Alcohol Oxidases of Kloeckera sp. and Hansenula polymorpha. Catalytic Properties and Subunit Structures

Abstract

1. Alcohol oxidase (alcohol : oxygen oxidoreductase) of a thermophilic methanol-utilizing yeast, Hunsenula polymorpha DL-1 , was isolated in crystalline form.2. This alcohol oxidase of H . polymorphu was more stable to heat than was the enzyme of Kloeckera sp. This difference in heat stability is compatible with the difference in growth temperatures for both yeasts.3. The crystalline alcohol oxidases of both yeasts oxidized the lower primary alcohols ((2-2 to C-4) as well as methanol. The apparent K,,, values for the methanol of Kloeckera and H. polymorpha enzymes were 0.44 and 0.23 mM, respectively. The enzymes could also oxidize formaldehyde to formate, and were inactivated by relatively low concentrations of hydrogen peroxide.4. The molecular weight for both enzymes was calculated to be about 670000. Each enzyme is composed of eight identical subunits (molecular weight 83000) and contains eight moles of FAD as the prosthetic group. The NH,-terminal and COOH-terminal amino acids of H . polymorpha enzyme were identified as alanine and phenylalanine, respectively. The octameric subunits model of each enzyme was confirmed by electron micrographs, which showed an octad aggregate, composed of two tetragons face to face.

Characterization of alcohol oxidase from Aspergillus ochraceus AIU 031

“…The K m value for ethylene glycol was smaller than that of AODs from Candida sp. and Pichia pastoris (Isobe and Nishise, 1994), and that for ethanol was larger than that of AODs from C. boidinii (Tani et al, 1972), H. polymorpha (presently named P. angusta; Kato et al, 1976) and Kloeckera sp. (presently named C. boi-2007 Alcohol oxidase from Aspergillus ochraceus 181 Fig.…”

“…The characteristics of those AODs and the regulation of the gene encoding AODs have been studied in detail (Couderc and Baratti, 1980;Kato et al, 1976;Ozimek et al, 2005;Tani et al, 1972Tani et al, , 1985. In the production of AODs by filamentous fungi, Ko et al have recently reported that Thermoascus aurantiacus NBRC 31963 inductively produced two AODs by incubation with the pectin medium, and characteristics of both enzymes have been revealed by his group (Ko et al, 2005).…”

Flavin‐dependent alcohol oxidase from yeast

“…Qualitatively inactivation by H 2 0 2 has also been shown with alcohol oxidases from Pichia pastoris [26] and Kloeckera sp. 2001 [27]. The Pichia enzyme can be reactivated by incubation with 1 mM GSH for 60 min at 25 "C [26], whereas the Cundida enzyme remains inactive under these conditions (data not shown).…”

Formaldehyde Oxidizing Enzymes and Genetically Modified Yeast Hansenula polymorpha Cells in Monitoring and Removal of Formaldehyde

“…Besides FdDH, FA can be easily oxidized by alcohol oxidase (AOX) (EC 1.1.3.13), an enzyme which is responsible in vivo for the first reaction of methanol metabolism in methylotrophic yeast (Klei van der et al, 1990). AOX is not an absolutely selective enzyme and oxidizes the hydrated form of FA to formic acid without any exogenous cofactor (Kato et al, 1976). The theoretical possibility of AOX using for FA assay is based on a known fact that FA exists in aqueous solutions in the hydrated form (95-99% of total concentration) which has a structural resemblance to methanol and can be oxidized by AOХ` with the subsequent formation of formic acid and hydrogen peroxide according to the following reactions: …”