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There are limited data on amperometric biosensors (ABSs) based on deiminases that produce ammonium as a byproduct of enzymatic reaction. The most frequently proposed biosensors utilizing such a mode are based on potentiometric transducers, which contain at least two enzymes in the bioselective layer; this complicates the procedure and increases the cost of analysis. Thus, the construction of a one-enzyme ABS is a practical problem. In our manuscript ABSs for the direct measurement of creatinine (Crn) and l-arginine (Arg), based on the recombinant bacterial creatinine deiminase (CDI) and arginine deiminase (ADI), are described. To choose the best chemosensor on ammonium ions, a number of nanoparticles (NPs) were synthesized and characterized using cyclic voltammetry. Hybrid Cu/Zn(Hg)S-NPs, having a good selectivity and an extremely high sensitivities towards ammonium ions (5660 A M -1  m -2  at +170 mV and 1870 A M -1  m -2  at -300 mV, respectively), was selected for the development of deiminase-based ABSs. The novel biosensors exhibited very high sensitivities (2660 A M -1  m -2 to Crn for CDI-ABS; 1570 A M -1  m -2 to Arg for ADI-ABS), broad linear ranges, low limits of detection, satisfactory storage stabilities and good selectivities towards natural substrates. The constructed CDI-ABS and ADI-ABS were tested on real samples of biological fluids and juices for Crn and Arg assay, respectively. High correlations of the obtained results with the reference methods were demonstrated for the target analytes., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Prussian blue analogs (PBAs) are well-known artificial enzymes with peroxidase (PO)-like activity. PBAs have a high potential for applications in scientific investigations, industry, ecology and medicine. Being stable and both catalytically and electrochemically active, PBAs are promising in the construction of biosensors and biofuel cells. The "green" synthesis of PO-like PBAs using oxido-reductase flavocytochrome b 2 is described in this study. When immobilized on graphite electrodes (GEs), the obtained green-synthesized PBAs or hexacyanoferrates (gHCFs) of transition and noble metals produced amperometric signals in response to H 2 O 2 . HCFs of copper, iron, palladium and other metals were synthesized and characterized by structure, size, catalytic properties and electro-mediator activities. The gCuHCF, as the most effective PO mimetic with a flower-like micro/nano superstructure, was used as an H 2 O 2 -sensitive platform for the development of a glucose oxidase (GO)-based biosensor. The GO/gCuHCF/GE biosensor exhibited high sensitivity (710 A M -1 m -2 ), a broad linear range and good selectivity when tested on real samples of fruit juices. We propose that the gCuHCF and other gHCFs synthesized via enzymes may be used as artificial POs in amperometric oxidase-based (bio)sensors.

Laccases are enzymes belonging to the family of blue copper oxidases. Due to their broad substrate specificity, they are widely used in many industrial processes and environmental bioremediations for removal of a large number of pollutants. During last decades, laccases attracted scientific interest also as highly promising enzymes to be used in bioanalytics. The aim of this study is to obtain a highly purified laccase from an efficient fungal producer and to demonstrate the applicability of this enzyme for analytics and bioremediation. To select the best microbial source of laccase, a screening of fungal strains was carried out and the fungus Monilinia fructicola was chosen as a producer of an extracellular enzyme. Optimal cultivation conditions for the highest yield of laccase were established; the enzyme was purified by a column chromatography and partially characterized. Molecular mass of the laccase subunit was determined to be near 35 kDa; the optimal pH ranges for the highest activity and stability are 4.5-5.0 and 3.0-5.0, respectively; the optimal temperature for laccase activity is 30°C. Laccase preparation was successfully used as a biocatalyst in the amperometric biosensor for bisphenol A assay and in the bioreactor for bioremediation of some xenobiotics., (© 2020 International Federation for Cell Biology.)

Alcohol oxidase (EC 1.1.3.13; AOX) is a flavoprotein that catalyzes the oxidation of primary short-chain alcohols to corresponding carbonyl compounds with a concomitant release of hydrogen peroxide. It is a key enzyme of methanol metabolism in methylotrophic yeasts, catalyzing the first step of methanol oxidation to formaldehyde.Here we describe the isolation and purification of AOX from the thermotolerant methylotrophic yeast Ogataea (Hansenula) polymorpha, and using this enzyme in enzymatic assay of ethanol, simultaneous analysis of methanol and formaldehyde, and in construction of amperometric biosensors selective to primary alcohols and formaldehyde.

Flavocytochrome b 2 (EC 1.1.2.3; L-lactate cytochrome: c oxidoreductase, FC b 2 ) from the thermotolerant methylotrophic yeast Ogataea polymorpha is a thermostable enzyme-prospective for a highly selective L-lactate analysis in the medicine, nutrition sector, and quality control of commercial products. Here we describe the construction of FC b 2 producers by overexpression of the CYB2 gene O. polymorpha, encoding FC b 2 , under the control of a strong alcohol oxidase promoter in the frame of plasmid for multicopy integration with the next transformation of recipient strain O. polymorpha C-105 (gcr1 catX) impaired in the glucose repression and devoid of catalase activity. The selected recombinant strain O. polymorpha "tr1" (gcr1 catX CYB2), characterized by eightfold increased FC b 2 activity compared to the initial strain, was used as a source of the enzyme. For purification of FC b 2 a new method of affinity chromatography was developed and purified preparations of the enzyme were used for the construction of the highly selective enzymatic kits and amperometric biosensor for L-lactate analysis in human liquids and foods.

The codA gene of Corynebacterium glutamicum PCM 1945 coding for a creatinine deiminase (CDI) (EC 3.5.4.21) has been amplified and cloned. The recombinant strain of Escherichia coli that overproduces the (His) 6 -tagged inactive CDI of C. glutamicum as inclusion bodies has been constructed. After solubilization of inclusion bodies in the presence of 0.3% N-lauroylsarcosine, the enzyme was renaturated and purified by a single-step procedure using metal-affinity chromatography. The yield of the (His) 6 -tagged CDI is ~30 mg from 1 L culture. The purified enzyme is sufficiently stable under the conditions designed and possesses an activity of 10-20 U/mg. The main characteristics of the tagged enzyme remained similar to that of the natural enzyme., (© 2020 International Federation for Cell Biology.)

A highly selective and sensitive enzymatic method for the quantitative determination of L-arginine (Arg) has been developed. The method is based on the use of recombinant bacterial arginine deiminase (ADI) isolated from the cells of a recombinant strain Escherichia coli and o-phthalaldehyde (OPA) as a chemical reagent. Ammonia, the product of the enzymatic digestion of Arg by ADI, reacts with OPA and forms in the presence of sulfite a product, which can be detected by spectrophotometry (S) and fluorometry (F). The linear concentration range for Arg assay in the final reaction mixture varies for ADI-OPA-F variant of the method from 0.35 μM to 24 μM with the detection limit of 0.25 μM. For ADI-OPA-S variant of the assay, the linearity varies from 0.7 μM to 50 μM with the detection limit of 0.55 μM. The new method was tested on real samples of wines and juices. A high correlation (R=0.978) was shown for the results obtained with the proposed and the reference enzymatic method., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Tumor cells often exhibit specific metabolic defects due to the aberrations in oncogene-dependent regulatory and/or signaling pathways that distinguish them from normal cells. Among others, many malignant cells are deficient in biosynthesis of certain amino acids and concomitantly exhibit elevated sensitivity to deprivation of these amino acids. Although the underlying causes of such metabolic changes are still not fully understood, this feature of malignant cells is exploited in metabolic enzymotherapies based on single amino acid, e.g., arginine, deprivation. To achieve efficient arginine depletion in vivo, two recombinant enzymes, bacterial arginine deiminase and human arginase I have been evaluated and are undergoing further development. This review is aimed to summarize the current knowledge on the application of arginine-degrading enzymes as anticancer agents and as bioanalytical tools for arginine assays. The problems that have to be solved to optimize this therapy for clinical application are discussed., (© 2014 International Federation for Cell Biology.)

A d-lactate-selective biosensor has been developed using cells' debris of recombinant thermotolerant methylotrophic yeast Hansenula polymorpha, overproducing d-lactate: cytochrome c-oxidoreductase (EC 1.1.2.4, d-lactate dehydrogenase (cytochrome), DlDH). The H. polymorpha DlDH-producer was constructed in two steps. First, the gene CYB2 was deleted on the background of the С-105 (gcr1 catX) strain of H. polymorpha impaired in glucose repression and devoid of catalase activity to avoid specific l-lactate-cytochrome c oxidoreductase activity. Second, the homologous gene DLD1 coding for DlDH was overexpressed under the control of the strong H. polymorpha alcohol oxidase promoter in the frame of a plasmid for multicopy integration in the Δcyb2 strain. The selected recombinant strain possesses 6-fold increased DlDH activity as compared to the initial strain. The cells debris was used as a biorecognition element of a biosensor, since DlDH is strongly bound to mitochondrial membranes. The cells' debris, prepared by mechanic disintegration of recombinant cells, was immobilized on a graphite working electrode in an electrochemically generated layer using an Os-complex modified cathodic electrodeposition polymer. Cytochrome c was used as additional native electron mediator to improve electron transfer from reduced DlDH to the working electrode. The constructed d-lactate-selective biosensors are characterized by a high sensitivity (46.3-61.6 A M(-1)m(-2)), high selectivity and sufficient storage stability., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A novel methylamine-selective amperometric bienzyme biosensor based on recombinant primary amine oxidase isolated from the recombinant yeast strain Saccharomyces cerevisiae and commercial horseradish peroxidase is described. Two amine oxidase preparations were used: free enzyme (AMO) and covalently immobilized on the surface of gold nanoparticles (AMO-nAu). Some bioanalytical parameters (sensitivity, selectivity, and storage stability) of the developed biosensors were investigated. The sensitivity for both sensors is high: 1450 ± 113 and 700 ± 30 A(-1) ·M(-1) ·m(-2) for AMO-nAu biosensor, respectively. The biosensors exhibit the linear range from 15 μM to 150 μM (AMO-nAu) and from 15 μM to 60 μM (AMO). The developed biosensor demonstrated a good selectivity toward methylamine (MA) (signal for dimethylamine and trimethylamine is less than 5% and for ethylamine 15% compared to MA output) and reveals a satisfactory storage stability. The constructed amperometric biosensor was used for MA assay in real samples of fish products in comparison with chemical method. The values obtained with both approaches different methods demonstrated a high correlation.

A highly selective and sensitive method for the quantitative determination of L-arginine (Arg) with a fluorescent detection of the reaction product has been developed. The method is based on the use of human liver arginase I isolated from a recombinant producer strain, yeast Hansenula polymorpha, and 2,3-butanedione monoxime, which is used to detect carbamide--the product of enzymatic reactions. The linear concentration range for determining Arg in the final reaction mixture varies from 0.2 to 250 μM, and the detection limit is 0.16 μM. Tests of the new method using commercial Arg-containing pharmaceutical preparations showed a high correlation (R = 1.0) of the results with the manufacturer's data and the results of other methods for Arg detection.

Arginase (EC 3.5.3.1; L-arginine amidinohydrolase) is a key enzyme of the urea cycle that catalyses the conversion of arginine to ornithine and urea, which is the final cytosolic reaction of urea formation in the mammalian liver. The recombinant strain of the yeast Saccharomyces cerevisiae that is capable of overproducing arginase I (rhARG1) from human liver under the control of the efficient copper-inducible promoter CUP1, was constructed. The (His)(6)-tagged rhARG1 was purified in one step from the cell-free extract of the recombinant strain by metal-affinity chromatography with Ni-NTA agarose. The maximal specific activity of the 40-fold purified enzyme was 1600 μmol min(-1) mg(-1) protein., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Purified human arginase I preparations homogeneous in SDS-PAAG test were obtained by the affinity chromatography on the synthesized sorbent L-arginine-macroporous glass. Some physico-chemical characteristics of the isolated arginase preparation have been estimated: thermo- and pH-stability, temperature- and pH-optima of the enzyme. The influence of some bivalent metal ions and other additives on enzymatic activity for stabilization of the enzyme and optimization of its storage conditions was studied.

Improvement of xylose fermentation is of great importance to the fuel ethanol industry. The nonconventional thermotolerant yeast Hansenula polymorpha naturally ferments xylose to ethanol at high temperatures (48-50 degrees C). Introduction of a mutation that impairs ethanol reutilization in H. polymorpha led to an increase in ethanol yield from xylose. The native and heterologous (Kluyveromyces lactis) PDC1 genes coding for pyruvate decarboxylase were expressed at high levels in H. polymorpha under the control of the strong constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH). This resulted in increased pyruvate decarboxylase activity and improved ethanol production from xylose. The introduction of multiple copies of the H. polymorpha PDC1 gene driven by the strong constitutive promoter led to a 20-fold increase in pyruvate decarboxylase activity and up to a threefold elevation of ethanol production.

L-Lactate cytochrome c oxidoreductase (flavocytochrome b2, FC b2) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker's yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b2 producers with overexpression of the H. polymorpha CYB2 gene, encoding FC b2. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (gcr1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b2 activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b2 producer characterized by a sixfold increased (to 3 micromol min(-1) mg(-1) protein in cell-free extract) activity of the enzyme.

A recombinant yeast clone, a derivative of the recipient Hansenula polymorpha strain NCYC 495, was chosen as an NAD and glutathione-dependent formaldehyde dehydrogenase overproducer. Optimal cultivation conditions for the highest yield of enzyme were established. A simple scheme for the isolation of formaldehyde dehydrogenase from the recombinant strain was proposed, and some characteristics of the purified enzyme were studied. An enzymatic method for formaldehyde assay based on formaldehyde dehydrogenase was developed and used for testing real samples.

Background: Accurate, rapid, and economic on-line analysis of ethanol is very desirable. However, available biosensors achieve saturation at very low ethanol concentrations and thus demand the time and labour consuming procedure of sample dilution., Results: Hansenula polymorpha (Pichia angusta) mutant strains resistant to allyl alcohol in methanol medium were selected. Such strains possessed decreased affinity of alcohol oxidase (AOX) towards methanol: the KM values for AOX of wild type and mutant strains CA2 and CA4 are shown to be 0.62, 2.48 and 1.10 mM, respectively, whereas Vmax values are increased or remain unaffected. The mutant AOX alleles from H. polymorpha mutants CA2 and CA4 were isolated and sequenced. Several point mutations in the AOX gene, mostly different between the two mutant alleles, have been identified. Mutant AOX forms were isolated and purified, and some of their biochemical properties were studied. An amperometric biosensor based on the mutated form of AOX from the strain CA2 was constructed and revealed an extended linear response to the target analytes, ethanol and formaldehyde, as compared to the sensor based on the native AOX., Conclusion: The described selection methodology opens up the possibility of isolating modified forms of AOX with further decreased affinity toward substrates without reduction of the maximal velocity of reaction. It can help in creation of improved ethanol biosensors with a prolonged linear response towards ethanol in real samples of wines, beers or fermentation liquids.

For the first time, spectrometric and electrochemical studies demonstrated the possibility of using artificial electron acceptors in reactions catalyzed by alcohol oxidase. We report kinetic parameters of homogenous catalytic oxidation of formaldehyde by organic redox compounds belonging to different structural classes (toluidine blue, methylene blue, 2,6-dichlorophenolindo-phenol, and p-benzoquinone) and replacing dioxygen in these reactions. p-Benzoquinone, having the highest redox potential, proved to be the most efficient artificial electron acceptor of all compounds studied.

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.

Screening of strains producing a stable form of L-lactate cytochrome c oxidoreductase (flavocytochrome b2, FC b2) was carried out among 14 yeast species. Enzyme activity was detected in polyacrylamide gel after the electrophoresis of cell-free extracts. The FC b2 of Hansenula polymorpha, Rhodotorula pilimanae, and Kluyveromyces lactis are characterized by high thermostability; in particular, the FC b2 of H. polymorpha retains its activity and tetrameric structure even after heating at 60 degrees C for 10 min. Constitutive synthesis of FC b2 was observed in H. polymorpha grown on either glucose, ethanol, or glycerol. L-Lactate induces de novo synthesis of FC b2, as proved by the use of cycloheximide, an inhibitor of protein synthesis.

A new alcohol oxidase-peroxidase method of determination of ethanol content in fermented musts and wine products is described and compared to conventional methods routinely used in winemaking. The sensitivity, accuracy, and reliability of this method were determined. The results of ethanol determination in fermented musts and wines correlated well with the data obtained by refractometry (correlation coefficient R = 0.9595, p < 0.0001) and densitometry (correlation coefficient R = 0.9384, p < 0.0001). This method is less time- and labor-consuming and allows simultaneous testing a series of wine samples.

It is necessary to create very specific and sensitive methods for assaying formaldehyde and methanol which are produced on the large scale and are very toxic and have mutagenic and carcinogenic action on living organisms. The methods for determination of formaldehyde, methanol and ethanol in the environment and fermentation products published and developed by the authors are reviewed in this paper. Most of the known methods are not sufficiently selective and sensitive and some of them are very expensive. Classical chemical, enzymatic, chemosensor and biosensor approaches used for methanol and formaldehyde assay are described. Enzymatic methods exploiting alcohol oxidase isolated from the mutant over-producing strain of methylotrophic yeast Hansenula polymorpha permit efficient determination of formaldehyde in industrial wastewaters. Enzymatic-chemical method based on the use of alcohol oxidase and 4-amino-5-hydrazine-3-mercapto-1,2,4-triazole (AHMT) allows simultaneous determination of methanol and formaldehyde. The technology of biosensor construction and their bioanalytical characteristics are described. Experimental data concerning amperometric and potentiometric biosensors based on the use of genetically modified cells of methylotrophic yeast Hansenula polymorpha are reviewed. The possibility to use alcohol oxidase-based biosensor for the assay of methanol in wastewater is demonstrated.

A new enzymo-chemical method for the simultaneous assay of methanol and formaldehyde in mixtures is described which exploits alcohol oxidase (AO) and aldehyde-selective reagent, 3-methyl-2-benzothiazolinone hydrazone (MBTH). The enzyme is used for methanol oxidation to formaldehyde and MBTH plays a double role: 1) at the first step of reaction, it forms a colorless azine adduct with pre-existing and enzymatically formed formaldehyde and masks it from oxidation by AO; 2) at the second step of reaction, non-enzymatic oxidation of azine product to cyanine dye occurs in the presence of ferric ions in acid medium. Pre-existing formaldehyde content is assayed by colorimetric reaction with MBTH without treating samples by AO, and methanol content is determined by a gain in a colored product due to methanol-oxidising reaction. Possibility of differential assay of methanol and formaldehyde by the proposed method has been proved for model solutions as well as for real samples of industrial waste and technical formaline. A threshold sensitivity of the assay method for both analytes is near 1 microM that responds to 30-32 ng analyte in 1 ml of reaction mixture and is 3.2-fold higher when compared to the chemical method with the use of permanganate and chromotropic acid. Linearity of the calibration curve is reliable (p < 0.0001) and standard deviation for parallel measurements for real samples does not exceed 7%. The proposed method, in contrast to the standard chemical approach, does not need the use of aggressive chemicals (concentrated sulfuric, phosphoric, chromotropic acids, permanganate), it is more simple in fulfillment and can be used for industrial wastes control and certification of formaline-contained stuffs.

Yeast Pichia guilliermondii strains L3 and L2, exposed to UV mutagenesis, produced over 80 mutants capable of growing on media containing 1.5 mM bichromate (Cr(VI)). The mutations making the strains resistant to Cr(VI) were dominant or semidominant. The mutants varied in Cr(VI) resistance, the degree of chromium accumulation in the cells (from 0.1 to 11.6 mg/g dry cells), and the degree of Cr(VI) reduction (from 50% to complete disappearance of bichromate from the culture liquid). Chromium accumulation in mutant cells depended on medium composition, Cr(VI) concentration, and the time of exposure to Cr(VI). The resistance to bichromate can be caused by various reasons: decrease in chromium absorption, altered ability to reduce Nr(VI), or damage of sulfate transport mechanisms.

A new method of visualization of the activity of flavocytochrome b2 (FC b2; L-lactate: ferricytochrome c oxidoreductase, EC 1.1.2.3) in electrophoretograms was developed, based on the interaction between ferrocyanide (generated during the enzymatic reaction) and Fe23+, resulting in the formation of intensely colored precipitates of Berlin blue. The main advantages of this method were its high sensitivity (less than 0.005 U FC b2 was detected within a suitable time period) and the stability of the dye formed. The method developed can be used for determining FC b2 activity in cell-free extracts (e.g., in the selection of FC b2 producers) and monitoring chromatographic purification of proteins, as well as in other cases associated with FC b2 assessment.

Cytochrome c from the methylotrophic yeast Hansenula polymorpha was isolated and purified to homogeneity for the first time. The final yield of the highly purified protein from 1.4 kg (wet weight) cells was about 20 mg. The hemoprotein has an apparent molecular mass of 12 kDa and isoelectric point (pI) of 9.3. The purified protein was characterized by electronic, EPR and NMR spectroscopies. The redox potential of the cytochrome, E degrees, measured by cyclic voltammetry measurements at neutral pH, is 0.302 V. Both NMR spectroscopy and electrochemical measurements confirm the presence in the solution of several acid-base equilibria, the most pronounced being characterized by a pK(a) of 8.3. The latter pK(a) was attributed to the detachment of the iron(III) ion-coordinated methionine and its replacement by a lysine residue. The electrochemically derived thermodynamic parameters for neutral and alkaline protein species (DeltaS degrees (rc) and DeltaH degrees (rc)) were obtained from the temperature dependence of the redox potential.

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., (Copyright 2000 John Wiley & Sons, Inc.)

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.

A new concept based on own experimental results and published data and concerning the involvement under extreme conditions of additional (nontraditional) mechanisms of formaldehyde, formate and hydrogen peroxide detoxification in methylotrophic yeast has been proposed. It has been shown that neutralization of toxic effect of formaldehyde and formate, except the known ways, includes a system of energy-dependent extrusion of formic acid into extracellular space. Detoxification of hydrogen peroxide, besides of catalase way, can be realized by means of cytochrome c peroxidase reaction. The proposed concept has been built on the basis of physiological and biochemical analysis of specially constructed mutants strains of methylotrophic yeast with certain metabolic damages. The selected mutants, used as the instrument in fundamental studies of detoxification processes, also have been successfully used for practical purposes in construction of cell biosensors and creation of enzymatic kits.

The dynamics of prostaglandin (PG) E2 synthesis by mouse peritoneal macrophages during the delivery of the basic substrate, arachidonic acid (AA), from different sources to the enzyme system of the cells was investigated. The dynamics of PGE2 synthesis in these cells was studied both after addition of exogenous AA and after stimulating the liberation of AA from intracellular pools with the calcium ionophore A23187. The kinetics of PGE2 synthesis when AA was supplied from intracellular and extracellular sources were absolutely different. PGE2 metabolism and the inactivation of the key enzyme of PG synthesis (PGH-synthase) during the reaction may be the regulating factors in the kinetics of PGE2 synthesis in the cells. For the different sources of AA in the cells, the rate constants of PGE2 consumption (k2) and PGH-synthase inactivation in the course of the reaction (kin) were calculated. The experimentally determined value of the apparent rate constant kin was identical to the theoretically calculated kin value for the case when AA was provided from an intracellular source. An observed deceleration in the PGE2 synthesis kinetics from exogenous AA is characterized by a 10-fold drop in the apparent kin and k2 values. The possibility of prostanoid synthesis regulation at the level of the traditional, constitutive isoenzyme PGH-synthase-1 is discussed.

Two types of alcohol-specific microbial/electrochemical biosensors have been developed using specially constructed mutant cells of the methylotrophic yeast Hansenula polymorpha. The cells were immobilized in a calcium alginate gel, and placed between two membranes on the surface of oxygen or hydrogen peroxide-electrodes. The O2 electrode based biosensor contained mutant cells with strongly elevated alcohol oxidase activity. The peroxide electrode based biosensor consisted of catalase-defective mutant cells which produce hydrogen peroxide in the presence of alcohol. Both types of mutant cells were used in permeabilized form in order to release some components of the cellular respiration system, thus increasing the selectivity of the cellular respiration response to alcohol (cell/O2-biosensor) Permeabilization also increased sensitivity of the signal and shortened the response time (cell/H2O2-biosensor). Cell/O2 biosensors were linear up to 1.2 mM for ethanol and 0.35 mM for methanol, cell/H2O2 biosensors were linear up to 4.0 mM for ethanol, and 1.2 mM for methanol. Results were reproducible, sample pretreatment was not required, and the sensors exhibited good operational and storage stability. The use of sucrose, dulcitol or inositol during the preparation of the sensors resulted in increased stability of cells during their liophilization and storage in the dried state. Both biosensors had similar selectivity towards alcohols in the order of methanol (100%), ethanol (21%), and formaldehyde (12%). No signal was observed with glucose or glycerol as substrates.

The changes in AA incorporation and release as well as prostanoid synthesis upon differentiation of human premonocytic cell line, U937, induced by three functionally diverse agents--phorbol ester (TPA), dimethyl sulfoxide (DMSO), and retinoic acid (RA) have been investigated. The rate of AA incorporation into the cells remained unchanged whereas a 3- to 6-fold increase in AA release upon stimulation with Ca(2+)-ionophore A23187 as compared to undifferentiated cells was observed. While undifferentiated cells were incapable to metabolise AA via the cyclooxygenase pathway all three types of differentiated U937 cells produced TxB2 and PGE2. Only TPA-differentiated cells responded with a 6-fold increase of prostanoid synthesis after A23187 stimulation, whereas in DMSO-differentiated cells prostanoid synthesis was slightly stimulated by A23187 and in RA-differentiated cells it was not stimulated at all. Thus, agonist-induced prostanoid synthesis in differentiated cells is dependent on the nature of differentiating agent and does not correlate with AA liberation.

Prostaglandins (PGs) have an important physiological role in the modulation of various cell immune functions. The main sources of PGs during immune responses are monocyte cells. We report here the ability of non-stimulated macrophages to synthesize prostanoids and show that peritoneal mouse macrophages synthesize PGE2, PGF2a and thromboxane B2, spleen macrophages produce PGE2 and PGF2a, and in a fresh medium this synthesis reaches a constant basal level in a few hours. We studied the kinetics of Con A-induced proliferation of murine splenocytes under the influence of a wide range of PGE2 concentrations (10(-14)-10(-7) M). The suppressive effect of PGE2 decreased when its concentration was lowered and disappeared at 10(-9) M PGE2 (this concentration corresponded to the basal level of non-stimulated macrophage synthesis of PGE2). Further lowering of the concentration became essential for the proliferation process once again, and at picomolar concentrations PGE2 caused a suppressive effect comparable with that for 10(-8) M PGE2. We also found that PGE2 significantly inhibited cell proliferation when it was added 1 h before the addition of mitogen, as compared with simultaneous mitogen addition. The effect was obtained for both low (10(-12) M) and high (10(-8) M) PGE2 concentrations. This phenomenon of PGE2 biphasic control of lymphocyte proliferation may play an important role in cellular homeostasis, in particular in immune cell function regulation.

Mutant strains of the methylotrophic yeast Hansenula polymorpha defective in catalase (cat) and in glucose repression of alcohol oxidase synthesis (gcr1) have been isolated following multiple UV mutagenesis steps. One representative gcr1 cat mutant C-105 grows during batch cultivation in a glucose/methanol medium. However, growth is preceded by a prolonged lag period. C-105 and other gcr1 cat mutants do not grow on methanol medium without an alternative carbon source. A large collection of second-site suppressor catalase-defective (scd) revertants were isolated with restored ability for methylotrophic growth (Mth+) in the absence of catalase activity. These Mth+ gcr1 cat scd strains utilize methanol as a sole source of carbon and energy, although biomass yields are reduced relative to the wild-type strain. In contrast to the parental C-105 strain, H2O2 does not accumulate in the methanol medium of the revertants. We show that restoration of methylotrophic growth in the suppressor strains is strongly correlated with increased levels of the alternative H2O2-destroying enzyme, cytochrome c peroxidase. Cytochrome c peroxidase from cell-free extracts of one of the scd revertants has been purified to homogeneity and crystallized.

Nonmethylotrophic (Candida maltosa and Saccharomyces cerevisiae) and methylotrophic (Hansenula polymorpha) yeast cells acidified their incubation media in the presence of formaldehyde. This was associated with the release of formate. We studied the formaldehyde-dependent production of formic acid and the enzymatic properties of these strains grown on media containing various carbon sources. The acidifying potential was considerably lower in formaldehyde dehydrogenase-deficient cells of mutant strains of H. polymorpha. The rates of acidification by C. maltosa and S. cerevisiae depended on the activity of their nonspecific aldehyde dehydrogenases. We suggest that accumulation of formate by yeast cells incubated in the presence of formaldehyde is caused by the total activity of formaldehyde dehydrogenase and nonspecific aldehyde dehydrogenase in methylotrophic yeasts or aldehyde dehydrogenase only in nonmethylotrophic yeasts. This is probably an additional mechanism for detoxification of formaldehyde.

The effect of low (10(-10)-10(-14 M) ibuprofen concentrations on the release of labeled arachidonic acid metabolites by mouse peritoneal macrophages containing a constitutive isoform of prostaglandin H synthase was investigated. It was found that during the activation the cells metabolized AA through the cyclooxygenase pathway, synthesizing PGE2 (110 +/- 10 ng per 10(6) cells), PGF(2 alpha) (120 +/- 15 ng per 10(6) cells), and TxB2 (48 +/- 5 ng per 10(6) cells). Incubation of the macrophages with 10(-12) M ibuprofen leads to a sharp increase of PGf2 and PGF(2 alpha) synthesis (315 +/- 84 and 320 +/- 20 ng per 10(6) cells, respectively). A microsomal preparation of macrophage PGH-synthase was compared with PGH-synthase of ram seminal vesicles and analogy of their properties was shown (Km = 5.3.10(-5) M, IC50, ibuprofen = 15 microM). Some particularities of the PGH-synthase consecutive isoform (PGH-synthase-1) cellular regulation are discussed.

The interest in the prostaglandin (PG) synthesis by animal cells today grows steadily because of the difficulties in obtaining them by any other way. Murine peritoneal macrophages can under certain conditions synthesize large amounts of PGs. The effect of well-known nonsteroidal anti-inflammatory drug ibuprofen on PG synthesis by the cells using a high-performance liquid chromatography (HPLC) method with fluorescence detection of 4-bromomethyl-7-methoxy-coumarin (BrMMC) derivatives was studied. In our case, the main metabolites were PGE2 and PGF2a. The PG synthesis activation effect was shown by ibuprofen concentrations in the 10(-10)-10(-14) M range with the maximum effect of 10(-12)M. In this case, the ibuprofen effect was comparable in value with the effect of well-known cell PG synthesis activator--calcium ionophore A23187. Although the exact mechanism of such an effect is not clear at the moment, at low concentration, ibuprofen itself is able to activate PG synthesis in murine peritoneal macrophages.

The effect of 10(-14)-10(-4)M ibuprofen and aspirin both on arachidonic acid metabolism in peritoneal murine macrophages and on the concanavalin A-induced proliferation of murine splenocytes were investigated. It was shown that 10(-7)-10(-4)M ibuprofen inhibits the arachidonic acid metabolism. On the other hand, 10(-12)-10(-11)M ibuprofen causes pronounced activation of arachidonic acid metabolism. The low concentration (10(-14)-10(-10)M) effects also take place when non-steroidal anti-inflammatory drugs influence other functions of the immune system: that is, they activate the splenocyte mitogen-induced proliferative response. These results are in accord with our suggestion that the low concentration effects of these drugs do not depend upon cell types and may have an important physiological significance.

A cell biosensor specific for formaldehyde was developed using double-mutant cells of the methylotrophic yeast Hansenula polymorpha A3-11. The activities of some of the enzymes in the metabolic pathway of the wild-strain cells were deliberately suppressed by introducing respective genetic blocks to optimize the selectivity and acidification rate. Mutant yeast cells produced in this way were immobilized in Ca-alginate gel on the gate of a pH-sensitive field effect transistor. The local acidification of the extracellular medium due to specific conversion of formaldehyde was recorded. The steady-state response time of the biosensor was 2-3 min, i.e., about 10 times shorter than the response time for the alcohol-specific cell biosensors described earlier. The linear dynamic range of the sensor's response corresponds to formaldehyde concentrations of 2 to 200 mM. The operational stability of the sensor was not less than 4 h. The biosensor demonstrated high specificity to formaldehyde with no response to several organic acids, methanol, and other alcohols, except for low sensitivity to ethanol. The influence of sample buffer capacity and pH on the sensor response, as well as thermostability, was investigated.

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.

Citrate and dihydroxyacetone inhibited [14C] incorporation from radioactive methanol to CO2 by washed cells of methylotrophic yeast Hansenula polymorpha grown in the media containing mixture of methanol with citrate or dihydroxyacetone, respectively. These results are discussed in connection with the earlier hypothesis on participation of the tricarboxylic acid cycle in the energy supply of the methylotrophic growth.

Studies have been made on the peroxidase activity of metmyoglobins in animals from various ecological groups--the horse Equus caballus, cattle Bos taurus, beaver Castor fiber, otter Lutra lutra, mink Mustela vison and dog Canis familiaris. It was found that the level of this activity in diving animals depends on the duration of their diving, whereas in terrestrial species--on the strength of muscular contraction.

Using ion-exchange and affinity chromatography and isoelectrofocusing, eight forms of pepsin with pI 1.6, 1.8, 2.1, 2.3, 2.6, 2.8, 3.2 and 3.6, were isolated from horse gastric juice. The molecular weights, amino acid composition, N-terminal sequence and functional activity of these multiple forms were determined. Partial primary structure of tryptic peptides of pepsin with pI 2.3 was investigated. The analyzed partial sequences of the forms with pI 1.8, 2.1, 2.3, and 2.6 have identical structures which differ from the amino acid sequence of pepsin with pI 3.2 by four substituents. In terms of their functional activity, horse pepsins differ only insignificantly. Presumably, the pepsins under study (at least the forms with pI 1.8, 2.1, 2.3, 2.6 and 3.2) arose comparatively recently as a result of duplication of the common precursor gene and exist at an early stage of structural and functional divergence. As far as their primary structure and functional properties are concerned, these pepsins are more related to pepsin A than to other isoenzymes of gastric aspartyl proteinases of mammalia, e. g., gastricsin or chymosin.

Considered are our own data and those found in literature on the properties of yeast mutants impaired in their ability to utilize methanol as sole carbon and energy source; hypotheses about the role of alcohol oxidase and citrate synthase in biogenesis of peroxisomes are proposed. It has been proved that formaldehyde reductase participates in the control of the formaldehyde level in the cell. Properties of mutants defective in the catabolite repression and inactivation of enzymes of methanol metabolism are described. The existence of several autonomous mechanisms of the catabolite repression of alcohol oxidase has been shown. It has been found, that the induction of glyoxysomal enzymes of C2-metabolism is repressed by methanol in the ecr1 mutant of Pichia pinus with the affected repression of alcohol oxidase by ethanol. Data are presented on the regulatory properties of the recently discovered acidification system of the medium induced by methanol. Such acidification occurs due to symport extrusion of protons and formate anions from the cells.

6 forms of pepsin are found in horse gastric juice. Amino acid sequence is determined of N-terminal (most variable) part of polypeptide chain of main pepsin components. Equine pepsines, which have pI 2.1 and 2.3, are found to have identical amino acid sequence at least for 31 amino acid residues. The same sequence is observed in the component with pI 2.6 for 10 first residues. The sequence of equine pepsin with pI 3.2 has 3 substitutions for 33 amino acids, when compared with pepsines having pI 2.1 and 2.3. The forms of equine pepsin studied are more similar than the other isoenzyme pair, human pepsin and gastricsin.

Chromatography of commercial rennet was studied on biospecific sorbents obtained by means of coupling of activated Sepharose 4B with epsilon-aminocapronyl-D-phenylalanine methyl ester and amide, epsilon-aminocapronyl-L-phenylalanyl-D-phenylalanine methyl ester, gramicidin S and N-2,4-dinitrophenylhexamethylenediamine. A mixture of two similar on their specificity enzymes chymosin and bovine pepsin was isolated from rennet by the chromatography on these sorbents. The individual enzymes might be isolated by chromatography on immobilized ribonuclease at pH 3,0, or by means of electrofocusing in pH gradient 4-6. Coloured inhibitor of acid proteases, N-diazoacetyl-N'-2,4-dinitrophenyl-ethylenediamine (DDE) is found to inactivate chymosin at pH 5,6 in the presence of Cu2+,one residue of the inhibitor being attached to the enzyme molecule. Unlike pig pepsin, chymosin is not inhibited with DDE at pH 4,7 and at the enzyme:DDE:Cu2+ ratio being 1:40:40. a synthesis of peptide sorbents is described.

The washed cells of Hansenula polymorpha and Pichia pinus grown on the medium with methanol rapidly acidify the medium during incubation with the mentioned alcohol or formaldehyde. It is found that proton extrusion is coupled with formate anion efflux. Acidification is proved to be energy-dependent process since it is inhibited by respiration poisons, uncouplers of oxidative phosphorylation, and by ATPase inhibitors.