[Thermodynamic behaviour and conformational changes of alcohol oxidase of yeasts Hansenula polymorpha].

We studied influence of heating, ethanol and sodium azide on the structural and conformational changes of the alcohol oxidase from yeast Hansenula polymorpha. The increase of fluorescence of alcohol oxidase -cofactor, flavin adenine dinucleotide, was revealed when heated to 60 degrees C while the enzymatic activity of alcohol oxidase remained unchanged. Differential scanning microcalorimetry revealed that ethanol stabilized the protein structure and increased the temperature of melting, Based on the data of circular dichroism, we concluded that the percentage of helicities in the secondary structure of alcohol oxidase was not influenced by both ethanol and sodium azide, however ethanol significantly modified the circular dichroism spectrum associated with the tertiary structure of alcohol oxidase.

Formation and distribution of modified FAD between isozymes of alcohol oxidase in the methylotrophic yeast pichia methanolica.

The content of modified FAD (mFAD) in isoforms of alcohol oxidase (AO) in the methylotrophic yeast Pichia methanolica MH4 is increased in the stationary growth phase of the culture or under lower dilution rates. The isoform with slower electrophoretic mobility has the higher mFAD content. The results of in vitro experiments and the occurrence of mFAD in AO-lacking mutants of Hansenula polymorpha DL-1 imply a biosynthetic origin of this cofactor. HPLC analysis of tryptic hydrolysates of the first and the ninth isoforms of AO from P. methanolica MH4 revealed two different types of subunits. Possible mechanism of mFAD formation and distribution between the AO isozymes is discussed.