Purification and characterization of intracellular and extracellular, thermostable and alkali-tolerant alcohol oxidases produced by a thermophilic fungus, Thermoascus aurantiacus NBRC 31693

Intracellular and extracellular alcohol oxidases (AOint and AOext) were purified from the liquid and solid cultures of a thermophilic fungus, Thermoascus aurantiacus NBRC 31693, as electrophoretically and isoelectrophoretically homogeneous proteins, respectively. Both enzymes contained a flavin adenine dinucleotide (FAD) cofactor and were stained with Schiff's reagent. The molecular weight of AOint was estimated to be about 320 kDa and its subunit was 75 kDa. The molecular weight of AOext was about 560 kDa, and it was composed of two types of subunits (75 kDa and 59 kDa). The pIs of AOint and AOext were 5.88 and 6.08, respectively. AOint and AOext were stable up to 60°C and 55°C, respectively. The enzymes were stable over a wide range of pH from 6 to 11. AOint oxidized short straight-chain alcohols (Km for methanol, 13.5 mM and Km for ethanol, 15.8 mM). On the other hand, AOext could oxidize secondary alcohols and aromatic alcohols (veratryl alcohol and benzyl alcohol) in addition to straight-chain alcohols (Km for methanol, 0.5 mM and Km for ethanol, 10.2 mM).