An enzyme electrode for on-line determination of ethanol and methanol

Since a stable alcohol oxidase with a high specific activity is not commercially available, we propose to produce and purify this enzyme from a strain of the yeast Hansenula polymorpha. This alcohol oxidase was immobilized into a gelatin matrix and its activity was estimated by a pO(2) sensor. The enzyme electrode obtained was then used in a continuous flow system to measure methanol or ethanol concentrations. The sample oxygen content dependence of the signal was minimized by the support properties. Measuring time for each sample were less than two minutes including response data treatment and rinsing step. The enzyme electrode response was set for ethanol from 0.5mM to 15mM and for methanol from 10mM to 300mM. On repeated use, the electrode signal for 10mM of ethanol was stable for at least 500 assays. Analysis have been performed in different beverages such as wine and beer, and the results compared to those obtained with classical methods of analysis.     

Receptor elements for biosensors in two ways of methylotrophic yeast immobilization

Receptor elements for biosensors based on Hansenula polymorpha NCYC 495 ln yeast cells for ethanol assay were developed using two ways of cell immobilization, i.e., physical adsorption on a glass fiber membrane and covalent binding on a modified nitrocellulose membrane. The linear diapason of ethanol assays for a biosensor based on yeast cells adsorbed on glass fiber was 0.05–1.18; for a biosensor based on yeasts immobilized on a nitrocellulose membrane, 0.2–1.53 mM. Receptor elements based on sorbed cells possessed 2.5 times higher long-term stability. The time response was 1.5 times less for cells immobilized using DEAE-dextran and benzoquinone. The results of ethyl alcohol assays using biosensors based on cells immobilized via adsorption and covalent binding, as well as using the standard areometric method, had high correlation coefficients (0.998 and 0.997, respectively, for the two ways of immobilization). The results indicate the possibility to consider the described models of receptor elements for biosensors as prototypes for experimental samples for practical use.     

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.     

An amperometric microbial biosensor development based on Candida tropicalis yeast cells for sensitive determination of ethanol

Different branchs of industry need to use ethanol in their production and some progress and not only the industry also to determine ethanol sensitively, accurately, fast and economical is very important. For the sensitive determination of ethanol a new amperometric biosensor based on Candida tropicalis cells, which contains alcohol oxidase enzyme, immobilized in gelatin by using glutaraldehyde was developed. In the study, before the microbial biosensor construction C. tropicalis cells were activated and cultured in a culture medium. By using gelatine and glutaraldehyde (0.1%) C. tropicalis cells obtained in logarithmic phase were immobilized and fixed on a pretreated teflon membrane of a dissolved oxygen probe. Ethanol determination is based on the assay of the differences on the respiration activity of the cells on the oxygenmeter in the absence and the presence of ethanol. The microbial biosensor response was depend linearly on ethanol concentration between 0.5 and 7.5 mM with 2 min response time. In the optimization studies of the microbial biosensor the most suitable microorganism amount were found as 4.42 mg cm(-2) and also phosphate buffer (pH:7.5; 50 mM) and 30 degrees C were obtained as the optimum working conditions. In the characterization studies of the microbial biosensor some parameters such as substrate specificity, interference effects of some substances on the biosensor response, operational and storage stability were carried out.     

Immobilization and characterization of Saccharomyces cerevisiae in crosslinked,prepolymerized polyacrylamide-hydrazide

Summary Baker's yeast (Saccharomyces cerevisiae) was immobilized in gels made of prepolymerized, linear, water soluble polyacrylamide, partially substituted with acylhydrazide groups. Gelation was effected by the addition of controlled amounts of dialdehydes (e.g. glyoxal). The immobilized yeasts retained full glycolytic activity. Moreover, the entrapped cells were able to grow inside the chemically corsslinked gel during continuous alcohol production. Glyoxal was found to be the most favourable crosslinking agent for this system. the system employed allowed for the free exchange of substrate and products. The gel surrounding the entrapped cells had no effect on temperature stability profile. On the other hand, substantial enhancement in survival of cells in presence of high ethanol concentrations was recorded for the entrapped yeast. The capability of the immobilized yeast to carry out continuous conversion of glucose to ethanol was demonstrated.     

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.     

Effect of selected alcohol dehydrogenase inhibitors on the human heart lactate dehydrogenase activity--an in vitro study

Metabolic acidosis complicates methanol, ethylene glycol and other alcohol intoxications. It is caused firstly by acid metabolites and secondly by the lactate elevation. The aim of the study was to evaluate the effect of alcohol dehydrogenase (ADH; EC 1.1.1.1) inhibitors and substrates: 4-methylpyrazole (4-MP), cimetidine, EDTA, ethanol and methanol on lactate dehydrogenase (LDH; EC 1.1.1.27) activity. The activity of LDH was determined spectrophotometrically in in vitro human heart homogenates with the mentioned compounds at 0.01, 0.1, 1.0 mM concentrations of 4-MP, cimetidine, EDTA, and 12.5, 25.0, 50.0 mM of ethanol and methanol. The LDH activity was significantly inhibited by 0.1 mM (p<0.05) and 1.0 mM (p<0.01) 4-MP and 1.00 mM EDTA (p<0.05). Higher LDH activity vs. control was observed in the samples incubated with all studied ethanol and methanol concentrations but these differences were not statistically significant. Thus, 4-MP was found to be the most effective inhibitor of LDH of all compounds tested. Therefore, such effect of 4-MP seems to be an additional advantage in methanol and ethylene glycol intoxications.     

Characterization of ψM1, a virulent phage of Methanobacterium thermoautotrophicum Marburg

We have studied the biogenesis and enzymic composition of microbodies in different yeasts during adaptation of cells to a new growth environment. After a shift of cells of Candida boidinii and Hansenula polymorpha from glucose to methanol/methylamine-containing media, newly synthesized alcohol oxidase and amine oxidase are imported in one and the same organelle together with catalase; as a consequence the cells contain one class of morphologically and enzymatically identical microbodies. Similar results were obtained when Candida utilis cells were transferred from glucose to ethanol/ethylamine-containing media upon which all cells formed microbodies containing amine oxidase and catalase.However, when methanol-limited cells of H. polymorpha were transferred from media containing ammonium sulphate to those with methylamine as the nitrogen source, newly synthesized amine oxidase was incorporated only in part of the microbodies present in these cells. This uptake was confined to the few smaller organelles generally present at the perimeter of the cells, which were considered not fully developed (immature) as judged by their size. Essentially similar results were obtained when stationary phase cells of C. boidinii or C. utilis — grown on methanol and ethanol plus ammonium sulphate, respectively — were shifted to media containing (m)ethylamine as the nitrogen source. These results indicate that mature microbodies may exist in yeasts which no longer are involved in the uptake of matrix proteins. Therefore, these yeasts may display heterogeneities in their microbody population.     

Immobilized Pseudomonas cepacia lipase for biodiesel fuel production from soybean oil

Enzymatic transesterification of soybean oil with methanol and ethanol was studied. Of the nine lipases that were tested in the initial screening, lipase PS from Pseudomonas cepacia resulted in the highest yield of alkyl esters. Lipase from Pseudomonas cepacia was further investigated in immobilized form within a chemically inert, hydrophobic sol-gel support. The gel-entrapped lipase was prepared by polycondensation of hydrolyzed tetramethoxysilane and iso-butyltrimethoxysilane. Using the immobilized lipase PS, the effects of water and alcohol concentration, enzyme loading, enzyme thermal stability, and temperature in the transesterification reaction were investigated. The optimal conditions for processing 10 g of soybean oil were: 35 degrees C, 1:7.5 oil/methanol molar ratio, 0.5 g water and 475 mg lipase for the reactions with methanol, and 35 degrees C, 1:15.2 oil/ethanol molar ratio, 0.3 g water, 475 mg lipase for the reactions with ethanol. Subject to the optimal conditions, methyl and ethyl esters formation of 67 and 65 mol% in 1h of reaction were obtained for the immobilized enzyme reactions. Upon the reaction with the immobilized lipase, the triglycerides reached negligible levels after the first 30 min of the reaction and the immobilized lipase was consistently more active than the free enzyme. The immobilized lipase also proved to be stable and lost little activity when was subjected to repeated uses.     

Close Correlation between Heat Shock Response and Cytotoxicity in Neurospora crassa Treated with Aliphatic Alcohols and Phenols

In Neurospora crassa the aliphatic alcohols methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutanol, ethylene glycol, glycerol, and allyl alcohol and the phenolic compounds phenol, hydroquinone, resorcinol, pyrogallol, phloroglucinol, sodium salicylate, and acetylsalicylic acid were analyzed with respect to their capacities to induce heat shock proteins (HSP) and to inhibit protein synthesis. Both the alcohols and phenols showed the greatest levels of HSP induction at concentrations which inhibited the overall protein synthesis by about 50%. The abilities of the different alcohols to induce the heat shock response are proportional to their lipophilicities: the lipophilic alcohol isobutanol is maximally inductive at about 0.6 M, whereas the least lipophilic alcohol, methanol, causes maximal induction at 5.7 M. The phenols, in general, show a higher capability to induce the heat shock response. The concentrations for maximal induction range between 25 mM (sodium salicylate) and 100 mM (resorcinol). Glycerol (4.1 M) shifted the concentration necessary for maximal HSP induction by hydroquinone from 50 to 200 mM. The results reveal that the induction of HSP occurs under conditions which considerably constrain cell metabolism. The heat shock response, therefore, does not represent a sensitive marker for toxicity tests but provides a good estimate for the extent of cell damage.     

Derepression of FAD Pyrophosphorylase and Flavin Changes during Growth of Kloeckera sp. No. 2201 on Methanol

A methanol-utilizing yeast Kloeckera sp. No. 2201, when grown with methanol as a sole carbon and energy source, accumulated about three times much flavin as those grown with glucose, ethanol, or glycerol. A high proportion of the total flavin was FAD in methanol-grown cells. A remarkable derepression of FAD pyrophosphorylase accompanied by an inducible formation of an FAD-dependent alcohol oxidase which catalyzes oxidation of methanol, the first step in the oxidation sequence, was observed during growth of the yeast on methanol. Significant elevations of riboflavin synthetase and flavokinase were also found. Formate, as well as methanol, effectively induced both FAD pyrophosphorylase and methanol-oxidizing enzymes (alcohol oxidase, formaldehyde dehydrogenase, formate dehydrogenase, and catalase). Observations with other methanol-utilizing yeasts also gave essentially same results. These results led to the conclusion that cellular flavin level might be under control with level of flavoprotein physiologically required.     

Contributions of immobilized and free cells of salt-tolerantZygosaccharomyces rouxii andCandida versatilis to the production of ethanol and 4-ethylguaiacol

Summary The contribution of immobilized cells and free cells released from gel beads to ethanol production by the salt-tolerant yeastsZygosaccharomyces rouxii andCandida versatilis, and 4-ethylguaiacol (4-EG) production byC. versatilis were investigated using an airlift reactor. The amounts of ethanol produced by free cells were about 65% and about 90% of total ethanol in the reactor forZ. rouxii andC. versatilis, respectively. It was found that immobilized cells gave a much lower specific productivity of ethanol (ethanol production per hour per cell) than free cells of both yeasts, especially ofC. versatilis. 4-EG was produced mainly by immobilized cells ofC. versatilis; the amount of 4-EG produced by free cells was about 20% of the total 4-EG, in contrast to the results of ethanol production. However, the specific productivity of 4-EG (4-EG production per hour per cell) by immobilized and free cells was fairly similar.     

Effects of metabolic inhibitors on the alcoholic fermentation by several yeasts in batch or in immobilized cell systems

Summary In previous papers it was shown that the bacterium Zymomonas mobilis might be an interesting alternative for industrial alcohol production from sugar, compared to Saccharomyces bayanus. Factors that might increase the glucose to ethanol conversion efficiency and which are in favour of the bacterium, are the production of less biomass and less by-products such as glycerol, succinic acid, butanediol, acetoin, and acetic acid. In order to reduce the synthesis of biomass three metabolic inhibitors were now studied: dinitrophenol, azide and arsenate. Their effects on the alcoholic fermentation in batch and in immobilized cell system were investigated, using three yeasts: Saccharomyces bayanus, Schizosacharomyces pombe, and Saccharomyces diastaticus. It was found that dinitrophenol in 0.1 mM concentration was effective in increasing the conversion of glucose to ethanol especially with Saccharomyces bayanus while azide in 0.1 mM concentration was better with Schizosaccharomyces pombe. In immobilized systems high steady state ethanol production from 15% glucose media was obtained by inclusion into the media of dinitrophenol or azide. Arsenate had less effect at the concentration used. Arsenate had less effect at the concentrations used. As a result ethanol productivity in g·l^-1·h^-1 was increased from around 70 in the absence of inhibitor to around 74 in the presence of dinitrophenol with Saccharomyces bayanus. With Schizosaccharomyces pombe the productivity was increased from around 65 in the absence of inhibitor to around 74 in the presence of azide. The specific ethanol productivity expressed as g ethanol formed per hour and per g viable cells was increased from 0.87 to 1.37 for Schizosaccharomyces pombe and from 1.02 to 1.66 for Saccharomyces bayanus.     

Lipase-catalyzed alcoholysis of triglycerides for the preparation of 2-monoglycerides

Rhizopus arrhizus lipase immobilized on celite was used to prepare isomerically pure 2-monoglycerides by alcoholysis of triglycerides in organic media. Reaction parameters such as choice of solvent, choice of alcohol, and alcohol concentration were studied. When 12.5 mM tripalmitin was used as substrate, methyl-tert-butyl ether was the best solvent for alcoholysis at water activity 0.11. Ethanol gave the highest yield (97%) at an optimal ethanol concentration of 200–300 mM. At higher alcohol concentrations, the enzyme activity was substantially lowered. The enzyme preparation showed high stability in repeated-batch reactions.     

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.     

Amperometric ethanol biosensor based on poly(vinyl alcohol)-multiwalled carbon nanotube-alcohol dehydrogenase biocomposite

A novel amperometric ethanol biosensor was constructed using alcohol dehydrogenase (ADH) physically immobilized within poly(vinyl alcohol)–multiwalled carbon nanotube (PVA–MWCNT) composite obtained by a freezing–thawing process. It comprises a MWCNT conduit, a PVA binder, and an ADH function. The measurement of ethanol is based on the signal produced by β-nicotinamide adenine dinucleotide (NADH), the product of the enzymatic reaction. The homogeneity of the resulting biocomposite film was characterized by atomic force microscopy (AFM). The performance of the PVA–MWCNT–ADH biocomposite modified glassy carbon electrode was evaluated using cyclic voltammetry and amperometry in the presence of NADH and in the presence of ethanol. The ethanol content in standard solutions was determined and a sensitivity of 196 nA mM⁻¹, a linear range up to 1.5 mM, and a response time of about 8 s were obtained. These characteristics allowed its application for direct detection of ethanol in alcoholic beverages: beer, red wine, and spirit.     

Formate production from methanol by formaldehyde dismutase coupled with a methanol oxidation system

Summary Formaldehyde dismutase was greatly stabilized by immobilization in a urethane prepolymer (PU-6). The immobilized enzyme exhibited stochiometrical dismutation of formaldehyde to methanol and formate in several repeated reactions. Conversion of methanol to formate occurred in a reaction with an immobilized enzyme system consisting of alcohol oxidase, catalase and formaldehyde dismutase, and with an intact cell-mixture of Hansenula polymorpha and Pseudomonas putida. Furthermore, the stability of the cell-mixture during repeated reactions was greatly improved by the immobilization, the 600 mM methanol added periodically being converted to formate in a 75% yield in 12 h. The immobilized cellsystem was also effective for the conversion of several aliphatic alcohols, C₁ to C₄, to the corresponding acids.     

Immobilization of yeast microbodies and the properties of immobilized microbody enzymes

Summary Yeast microbodies isolated from methanol-grown cells of Kloeckera sp. No. 2201 were immobilized by two types of entrapping techniques: photocrosslinking of liquid oligomers of suitable photosensitive resins and crosslinking of albumin molecules with glutaraldehyde. The apparent activities of catalase, alcohol oxidase, and D-amino acid oxidase in the gel-entrapped microbodies were 40–50, 70–80, and ca. 50% respectively as compared with those in the free microbodies. Alcohol oxidase in the immobilized microbodies, similarly to that in free ones, oxidized methanol, ethanol, n-propanol, n-butanol, n-amyl alcohol, and benzyl alcohol. Some properties of catalase and alcohol oxidase in the microbodies immobilized by the above-mentioned techniques were studied in comparison with those of the enzymes in the free microbodies.     

Efficient bioconversion of ethanol to acetaldehyde using a novel mutant strain of the methylotrophic yeast Hansenula polymorpha

We report the isolation of mutant strains of the methylotrophic yeast Hansenula polymorpha that are able to efficiently oxidize ethanol to acetaldehyde in an intact cell system. The oxidation reaction is catalyzed by alcohol oxidase (AOX), a key enzyme in the methanol metabolic pathway that is typically present only in H. polymorpha cells growing on methanol. At least three mutations were introduced in the strains. Two of the mutations resulted in high levels of AOX in glucose-grown cells of the yeast. The third mutation introduced a defect in the cell's normal ability to degrade AOX in response to ethanol, and thus stabilizing the enzyme in the presence of this substrate. Using these strains, conditions for bioconversion of ethanol to acetaldehyde were examined. In addition to pH and buffer concentration, we found that the yield of acetaldehyde was improved by the addition of the proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF) and by permeabilization of the cells with digitonin. Under optimal shake-flask conditions using one of the H. polymorpha mutant strains, conversion of ethanol to acetaldehyde was nearly quantitative.     

Direct injection high-performance liquid chromatographic method with electrochemical detection for the determination of ethanol and methanol in plasma using an alcohol oxidase reactor

A highly sensitive reversed-phase high-performance liquid chromatographic assay for ethanol and methanol in plasma, using a post-column enzymic reactor with electrochemical detection, has been developed. The alcohols, separated on the column, were converted by immobilized alcohol oxidase into their respective aldehydes with formation of stoichiometric amounts of hydrogen peroxide, detected via oxidation at a platinum electrode. As the chromatographic column, two glass cartridges (150 mm × 3 mm I.D.) in series, packed with 10-μm HEMA-S 1000^® packing, were used. Alcohol oxidase from Candida boidinii was immobilized onto HEMA-BIO 1000 VS-L (10 μm), packed in a 30 mm × 3 mm I.D. glass cartridge. The reaction product, hydrogen peroxide, was detected with an amperometric detector with a platinum electrode, operated at +500 mV vs. an Ag/AgCl reference electrode. A 20-μl volume of ten-fold diluted plasma was injected without any pre-treatment. Under the described conditions, methanol and ethanol were well resolved from each other and from the “front” of the chromatogram. The limit of detection was ca. 2.5 nmol for ethanol and 0.6 nmol for methanol in plasma, at a signal-to-noise ratio of 3. Excellent linearity was observed for ethanol, in the range 0.125–4 μg injected (r = 0.9999). In contrast, the response for methanol was markedly non-linear above 500 μg injected, presumably owing to progressive saturation of the reactor. The precision and accuracy of the assay were satisfactory, as was the reactor life (one month).