Amperometric biosensors for L-arginine and creatinine assay based on recombinant deiminases and ammonium-sensitive Cu/Zn(Hg)S nanoparticles.

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.

Novel arginine deiminase-based method to assay L-arginine in beverages.

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.

[L-arginine assay with the use of arginase I].

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.

[Human arginase I from the recombinant yeast Hansenula polymorpha: isolation and characterization of the enzyme].

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.

[Construction of flavocytochrome b2-overproducing strains of the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta)].

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.

[Oxygen can be replaced by artificial electron acceptors in reactions catalyzed by alcohol oxidase].

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.

[Screening of yeasts producing stable L-lactate cytochrome c oxidoreductase and regulation of enzyme synthesis].

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.

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.

[Oxidase-peroxidase method for determination of ethanol in fermented musts and wine products].

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.

[Modern methods for formaldehyde, methanol and ethanol analysis].

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.

[Selection and properties of mutant yeast Pichia guilliermondii strains resistant to chromium (VI)].

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 enzymo-chemical method for simultaneous assay of methanol and formaldehyde.

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.

[New method for detection of enzymatic activity of flavocytochrome b2 in electrophoregrams]. / Novyi metod obnaruzheniia fermentativnoi aktivnosti flavotsitokhroma b2 na élektroforegrammakh.

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.

Isolation and physico-chemical characterization of a cytochrome c from the methylotrophic yeast Hansenula polymorpha.

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.

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.

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.

[Alternative mechanisms of detoxication of formaldehyde, formate, and hydrogen peroxide in methylotrophic yeasts]. / Al'ternatyvni mekhanizmy detoksikatsiï formal'degidu, formiatu ta peroksidu vodniu u metylotrofnykh drizhdzhiv.

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.

Arachidonic acid regulation of prostanoid synthesis in macrophages.

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.