Immobilization Of Alcohol Dehydrogenase Research Articles

Immobilization of the Alcohol Dehydrogenase Enzyme on TiO2 Nanotubes for Application in Microfluidic Fuel Cell

Microfluidic fuel cells are a low-power demand fuel cells which work without the need of a physical membrane and have a wide range of potential applications. Biological catalysts such as immobilized enzymes can be used in electrodes to carry out the oxidation processes of different organic fuels using the immobilization technology of enzymes; they are carried out under moderate conditions of pH and temperature, and the specificity of the catalytic reactions. The alcohol dehydrogenase enzyme has been used for the development of bioanodes for the oxidation of different alcohols such as ethanol and methanol. In this work, the immobilization of this enzyme was carried out using nanotubes of titanium dioxide for the development of electrodes for an air-breathing microfluidic fuel cell. TiO2 nanotubes has excellent properties such as pH resistance, superior mechanical strength, good biocompatibility making it a great candidate for the process of immobilization of the enzyme alcohol dehydrogenase. TiO2 nanotubes were fabricated by electrochemical anodic oxidation on Ti foils. The foils were pre-treated with sand-paper, thereafter immersed in ethanol, placed in ultrasonic bath, dried under N2 flow. An ethylene glycol-based solution with a concentration of 0.1M NH4F (96% purity, Alfa-Aesar) and 2% w/w deionized water was used as electrolyte. The anodization times used was 1 h with a voltage step of 60 V via a power source. Using this nanostructure, the enzymatic electrode was constructed using a catalityc ink with enzyme, tetrabutylammonium bromide and Nafion to carry out the immobilization process. We carried out the characterization of the electrode developed by means of different electrochemical and kinetic techniques, where it was demonstrated that the enzyme is present and active in the electrode, as well as its capacity to oxidize ethanol. Then the bioanode was evaluated in the microfluidic device also using an inorganic cathode of Pt / C, obtaining a good performance over an open circuit potential greater than 0.93V.

Surface modification of polysulfone membranes applied for a membrane reactor with immobilized alcohol dehydrogenase

Commercially available polysulfone (PSf) membranes with a polypropylene backing are used across a broad range of applications. However, the natural properties of the PSf surface sometimes limit their application. Here we present, how the surface of supported membranes can be heterogeneously activated by lithiation followed by functionalization with acid chlorides at 0 °C, permitting modification of commercial PSf membranes without compromising the mechanical integrity of the membrane. Post-functionalization polymer grafting was illustrated through both, a “grafting from” approach by surface initiated atom transfer radical polymerization (SI-ATRP) and by a “grafting to” approach exploiting Cu(I) catalyzed 1,3-cycloadditions of alkynes with azides (CuAAC) introducing hydrophilic polymers onto the membrane surface. Poly(1-vinyl imidazole) (pVim) grafted membranes were exploited as support for immobilization of alcohol dehydrogenase (ADH) in a biocatalytic membrane reactor (BMR) and demonstrated substantial improvements in terms of operational enzyme stability compared to immobilization onto pristine membranes.

Product Analysis of Operating an Ethanol/O2 Biofuel Cell Shows the Synergy between Enzymes within an Enzymatic Cascade

We report the preparation of mono and bi-enzymatic bioanodes immobilizing dehydrogenase enzymes at the surface of a carbon support aiming to follow the 2 and 4 electron transfer of ethanol oxidation by HPLC. Enzymatic immobilization of ADH and AlDH enzymes at the bioanodes was performed using Nafion membranes modified with tetrabutylammonium salt. The monoenzymatic film with immobilized alcohol dehydrogenase (ADH) provided, at pH 8.50, power densities of 29.5 ± 3.4 μW cm−2; however, addition of the second enzyme (AlDH) aiming to transfer 4 electrons furnished values as high as 186.0 ± 19.4 μW cm−2. These results indicate that as the product of the first oxidation is consumed by the second enzyme it inhibits the first enzyme less thus, furnishing better performance for the bienzymatic electrodes. Long-term electrolysis (12 hours) of ethanol followed by HPLC-UV/RID system confirms our electrochemical finding demonstrating formation of 2 and 4 electrons products, e.g., acetaldehyde (6.6 mmol L−1) and acetate (0.72 mmol L−1) when a two dehydrogenase enzyme system was employed. The faradaic efficiency was above 60% for the bi-enzymatic system. We demonstrate that simultaneous product identification and mass balance analysis is an important tool to follow the synergy between the enzymes within the enzymatic cascade.

Enhanced long-chain fatty alcohol oxidation by immobilization of alcohol dehydrogenase from S. cerevisiae.

This work reports on the oxidation of long-chain aliphatic alcohols catalyzed by a stabilized alcohol dehydrogenase from S. cerevisiae (yeast alcohol dehydrogenase (YADH)). In particular, the oxidation of the fatty alcohol tetracosanol (C24H50O) to yield lignoceric acid (C23H47COOH) was studied. The immobilization of YADH onto glyoxyl agarose supports crosslinked with a polymer (polyethylenimine) produced a highly stable catalyst (60-fold higher than the soluble enzyme at 40°C). Aliphatic alcohols with different chain lengths (ranging from 2 to 24 carbons) were studied as substrates for YADH. The activity of YADH with aliphatic alcohols with a chain length higher than five carbon atoms is reported for the first time. The activities obtained with the immobilized YADH were all similar in magnitude, even with long-chain fatty alcohols such as docosanol and tetracosanol. As far as the oxidation of tetracosanol is concerned, the best values of reaction rate and substrate conversion were obtained at pH=8.2 and T=58°C. At these conditions, the soluble enzyme inactivated rapidly, precluding its use in batch reaction. However, using the immobilized YADH, up to three sequential reaction batches were performed by recovering the catalyst after each batch. Several applications in the green oleochemical industry, e.g., for making plasticizers, lubricants, detergents, and personal care products, may benefit from having novel and stable biocatalysts able to oxidize long-chain fatty alcohols.

Effective immobilization of alcohol dehydrogenase on carbon nanoscaffolds for ethanol biofuel cell

An efficient approach for immobilizing alcohol dehydrogenase (ADH) while enhancing its electron transfer ability has been developed using poly(2-(trimethylamino)ethyl methacrylate) (MADQUAT) cationic polymer and carbon nanoscaffolds. The carbon nanoscaffolds were comprised of single-walled carbon nanotubes (SWCNTs) wrapped with reduced graphene oxide (rGO). The ADH entrapped within the MADQUAT that was present on the carbon nanoscaffolds exhibited a high electron exchange capability with the electrode through its cofactor β-nicotinamide adenine dinucleotide hydrate and β-nicotinamide adenine dinucleotide reduced disodium salt hydrate (NAD+/NADH) redox reaction. The advantages of the carbon nanoscaffolds used as the support matrix and the MADQUAT employed for the entrapment of ADH versus physisorption were demonstrated via cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Our experimental results showed a higher electron transfer, electrocatalytic activity, and rate constant for MADQUAT entrapped ADH on the carbon nanoscaffolds. The immobilization of ADH using both MADQUAT and carbon nanoscaffolds exhibited strong potential for the development of an efficient bio-anode for ethanol powered biofuel cells.

Enhanced NADH Oxidation Using Polytyramine/Carbon Nanotube Modified Electrodes for Ethanol Biosensing

AbstractPolytyramine (PT) has been electro‐deposited onto multi‐walled carbon nanotube (MWCNT) modified glassy carbon (GC) electrodes via oxidation of tyramine in 0.1 M H3PO4 by cycling the potential over the range of −400 mV to 1300 mV (versus Ag/AgCl). The reactivity of the resulting chemically‐modified electrodes was characterized using cyclic voltammetry in the presence and absence of reduced nicotinamide adenine dinucleotide (NADH). The modified electrodes displayed electrochemical activity due to the formation of quinone species and were catalytically active towards NADH oxidation by lowering the oxidation peak potential by 170 mV compared to the value of the MWCNT modified electrode with a peak potential of 180±10 mV (versus Ag/AgCl). The MWCNT/PT surface was further characterized using SEM and XPS methods, which indicated that a thin polymeric film had been formed on the electrode surface. The present work demonstrates the advantage of using PT as a platform that combines both the immobilization of alcohol dehydrogenase (ADH) and the mediation of NADH oxidation at a low overpotential essential to the design of high performance ethanol biosensors, all within an easily electropolymerizable film. The resulting biosensor displayed an ethanol sensitivity of 4.28±0.06 μA mM−1 cm−2, a linear range between 0.1 mM and 0.5 mM and a detection limit of 10 μM.

Cover Picture: Immobilization of Alcohol Dehydrogenase from E. coli onto Mesoporous Silica for Application as a Cofactor Recycling System (ChemCatChem 7/2017)

Cover Picture: Immobilization of Alcohol Dehydrogenase from <i>E. coli</i> onto Mesoporous Silica for Application as a Cofactor Recycling System (ChemCatChem 7/2017)

Immobilization of Alcohol Dehydrogenase from E. coli onto Mesoporous Silica for Application as a Cofactor Recycling System

AbstractThe front cover artwork for Issue 7/2017 is provided by M. Dreifke, F. J. Brieler and M. Fröba, University of Hamburg. The image shows a single spherical cell of a mesoporous cellular siliceous foam with an immobilized alcohol dehydrogenase (ADH). The ADH is able to catalyze the oxidation of 2‐propanol to acetone with concomitant reduction of NADP+ to NADPH. However, in the presence of a sufficient concentration of acetone, the back reaction accompanied by the oxidation of NADPH to NADP+ can be introduced too. See the Full Paper itself at https://doi.org/10.1002/cctc.201601288.

Rapid In Situ Immobilization of Enzymes in Metal–Organic Framework Supports under Mild Conditions

AbstractThe use of a metal–organic framework (MOF) as a support for the in situ immobilization of enzymes was explored. The MOF support, a Basolite F300‐like material, was prepared from FeCl3 and the tridentate linker trimesic acid. Immobilization of alcohol dehydrogenase, lipase, and glucose oxidase was performed in situ under mild conditions (aqueous solution, neutral pH, and at room temperature) in a rapid and facile manner with retention of activity for at least 1 week. The catalytic activities of lipase and glucose oxidase were similar to the activities of the free enzymes; with alcohol dehydrogenase, there was a substantial decrease in activity on immobilization that may arise from diffusion limitations. The approach demonstrates that a MOF material, prepared from cheap and commercially available materials, can be successively utilized to prepare stable and catalytically active biocatalysts in a rapid and facile manner.

Immobilization of Alcohol Dehydrogenase from E. coli onto Mesoporous Silica for Application as a Cofactor Recycling System

AbstractAlcohol dehydrogenase (ADH) from Escherichia coli (E. coli) was electrostatically immobilized onto the surface of aminopropyl modified mesoporous cellular siliceous foams (MCF‐NH2). To ascertain the optimal pH for the immobilization, several pH values (pH 6.5, 7.0, 7.5) were examined. The MCF‐NH2 amount applied for immobilization was also varied to identify the most suitable enzyme to host ratio. ADH uptakes, long‐term stabilities, and leaching behavior were monitored, and the forward and back reactions were studied. ADH activity after 24 h of immobilization decreased with increasing pH. Independent of the pH, the best long‐term stabilities were obtained with an enzyme to silica ratio of 1:10. The back reaction is always preferred and is thus much faster. Michaelis–Menten kinetics of the free and the immobilized ADH of the forward as well as the back reaction were elaborated for each immobilization pH and by employing an ADH to MCF‐NH2 ratio of 1:10. Turnover numbers and the enzyme efficiencies (EE) were determined. From the kinetic aspects the immobilization was most promising when using a pH 6.5 buffer (forward reaction) or pH 7.0 buffer (back reaction) for immobilization. Thus, depending on the coenzyme that needs to be recycled (NADP+ or NADPH) the pH of the immobilization has to be selected.

Reactive Green 19 modified poly(HEMA) nanostructures for alcohol dehydrogenase immobilization

Reactive Green 19 modified poly(HEMA) nanostructures for alcohol dehydrogenase immobilization In this presented work, poly(HEMA) nanostructures were prepared by using surfactant free emulsion polymerization technique. Magnetic character was introduced to the polymer by simple adding of Fe 3 O 4 to the polymerization medium while polymerization taken place. These magnetic nanostructures were modified with Reactive Green 19 dye by nucleophilic substitution reaction. Synthesized dye attached poly(HEMA) nanostructures were characterized by FTIR, ESR, AFM and EDX analysis. These nanostructures were used for immobilization of alcohol dehydrogenase by the help of dye affinity interaction. Effects of medium pH and temperature, and substrate concentration on the activity of the alcohol dehydrogenase were investigated. Optimum pHs for free and immobilized alcohol dehydrogenase were found to be 8.0 (in phosphate buffer), while optimum temperature of both enzyme types was found to be 25 °C. Kinetic parameters of free and immobilized enzyme were also determined. Both K m ad V max values of the immobilized alcohol dehydrogenase were found to be higher than that of free enzyme. Also, thermal, operational and storage stability of immobilized enzyme were investigated and it was found that immobilized preparation showed more stability than free one. Immobilized preparation worked 10 successive reuse with a little decrease in its activity.

Single-walled carbon nanotubes covalently functionalized with polytyrosine: A new material for the development of NADH-based biosensors

We report for the first time the use of single-walled carbon nanotubes (SWCNT) covalently functionalized with polytyrosine (Polytyr) (SWCNT-Polytyr) as a new electrode material for the development of nicotinamide adenine dinucleotide (NADH)-based biosensors. The oxidation of glassy carbon electrodes (GCE) modified with SWCNT-Polytyr at potentials high enough to oxidize the tyrosine residues have allowed the electrooxidation of NADH at low potentials due to the catalytic activity of the quinones generated from the primary oxidation of tyrosine without any additional redox mediator. The amperometric detection of NADH at 0.200V showed a sensitivity of (217±3)µAmM−1cm−2 and a detection limit of 7.9nM. The excellent electrocatalytic activity of SWCNT-Polytyr towards NADH oxidation has also made possible the development of a sensitive ethanol biosensor through the immobilization of alcohol dehydrogenase (ADH) via Nafion entrapment, with excellent analytical characteristics (sensitivity of (5.8±0.1)µAmM−1cm−2, detection limit of 0.67µM) and very successful application for the quantification of ethanol in different commercial beverages.

Immobilization of dehydrogenase onto epoxy-functionalized nanoparticles for synthesis of (R)-mandelic acid

Epoxy functionalized magnetic Fe3O4@SiO2 nanoparticles were successfully prepared and characterized by Fourier-transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). The prepared nanoparticles were used for immobilization of alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae by covalent attachment. The optimal immobilization conditions were obtained as follows: enzyme/support 4.49mg/g, pH 8.0, buffer concentration 0.05M, time 12h and temperature 30°C. Under these conditions, a high immobilization yield and efficiency of above 92% were obtained after the optimization. Broad pH tolerance and high thermostability were achieved by the immobilization. The immobilized ADH retained about 84% initial activity after five cycles. Kinetic parameters Vmax and Km of free and immobilized ADH were determined as 56.72μM/min, 44.27μM/min and 11.54mM, 31.32mM, respectively. (R)-mandelic acid synthesis with the immobilized ADH was carried out, and the yield of (R)-mandelic acid was as high as 64%. These results indicate that the ADH immobilized onto epoxy-functionalized nanoparticles is an efficient and simple way for preparation of stable ADH, and the immobilized ADH has potential applications in the production of (R)-mandelic acid.

流通気相型バイオリアクターに用いる固定化アルコール脱水素酵素の調製条件

流通気相型バイオリアクターに用いる固定化アルコール脱水素酵素の調製条件

An Ethanol Biosensor Based on Simple Immobilization of Alcohol Dehydrogenase on Fe3O4@Au Nanoparticles

AbstractAn ethanol biosensor based on alcohol dehydrogenase (ADH) attached to Au seeds decorated on magnetic nanoparticles (Fe3O4@Au NPs) is presented. ADH was immobilized on Fe3O4@Au NPs, which were subsequently fixed by a magnet on a carbon paste electrode modified with 5 % (m : m) MnO2. Optimum conditions for the amperometric determination of ethanol with the biosensor were as follows: working potential +0.1 V (vs. Ag/AgCl); supporting electrolyte: 0.1 M phosphate buffer solution at pH 6.8 containing 0.25 mM of the coenzyme (NAD+); working electrode: carbon paste with magnetically attached Fe3O4@Au NPs (0.012 mg ⋅ cm−2 electrode area) with immobilized alcohol dehydrogenase (120 units per cm2 of electrode area). Linearity between signal and concentration was found for the range from 0.1 to 2.0 M ethanol (r2=0.995) with a detection limit of 0.07 M, a sensitivity of 0.02 µA ⋅ mM−1 ⋅ cm−2, a reproducibility of 4.0 % RSD, and a repeatability of 2.7 % RSD. The results for the determination of ethanol in alcoholic beverages showed good agreement with gas chromatography (GC) with recovery of 96.0 – 108.8 %.

Immobilization of alcohol dehydrogenase onto metal-chelated cryogels

In this presented work, poly(HEMA–GMA) cryogel was synthesized and used for the immobilization of alcohol dehydrogenase. For this, synthesized cryogels were functionalized with iminodiacetic acid and chelated with Zn2+. This metal-chelated cryogels were used for the alcohol dehydrogenase immobilization and their kinetic parameters were compared with free enzyme. Optimum pH was found to be 7.0 for both immobilized and free enzyme preparations, while temperature optima for free and immobilized alcohol dehydrogenase was 25 °C. Kinetic constants such as Km, Vmax, and kcat for free and immobilized form of alcohol dehydrogenase were also investigated. kcat value of free enzyme was found to be 3743.9 min−1, while kcat for immobilized enzyme was 3165.7 min−1. Thermal stability of the free and immobilized alcohol dehydrogenase was studied and stability of the immobilized enzyme was found to be higher than free form. Also, operational stability and reusability profile of the immobilized alcohol dehydrogenase were investigated. Finally, storage stability of the free and immobilized alcohol dehydrogenase was studied, and at the end of the 60 days storage, it was demonstrated that, immobilized alcohol dehydrogenase was exhibited high stability than that of free enzyme.

Enhanced Stability and Reusability of Alcohol Dehydrogenase Covalently Immobilized on Magnetic Graphene Oxide Nanocomposites.

Graphene oxide (GO) has a unique planar structure and contains many functional groups. As a functional material, it can be functionalized with biomolecules and nanomaterials for various applications. In this study, Magnetic GO (MGO) nanocomposites were synthesized according to covalent binding of amino Fe3O4 nanoparticles onto the GO surface and the as-made nanocomposites were successfully applied as supports for the immobilization of alcohol dehydrogenase (ADH). Compared with free ADH and Fe3O4 nanoparticles immobilized ADH (MNP-ADH), the MGO immobilized ADH (MGO-ADH) exhibited a wider pH stability range and a better thermal stability. Furthermore, the MGO-ADH exhibited better storage stability and reusability than MNP-ADH after recovered by magnetic separations. The MGO-ADH maintained 35.1% activity after 20 days storage and lost about 20.4% activity after ten times usage. The Michaelis constant (Km) of MGO-ADH was close to that of free ADH. The results showed the MGO nanocomposites were appropriate for the immobilization of enzyme. As a novel support, MGO nanocomposites effectively increased the stability of enzyme, allowed the reuse or continuous use of enzymes and therefore improved the potential use in practical.

Reduced pressure gas phase bioreactor as a tool for stereoselective reduction catalyzed by alcohol dehydrogenase from Parvibaculum lavamentivorans

This study presents an immobilized alcohol dehydrogenase reaction in the gas phase under reduced pressure. The catalytic ability (activity, stereoselectivity and productivity) of alcohol dehydrogenase from Parvibaculum lavamentivorans was investigated as a function of various factors for process design. The reduction of 4-methyl-2-pentanone to (R)-4-methyl-2-pentanol with concomitant regeneration of cofactor using 2-propanol as cosubstrate was employed as a model reaction. Under reduced pressure the catalytic ability of the immobilized enzyme was efficiently exhibited, similar to that under standard pressure. On the other hand, the dependence of the water and substrate activities as well as the reaction temperature on the catalytic ability of the immobilized enzyme remained almost the same, compared to the standard pressure. Based on the kinetic parameters (maximum reaction rate and Michaelis constant) estimated, the enzyme activity was found to be effectively maintained under reduced pressure. These findings indicate that the reduced pressure gas phase reactor is capable of proceeding enzyme reaction for chiral compound production. However, as the future scope, the reaction system which proceeds with the catalytic ability of enzyme enhanced more in the reduced pressure condition needs to be widely investigated.

Paper-Based, Multi-Fueled Enzymatic Fuel Cell with Passive Microfluidic Flow

Enzymatic fuel cell (EFC) technology offers several advantages over the conventional electrochemical power sources: higher energy density, low-cost, environmentally-friendly and renewable biocatalysts, room temperature and pH neutral operating environment, and fuel flexibility via a variety of renewable fuels (e.g. sugars and alcohols). The development of a flexible, paper-based system can significantly increase the utility of the device.The developed system was based on microfluidic passive evaporative pump action provided by the paper and ensured continuous delivery of fuel to the electrodes. The multi-enzymatic anode, consisting of several oxidative enzymes, was capable of simultaneously or separately converting glucose and/or ethanol fuels to electrical energy. Coupled with an air-breathing enzymatic cathode the complete cell produced 800 µW /cm2 at 0.3V.Employing our previously developed carbon nanotube (CNT) based ink we developed a series of enzymatic anodes: with immobilized glucose dehydrogenase (GDH); with immobilized alcohol dehydrogenase (ADH); and with co-immobilized GDH and ADH enzymes. Additionally, in order to investigate multi-oxidation of a single fuel (ethanol) anodes were developed with co-immobilized ADH and aldehyde dehydrogenase (AlDH). Half-cell experiments were performed on the various enzymatic anodes to determine limits of performance as well as stability and compatibility with non-complementary fuels. Results are depicted in Figure 1.Bilirubin oxidase (BOx) air-breathing cathode was also developed, characterized and optimized for best performance. The composite cathode consisted of three layers: Toray paper (TP) current collector layer; Teflon-treated carbon black (XC-35) gas diffusional layer, and high conductivity CNT paper catalytic layer. All three layers were fused together in a hydraulic press at 500 psi. BOx was tethered to the surface of the catalytic layer through the use of bi-functional chemical linker (1-Pyrenebutanoic acid, succinimidyl ester, PBSE), which forms peptide bonds with the enzyme through the succinimidyl moiety and π-π stacks on CNTs through the pyrene moiety. Such approach resulted in increased stability of the enzyme at the electrode surface when compared to physical adsorption deposition method.Once the individual components of the fuel cell were developed and optimized, the complete EFC was constructed and tested. The GDH-based and ADH/AlDH-based systems were analyzed in respective fuels. The experimental power curve for the GDH EFC is illustrated in Figure 2. Finally, in order to demonstrate actual application, three paper-based EFCs were connecter in series and immersed in Gatorade® (fuel). This system was then employed to power a digital clock continuously for several days (Figure 3).

Electrochemical and Photoelectrochemical Sensing of NADH and Ethanol Based on Immobilization of Electrogenerated Chlorpromazine Sulfoxide onto Graphene‐CdS Quantum Dot/Ionic Liquid Nanocomposite

AbstractHere, a simple one‐step solvothermal procedure was employed to synthesize a nanocomposite containing graphene‐nanosheets and CdS quantum dots (GNs‐CdS QDs). The electrochemical oxidation of chlorpromazine (CPZ) to chlorpromazine‐sulfoxide (CPZ‐SO) onto a GNs‐CdS QDs/ionic liquid (IL) nanocomposite modified glassy carbon (GC) electrode give rise to redox‐active products which showed excellent electrocatalytic and photoelectrocatalytic activity toward NADH oxidation at reduced overpotential. A linear response up to 200 µM was obtained for photoamperometric determination of NADH with detection limit 1 µM. Immobilizing alcohol dehydrogenase(ADH) onto the modified electrode via a simple cross linking procedure, the photoelectrochemical capability of the proposed system toward ethanol biosensing was clearly shown.