Pavlishko H. Oxidases of methylotrophic yeast and fungi: characterization and bioanalytical application.

The objeсt - oxidases of methylotrophic yeast and fungi of bioanalytical importance. The aim: screening of microbial producers of oxidases of the bioanalytical importance (alcohol oxidase and glycerol oxidase), isolation and characterization, and development of the new enzymatic and biosensor methods based on alcohol oxidase for analysis of ethanol, methanol and formaldehyde. Methods of studies: screening of producer strains, determination of enzyme activities, electrophoresis in polyacrylamide gel, ion echange chromathography, absorbation spectroscopy, izoelectrofocusation, chemical, enzymatic and biosensor analysis. The thesis are devoted to the isolation, physico-chemical, enzymological characterization and bioanalytical application of alcohol oxidase from methylotrophic yeast Hansenula polymorpha. Another tasks of the work were screening some fungi as possible producers of glycerol oxidase, its isolation and initial investigation of its structure and enzymological characteristics. The effectivescheme of preparative isolation of highly purified alcohol oxidase from mutant cells of methylotrophic yeast H. polymorpha. was developed. Some physico-chemical and enzymological characteristics of the enzyme were investigated. A new enzymo-chemical method for ethanol assay was developed. Using alcohol oxidase and 3-methyl-2-benzothiazolinone hydrazine, a new enzymo-chemical method for selective assay of methanol and formaldehyde in a mixture was proposed. A new prototype of amperometric biosensor based on highly purified alcohol oxidase from H. polymorpha, horseradish peroxidase and osmium-containing redox-paint was developed. The main characteristics of the sensor were investigated and possibility of its application for assay of alcohol in wines was approved. Field of application: biochemistry of microorganisms, analytical biotechnology, production of alcoholic beverages.