biocatalysts AldDH/MSU-H and ADH/MSU-H were used to perform the reduction reaction and it was seen that after five reaction cycles the residual activity was greater than 20% in both cases.ConclusionThe ADH and AldDH enzymes have been successfully immobilized on mesoporous siliceous supports, considerably increasing their thermal stability and being able to reuse them for several reaction cycles. The use of this immobilization and these supports is adaptable to a wide variety of enzymes.Graphical

One-Pot Purification and Immobilization of Phenylalanine Dehydrogenase from Bacillus nanhaiensi by Functional Reduced Graphene Oxide.

The one-pot immobilization of halophilic phenylalanine dehydrogenase from marine microorganism with metal ions modified reduced graphene oxide (CRGO) material was studied. Phenylalanine dehydrogenase was from Bacillus nanhaiensi and expressed with a C-terminal His-tag. Investigation of CRGO, CRGO-PEI, CRCO-Mn, and CRGO-PEI-Mn for one-pot purification and immobilization of phenylalanine dehydrogenase from crude enzyme solution was carried out. Enzyme activity yield rate achieved 80.0% by immobilization with CRCO-Mn, and the loading capacity was 6.7mg/mg. Manganese ion coordination greatly improved the selectivity of the CRGO for the target His-tagged enzyme. Furthermore, the effect of NaCl concentration on the immobilization was investigated, which the loading capacity of CRGO-PEI and CRGO-Mn-PEI was increased by 10.7% and 30.6% with 1M NaCl, respectively. The adsorption curves of crude enzyme one-pot immobilized by CRGO-Mn and purified enzyme immobilized by CRGO-Mn were similar. Therefore, one-pot immobilization strategy is promising for industrial application with advantages such as high efficiency and low cost, which shorten the pipelines for enzyme discovery towards industrial applications through the establishing of marine enzyme collections.

Effective immobilization of lactate dehydrogenase onto mesoporous silica.

This study presents that covalent immobilization technique has been utilized for the immobilization of l-lactate dehydrogenase (l-LDH) from porcine on mesoporous silica. To develop mesoporous silica as support material for use in l-LDH immobilization, the particle surfaces were functionalized with 3-aminopropyltrimethoxysilane and further conjugated with glutaraldehyde. The effect of some parameters such as glutaraldehyde concentration, immobilization pH, initial enzyme concentration, and immobilization time was investigated and the optimum conditions for these parameters were determined as 1% (w/v), pH 8.0, 1mg/ml, and 120min, respectively. The maximum working pH and temperature for the oxidation of lactate to pyruvate reaction were determined as 10.0 and 35°C for free and 9.0 and 40°C for immobilized l-LDH, respectively. The kinetic parameters (Km and Vmax ) of l-LDH for the oxidation of lactate to pyruvate reaction were examined as 1.02 mM and 7.58 U/mg protein for free and 0.635mM and 1.7 U/mg protein for immobilized l-LDH, respectively. Moreover, the immobilized l-LDH was 1.3-fold more stable than free l-LDH at 25°C according to calculated t1/2 values. The immobilized l-LDH retained 80% of its initial activity in a batch reactor after 14 reuses.

Fabrication of a biocathode for formic acid production upon the immobilization of formate dehydrogenase from Candida boidinii on a nanoporous carbon

The immobilization of the non-metallic enzyme formate dehydrogenase from Candida boidinii (CbFDH) into a nanoporous carbon with appropriate pore structure was explored for the bioelectrochemical conversion of CO2 to formic acid (FA). Higher FA production rates were obtained upon immobilization of CbFDH compared to the performance of the enzyme in solution, despite the lower nominal CbFDH to NADH (β-nicotinamide adenine dinucleotide reduced) cofactor ratio and the lower amount of enzyme immobilized. The co-immobilization of the enzyme and a rhodium complex as mediator in the nanoporous carbon allowed the electrochemical regeneration of the cofactor. Preparative electrosynthesis of FA carried out on biocathodes of relatively large dimensions (ca. 3 cm × 2 cm) confirmed the higher production rate of FA for the immobilized enzyme. Furthermore, the incorporation of a Nafion binder in the biocathodes did not modify the immobilization extent of the CbFDH in the carbon support. Coulombic efficiencies close to 46% were obtained for the electrosynthesis carried out at −0.8 V for the biocathodes prepared using the lowest Nafion binder content and the co-immobilized enzyme and rhodium redox mediator. Although these values may yet be improved, they confirm the feasibility of these biocathodes in larger scales (6 cm2) beyond most common electrode dimensions reported in the literature (ca. a few mm2).

Novel nanobiosensor design to detect cholic acid using multiwalled carbon nanotube/TiO2 nanoparticle for 3α-Hydroxysteroid dehydrogenase immobilization

Determination of cholic acid concentration is a useful method to monitor liver diseases. We propose a rapid and simple method for measuring cholic acid. The development of a cholic acid electrochemical biosensor is described that is based on the modification of glassy carbon electrode surface using a mixture of carboxylated multiwalled carbon nanotube and titanium dioxide nanoparticles in chitosan solution and immobilization of 3α-hydroxysteroid dehydrogenase. The modification process of the sensing surface was characterized by Fourier transform infrared spectroscopy, Energy Diverse X-ray Spectrometry, Field Emission Scanning Electron Microscopy, and voltammetry techniques. A good correlation was demonstrated between cholic acid concentration and the peak currents in the presence of nicotinamide adenine dinucleotide. Using carboxylated multiwalled carbon nanotube and titanium dioxide nanoparticles for electrode modification showed more effective area than unmodified electrode at optimum pH of 6. Two linear ranges were obtained at 7.1 - 42.7, and 70.9-476.2 nM of cholic acid. Also, the detection limit was 6 nM and the sensitivities of the two ranges were obtained 956.9 and 28.7 µA/µM.

Reagentless D-Tagatose Biosensors Based on the Oriented Immobilization of Fructose Dehydrogenase onto Coated Gold Nanoparticles- or Reduced Graphene Oxide-Modified Surfaces: Application in a Prototype Bioreactor.

As electrode nanomaterials, thermally reduced graphene oxide (TRGO) and modified gold nanoparticles (AuNPs) were used to design bioelectrocatalytic systems for reliable D-tagatose monitoring in a long-acting bioreactor where the valuable sweetener D-tagatose was enzymatically produced from a dairy by-product D-galactose. For this goal D-fructose dehydrogenase (FDH) from Gluconobacter industrius immobilized on these electrode nanomaterials by forming three amperometric biosensors: AuNPs coated with 4-mercaptobenzoic acid (AuNP/4-MBA/FDH) or AuNPs coated with 4-aminothiophenol (AuNP/PATP/FDH) monolayer, and a layer of TRGO on graphite (TRGO/FDH) were created. The immobilized FDH due to changes in conformation and spatial orientation onto proposed electrode surfaces catalyzes a direct D-tagatose oxidation reaction. The highest sensitivity for D-tagatose of 0.03 ± 0.002 μA mM−1cm−2 was achieved using TRGO/FDH. The TRGO/FDH was applied in a prototype bioreactor for the quantitative evaluation of bioconversion of D-galactose into D-tagatose by L-arabinose isomerase. The correlation coefficient between two independent analyses of the bioconversion mixture: spectrophotometric and by the biosensor was 0.9974. The investigation of selectivity showed that the biosensor was not active towards D-galactose as a substrate. Operational stability of the biosensor indicated that detection of D-tagatose could be performed during six hours without loss of sensitivity.

Immobilization of aldehyde dehydrogenase on montmorillonite using polyethyleneimine as a stabilization and bridging agent

In the following study the tetrameric enzyme, aldehyde dehydrogenase (ALDH - 228 kDa), was immobilized on montmorillonite using polyethyleneimine (PEI) as a bridging agent. Previous studies have shown that small enzymes such as laccases and peroxidases exhibit increased stability and enhanced activity over time when immobilized on the surface of clay minerals. However, low-cost immobilization methods for high MW, multimeric enzymes, are lacking because these enzymes are prone to disassociation and deactivation upon adsorption. Herein, ALDH was successfully immobilized by pre-stabilization with a polyelectrolyte prior to adsorption on the surface of montmorillonite. The resulting complex was characterized in terms of enzyme conformation and loading using zeta potential, SEM, FTIR, and TGA measurements. Aldehyde transformation activity was measured by following benzaldehyde transformation to benzoic acid and NADH formation. An optimal ALDH:PEI ratio of 4:1 (w/w) was identified, yielding the highest activity at the adsorbed state due to conformational stability. The adsorption isotherm best fit the dual Langmuir-Freundlich model and revealed a significantly high loading capacity of 6.2 mg protein per mg montmorillonite. Rate experiments showed that although activity was maintained at the adsorbed state, the immobilization slowed aldehyde transformation, probably due to diffusion limitations caused by aggregation and heterogeneity of the material. Overall, the results suggest that this method has the potential to efficiently immobilize large multimeric proteins. This in turn can help solve separation issues and advance the design of efficient platforms for bio-catalysis and bioremediation technologies.