ABSTRACT
Enzymatic method for formaldehyde assay, based on formaldehyde dehydrogenase from the recombinant strain of the yeast Hansenula polymorpha, was developed. Linear detection range for the proposed method was estimated to be from 0.01 to 0.05 mM, and sensitivity of the assay (or detection limit) was determined as 0.007 mM. In comparison with the known methods, the described procedure is rather simple: the method does not require transformation of formaldehyde into chemical adduct for the extraction of the target analyte from the tested sample. As compared to chemical methods, the analysis time is shorter and some dangerous operations (e.g. heating in strong acid) are not required. The developed method is approved on the real samples which contained formaldehyde, and data are well correlated with the results of the known chemical methods.
Subject(s)
Aldehyde Oxidoreductases/chemistry , Formaldehyde/analysis , Pichia/enzymology , Recombinant Proteins/chemistry , Aldehyde Oxidoreductases/genetics , Formaldehyde/chemistry , Genetic Engineering , Oxidation-Reduction , Pichia/genetics , Recombinant Proteins/geneticsABSTRACT
A new oxidase-coupled colorimetric method for analysis of L-lactate in biological fluids has been developed without use of peroxidase. The method is based on lactate oxidase-catalysed transformation of lactate to pyruvate which is determined photometrically in the next dye-producing reaction of 3-methyl-2-benzothiazolinone hydrazone (MBTH) in the presence of ferric ions. Sensitivity of the method is estimated as 0.1 micromole of analyte in 4-ml of reaction mixture. Linearity is observed in the range 0.1-1.0 micromole of L-lactate in sample (r = 0.99943; p < 0.0001). The developed method has been adapted for assay of L-lactic acid in kefirs and yogurts.
Subject(s)
Lactic Acid/metabolism , Mixed Function Oxygenases/metabolism , Catalysis , Colorimetry , Sensitivity and SpecificityABSTRACT
An extended definition of the term "metabolic engineering" is given and main spheres of its using in fundamental studies and modern biotechnology are discussed in this article. Emphasis is made on specific using the approaches of metabolic engineering in construction of the cell elements of sensors based on the use of mutant and chemically modified cells of methylotrophic yeasts. This investigation is designed in the laboratory of Biochemical Genetics of the Division of Cell Regulatory Systems, A. V. Palladin Institute of Biochemistry. Genetic and chemical modifications have allowed to provide some directed changes in cell sensoring output toward methanol, ethanol and formaldehyde that result in enhanced selectivity and shortened time-output of the corresponding potentiometric and amperometric sensors.
Subject(s)
Biomedical Engineering , Biosensing Techniques , Cell Biology , Electrochemistry , MetabolismABSTRACT
The sensitive method for peroxidative assays of hydrogen peroxide (up to 5 nmole in 1 ml) and oxidases' substrates (glucose, ethanol) has been developed by use of a non-cancerogenic chromogen--3,3', 5,5'-tetramethylbenzidine (TMB), horseradish peroxidase and corresponding oxidases. The proposed modification of the known method consists in using decreased concentration of TMB (0.05-0.1 mM) and stopping the reaction by adding of acid to pH 1.4-2.1. This approach allowed to enhance the sensitivity of assays of corresponding analytes (7-10 times) and decrease the costs of analysis.
Subject(s)
Ethanol/analysis , Glucose/analysis , Hydrogen Peroxide/analysis , Oxidoreductases , Benzidines/metabolism , Chromogenic Compounds , Horseradish Peroxidase/metabolism , Hydrogen-Ion ConcentrationABSTRACT
The photometric method is suggested for ethanol analysis by means of purified alcohol oxidase isolated from a wild strain of methylotrophic yeast Hansenula polymorpha. Three different chromogenic systems for peroxidative oxidation together with alcohol oxidase reaction have been used. For the first time the intact cells of the catalase-negative mutant as a source of alcohol oxidase are employed in the photometric alcohol assay. The prospects for application of the latter method for practical purposes are discussed.
