A new amperometric enzyme electrode for alcohol determination

A new enzyme electrode for the determination of alcohols was developed by immobilizing alcohol oxidase in polvinylferrocenium matrix coated on a Pt electrode surface. The amperometric response due to the electrooxidation of enzymatically generated H(2)O(2) was measured at a constant potential of +0.70 V versus SCE. The effects of substrate, buffer and enzyme concentrations, pH and temperature on the response of the electrode were investigated. The optimum pH was found to be pH 8.0 at 30 degrees C. The steady-state current of this enzyme electrode was reproducible within +/-5.0% of the relative error. The sensitivity of the enzyme electrode decreased in the following order: methanol>ethanol>n-butanol>benzyl alcohol. The linear response was observed up to 3.7 mM for methanol, 3.0 mM for ethanol, 6.2 mM for n-butanol, and 5.2 mM for benzyl alcohol. The apparent Michaelis-Menten constant (K(Mapp)) value and the activation energy, E(a), of this immobilized enzyme system were found to be 5.78 mM and 38.07 kJ/mol for methanol, respectively.     

Development of post-column enzymic reactors with immobilized alcohol oxidase for use in the high-performance liquid chromatographic assay of alcohols with electrochemical detection

The development of a very sensitive, direct injection high-performance liquid chromatographic method, using a post-column reactor with immobilized alcohol oxidase, was undertaken with the aim of determining methanol and ethanol levels in microlitre volumes of biological samples. After reversed-phase chromatography to separate methanol and ethanol, the analytes were enzymically converted into the respective aldehydes with formation of stoichiometric amounts of hydrogen peroxide, which could be measured via electrochemical oxidation at a platinum electrode. Some problems were encountered in the development of solid-phase enzymic reactors, using a delicate enzyme, that is prone to lose activity, such as alcohol oxidase. Owing to the slightly alkaline pH required for the optimum activity of alcohol oxidase, polymeric columns seemed to be preferable for the chromatography. HEMA copolymer was chosen as the stationary phase, but the methanol and ethanol peaks eluted close together and posed severe problems of limiting post-column band spreading. Reactors based on coarse supports for enzyme immobilization gave unacceptable band spreading, causing the methanol and ethanol peaks to overlap. On the other hand high-performance liquid chromatographic packings maintained the efficiency of the chromatographic separation, quite independently of the reactor volume. Polymeric supports proved superior to silicas in maintaining the enzyme activity. However, relevant changes in the enzyme substrate specificity were observed after immobilization.     

On-line monitoring of methanol in n-hexane by an organic-phase alcohol biosensor

An organic-phase alcohol biosensor has been developed by co-entrapping alcohol oxidase and horseradish peroxidase within an ionotropy polymer hydrogel matrix fabricated from silica gel particles, hydroxyethyl carboxymethylcellulose, an adduct of 3-methoxy-4-ethoxybenzaldehyde and 4-tert-butylpyridinium acetohydrazone, and octadecylsilica particles. The viability of the immobilised enzymes for the biocatalytic reaction of methanol in n-hexane was comparatively studied by using a bulk cell or a volume-changeable flow-through cell coupled with an oxygen optical transducer. It was found that the microenvironment around the enzyme, the deterioration property of the enzyme, the substrate throughput and the mass transfer process of the reactant in the bioreactor were the crucial parameters affecting the performance of the alcohol organic-phase biosensor. Our optimal biosensor was constructed from a flow-through cell packed with small particles of immobilised enzymes and it could maintain the biocatalytic reaction at high and stable rate for on-line detection of methanol in n-hexane under flow operation mode. The biosensor had an analytical working range of 2.3-90 mM methanol in n-hexane. The response times (t95) were 4.5 and 7.5 min for 60 and 10 mM methanol, respectively. The operational lifetime of the biosensor was more than 45 assays and the shelf lifetime was longer than 2 weeks. The biosensor has been successfully applied to determine the methanol content in a commercial gasoline-methanol blend sample with good recovery.     

Immobilization of tyrosinase and alcohol oxidase in conducting copolymers of thiophene functionalized poly(vinyl alcohol) with pyrrole

Immobilization of tyrosinase and alcohol oxidase is achieved in the copolymer of pyrrole with vinyl alcohol with thiophene side groups (PVATh-co-PPy) which is a newly synthesized conducting polymer. PVATh-co-PPy/alcohol oxidase and PVATh-co-PPy/tyrosinase electrodes are constructed by the entrapment of enzyme in conducting copolymer matrix during electrochemical copolymerization. For tyrosinase and alcohol oxidase enzymes, catechol and ethanol are used as the substrates, respectively. Kinetic parameters: maximum reaction rates (V(max)) and Michaelis-Menten constants (K(m)) are obtained. V(max) and K(m) are found as 2.75 micromol/(minelectrode) and 18 mM, respectively, for PVATh-co-PPy/alcohol oxidase electrode and as 0.0091micromol/(minelectrode) and 40 mM, respectively, for PVATh-co-PPy/tyrosinase electrode. Maximum temperature and pH values are investigated and found that both electrodes have a wide working range with respect to both temperature and pH. Operational and storage stabilities show that although they have limited storage stabilities, the enzyme electrodes are useful with respect to operational stabilities.     

Biocompatible glucose sensor prepared by modifying protein and vinylferrocene monomer composite membrane

This paper proposes a very simple procedure for preparing a biocompatible sensor based on a protein (bovine serum albumin, BSA), enzyme and vinylferrocene (VF) composite membrane modified electrode. The membrane was prepared simply by first casting vinylferrocene and then coating it with BSA and glucose oxidase immobilised with glutaraldehyde. The sensor response was independent of dissolved oxygen concentration from 3 to 10 ppm and showed good stability for serum sample measurement, unlike the commonly used BSA/enzyme modified electrode. The sensor response was almost unchanged over the measurement time (>10 h) whereas the responses of a BSA and glucose oxidase modified platinum electrode and an osmium-polyvinylpyridine wired horseradish peroxidase modified electrode (Ohara et al., 1993) fell to 68% of their initial value in a serum sample containing 10mM glucose.     

Isolation and Characterization of a Catabolite Repression-Insensitive Mutant of a Methanol Yeast, Candida boidinii A5, Producing Alcohol Oxidase in Glucose-Containing Medium

Mutants exhibiting alcohol oxidase (EC 1.1.3.13) activity when grown on glucose in the presence of methanol were found among 2-deoxyglucose-resistant mutants derived from a methanol yeast, Candida boidinii A5. One of these mutants, strain ADU-15, showed the highest alcohol oxidase activity in glucose-containing medium. The growth characteristics and also the induction and degradation of alcohol oxidase were compared with the parent strain and mutant strain ADU-15. In the parent strain, initiation of alcohol oxidase synthesis was delayed by the addition of 0.5% glucose to the methanol medium, whereas it was not delayed in mutant strain ADU-15. This showed that alcohol oxidase underwent repression by glucose. On the other hand, degradation of alcohol oxidase after transfer of the cells from methanol to glucose medium (catabolite inactivation) was observed to proceed at similar rates in parent and mutant strains. The results of immunochemical titration experiments suggest that catabolite inactivation of alcohol oxidase is coupled with a quantitative change in the enzyme. Mutant strain ADU-15 was proved to be a catabolite repression-insensitive mutant and to produce alcohol oxidase in the presence of glucose. However, it was not an overproducer of alcohol oxidase and, in both the parent and mutant strains, alcohol oxidase was completely repressed by ethanol.     

Cellular microbiosensors for methanol and ethanol determination on the basis of pH-sensitive field transistors]

Cellular sensors for methanol and ethanol determination were developed using immobilized mutant cells of methylotrophic yeasts Hansenula polymorpha and Pichia pinus (able to extrude protons in the presence of alcohol) and pH-sensitive field effect transistors (pH-SFETs). The intact cells of yeasts were immobilized in Ca-alginate gel to obtain a biomembrane. The minimal detectable response was obtained to approximately 0.5 mM of methanol and ethanol, a linear dependence of biosensor's response on the logarithmic alcohol concentration was observed in the range from 5 to 100 mM for both types of alcohol. The prospects for application of biosensors to determine alcohols in the analytical practice are discussed.     

Purification and properties of alcohol oxidase from Poria contigua.

1. Alcohol oxidase (alcohol:oxygen oxidoreductase) was purified 22-fold from the brown rot fungus Poria contigua. The final enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis, and by sedimentation in an ultracentrifuge. The molecular weight was calculated to be 610000 +/- 5000 from sedimentation equilibrium experiments. Electrophoresis in sodium dodecylsulfate gels and electron microscopic analysis indicate that the enzyme is an octamer composed of eight probably identical subunits, each having a molecular weight of 79 000. The enzyme contains eight mol FAD/mol as the prosthetic group. 2. This alcohol oxidase oxidizes not only methanol but also lower primary alcohols (C2-C4), 2-propin-1-ol and formaldehyde. The apparent Km value for methanol is 0.2 mM, and that for formaldehyde 6.1 mM. Sodium azide was found to be a competitive inhibitor with respect to methanol. 3. The enzyme from the fungus Poria contigua is immunologically different from the alcohol oxidase isolated from the methanol-utilizing yeast Candida boidinii. Furthermore antiserum raised against this enzyme did not cross-react with the alcohol oxidase from the white rot fungus Polyporus obtusus.     

Alcohol biosensing by polyamidoamine (PAMAM)/cysteamine/alcohol oxidase‐modified gold electrode

A highly stable and sensitive amperometric alcohol biosensor was developed by immobilizing alcohol oxidase (AOX) through Polyamidoamine (PAMAM) dendrimers on a cysteamine‐modified gold electrode surface. Ethanol determination is based on the consumption of dissolved oxygen content due to the enzymatic reaction. The decrease in oxygen level was monitored at ?0.7 V vs. Ag/AgCl and correlated with ethanol concentration. Optimization of variables affecting the system was performed. The optimized ethanol biosensor showed a wide linearity from 0.025 to 1.0 mM with 100 s response time and detection limit of (LOD) 0.016 mM. In the characterization studies, besides linearity some parameters such as operational and storage stability, reproducibility, repeatability, and substrate specificity were studied in detail. Stability studies showed a good preservation of the bioanalytical properties of the sensor, 67% of its initial sensitivity was kept after 1 month storage at 4°C. The analytical characteristics of the system were also evaluated for alcohol determination in flow injection analysis (FIA) mode. Finally, proposed biosensor was applied for ethanol analysis in various alcoholic beverage as well as offline monitoring of alcohol production through the yeast cultivation. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Biofunctional nanocomposite of carbon nanofiber with water-soluble porphyrin for highly sensitive ethanol biosensing

A biofunctional hybrid nanocomposite of carbon nanofiber (CNF) with water-soluble iron(III) meso-tetrakis(N-methylpyridinum-4-yl) porphyrin (FeTMPyP) was designed via non-covalent interaction for preparation of highly sensitive ethanol biosensor. The prepared nanocomposite showed good dispersion in water and was characterized with steady-state electronic absorption spectroscopy and scanning electron microscope. The nanocomposite combined the good conductivity of CNF and the excellent catalytic activity of both CNF and FeTMPyP toward the reduction of dissolved oxygen, producing a method for amperometric detection of oxygen ranging from 6.5 nM to 6.4 microM at a low overpotential. The nanocomposite modified electrode was further used for assembly of alcohol oxidase to construct an amperometric biosensor for ethanol. The biosensor showed rapid and highly sensitive response to ethanol with a linear range from 2.0 microM to 112 microM. The immobilized alcohol oxidase also showed its direct electrochemistry. The biofunctional nanocomposite provides a new way to not only construct the highly sensitive biosensors but also mimic the catalytic activity of enzyme in the life process.     

Enzyme electrode for specific determination of L-lysine

L-Lysine alpha-oxidase from Trichoderma viride Y244-2 is immobilized in a gelatin support and fixed on a pO(2) sensor. The enzyme electrode obtained is used in a continuous flow system in order to measure the concentration of L-lysine in a fermentor. The sample oxygen-content dependance of the signal is minimized because of the enzyme support properties. The enzyme electrode response is set for lysine concentration from 0.2mM to 4mM. The specificity of lysine is tested with other amino acids. The enzyme membrane for lysine electrode can be used 3000 times or stored six months with good stability.     

Urate oxidase electrode based on dissolved oxygen probe for urine uric acid determination

A biosensor for the specific determination of uric acid in urine was developed using urate oxidase (EC 1.7.3.3) in combination with a dissolved oxygen probe. Urate oxidase was immobilized with gelatin by means of glutaraldehyde and fixed on a pretreated teflon membrane to serve as enzyme electrode. The electrode response was maximum when 50 mM glycine buffer was used at pH 9.2 and 35 degrees C. The enzyme electrode response depends linearly on uric acid concentration between 5-40 microM with a response time of 5 min. The enzyme electrode is stable for more than 2 weeks and during this period over 35 assays were performed.     

Polyvinylferrocenium modified Pt electrode for anaerobic glucose monitoring

An amperometric enzyme electrode for the determination of glucose under anaerobic solution conditions was developed by immobilizing glucose oxidase and then by adsorbing ferrocene in polyvinylferrocenium matrix coated on a Pt electrode surface. The amperometric response due to the electrooxidation of ferrocene that the reduced flavin adenine dinucleotide centers of glucose oxidase was measured at a constant potential. The response characteristics of the enzyme electrode were investigated. The effects of the thickness of the polymeric film, the amount of the enzyme immobilized, the amount of the mediator, the glucose concentration, the applied potential, operating pH and temperature on the response of the enzyme electrode were studied. The response time and the optimum pH were found to be 30-40 s and pH 7.4 at 25 degrees C, respectively. The linear response was observed up to 5.0 mM glucose concentration that the produced detectable current was 0.0075 mM glucose concentration. The activation energy (E(a)) of immobilized enzyme reaction was calculated to be 41.3 kJ mol(-1) from the Arrhenius plot. The apparent Michaelis-Menten constant (K(Mapp)) was found to be 6.05 mM glucose according to the Lineweaver-Burk graph of the Michaelis-Menten equation under the optimum conditions. The interference signal due to the most common electrochemical interfering species was also evaluated.     

Ascorbate oxidase based amperometric biosensor for organophosphorous pesticide monitoring.

An amperometric principle based biosensor containing tissues of cucumber, rich in ascorbic acid oxidase, was used for the detection of organophosphorous (OP) pesticide ethyl paraoxon, which inhibits the activity of ascorbic acid oxidase. The optimal concentration of ascorbic acid used as substrate was found to be 5.67 mM. The biosensor response was found to reach steady state within 2 min. A measurable inhibition (> 10%) was obtained with 10 min incubation of the enzyme electrode with different concentrations of the pesticide. There was a linear relationship between the percentage of inhibition of the enzyme substrate reaction and the pesticide (ethyl paraoxon) concentration in the range 1-10 ppm with a regression value 0.9942.     

Application of a biosensor for monitoring of ethanol

An alcohol biosensor for the measurement of ethanol has been developed. It comprises an alcohol oxidase/chitosan immobilized eggshell membrane and a commercial oxygen sensor. Ethanol determination is based on the depletion of dissolved oxygen content upon exposure to ethanol solution. The decrease in oxygen level was monitored and related to the ethanol concentration. The biosensor response depends linearly on ethanol concentration between 60 microM and 0.80 mM with a detection limit of 30 microM (S/N=3) and 1 min response time. In the optimization studies of the enzyme biosensor the most suitable enzyme and chitosan amounts were found to be 1.0 mg and 0.30% (w/v), respectively. The phosphate buffer (pH 7.4, 25 mM) and room temperature (20-25 degrees C) were chosen as the optimum working conditions. In the characterization studies of the ethanol biosensor some parameters such as interference effects, operational and storage stability were studied in detail. The biosensor was also tested with various wine samples. The results of this newly developed biosensor were comparable to the results obtained by a gas chromatographic method.     

Development of highly selective and stable potentiometric sensors for formaldehyde determination

Two types of biosensors selective to formaldehyde have been developed on the basis of pH-sensitive field effect transistor as a transducer. Highly or partially purified alcohol oxidase (AOX) and the permeabilised cells of methylotrophic yeast Hansenula polymorpha (as a source of AOX) have been used as sensitive elements. The response time in steady-state measurement mode is in the range of 10-60 s for the enzyme-based sensors and 60-120 s for the cell-based sensor. When measured in kinetic mode the response time of all biosensors developed was less than 5 s. The linear dynamic range of the sensor output signals corresponds to 5-200 mM formaldehyde for highly and partially purified alcohol oxidase, and 5-50 mM formaldehyde for the cells. The operational stability of the biosensors is not less than 7 h, and the relative standard deviation of intra-sensor response is approximately 2 and 5% for the enzyme- and cell-based sensors, respectively. When stored at 4 degrees C, the enzyme and cell sensor responses have been found stable for more than 60 and 30 days, respectively. Both types of biosensors demonstrate a high selectivity to formaldehyde with no potentiometric response to primary alcohols, including methanol, or glycerol and glucose. The possible reasons of such unexpected high selectivity of AOX-based FET-sensors to formaldehyde are discussed. The influence of the biomembrane composition and the effect of different buffers on the sensor response to formaldehyde are also discussed.     

Purification and characterization of alcohol oxidase from a genetically constructed over-producing strain of the methylotrophic yeast Hansenula polymorpha

Alcohol oxidase (AOX) has been purified 8-fold from a genetically constructed over-producing strain of the methylotrophic yeast Hansenula polymorpha C-105 (gcr1 catX) with impaired glucose-induced catabolite repression and completely devoid of catalase. The final enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis and HPLC. Some physicochemical and biochemical properties of AOX were studied in detail: molecular weight (approximately 620 kD), isoelectric point (pI 6.1), and UV-VIS, circular dichroism (CD), and fluorescence spectra. The content of different secondary structure motifs of the enzyme has been calculated from the CD spectra using a computer program. It was found that the native protein contains about 50% alpha-helix, 25% beta-sheet, and about 20% random structures. The kinetic parameters for different substrates, such as methanol, ethanol, and formaldehyde, were measured using a Clark oxygen electrode. The rate of enzymatic oxidation of formaldehyde by alcohol oxidase from H. polymorpha is only twice lower compared to the best substrate of the enzyme, methanol.     

The highly stable alcohol dehydrogenase of Thermomicrobium roseum: purification and molecular characterization

An alcohol dehydrogenase (ADH) was purified to electrophoretic homogeneity from an extremely thermophilic bacterium, Thermomicrobium roseum. The native enzyme was found to be a homo-dimer of 43-kDa subunits. The pI of the enzyme was determined to be 6.2, while its optimum pH is 10.0. The enzyme oxidized mainly primary aliphatic alcohols and exhibited high substrate specificity towards ethanol, n-propanol and crotyl alcohol. The highest reaction rate was observed when ethanol was used as substrate and the K(m) value of the enzyme for ethanol was 24.2 mM. Pyrazole notably inhibited the enzymatic activity. The enzyme had the optimal temperature of 70 degrees C and was highly stable against high temperature.     

A novel amperometric bienzymatic biosensor based on alcohol oxidase coupled PVC reaction cell and nanomaterials modified working electrode for rapid quantification of alcohol

Abstract

A new amperometric sensor has been fabricated for sensitive and rapid quantification of ethanol. The biosensor assembly was prepared by covalently immobilizing alcohol oxidase (AOX) from Pichia pastoris onto chemically modified surface of polyvinylchloride (PVC) beaker with glutaraldehyde as a coupling agent followed by immobilization of horseradish peroxidase (HRP), silver nanoparticles (AgNPs), chitosan (CHIT), carboxylated multi-walled carbon nanotubes (c-MWCNTs) and nafion (Nf) nanocomposite onto the surface of Au electrode (working electrode). Owing to properties such as chemical inertness, light weight, weather resistance, corrosion resistance, toughness and cost-effectiveness, PVC membrane has attracted a growing interest as a support for enzyme immobilization in the development of biosensors. The amperometric biosensor displayed optimum response within 8?s at pH 7.5 and 35°C temperature. A linear response to alcohol in the range of 0.01mM–50?mM and 0.0001?µM as a minimum limit of detection was displayed by the proposed biosensor with excellent storage stability (190?days) at 4°C. The sensitivity of the sensor was found to be 155?µA mM^?1?cm^?2. A good correlation (R²?=?0.99) was found between alcohol level in commercial samples as evaluated by standard ethanol assay kit and the current biosensor which validates its performance.     

Conversion of methanol to formic acid through the coupling of the enzyme reactions of alcohol oxidase,catalase and formaldehyde dismutase

Summary A novel process for the production of formic acid from methanol has been developed that involves the coupled reactions of the three enzymes, alcohol oxidase, catalase and formaldehyde dismutase. In this process, methanol is oxidized to formaldehyde by alcohol oxidase and catalase, followed by the formaldehyde dismutase reaction that leads to the formation of methanol and formic acid. Ultimately, the substrate methanol (100 to 200 mM) is completely converted to formic acid, by the recycling of the consecutive enzyme reactions.