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 (AO int and AO ext) 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 AO int was estimated to be about 320 kDa and its subunit was 75 kDa. The molecular weight of AO ext was about 560 kDa, and it was composed of two types of subunits (75 kDa and 59 kDa). The pIs of AO int and AO ext were 5.88 and 6.08, respectively. AO int and AO ext were stable up to 60 degrees C and 55 degrees C, respectively. The enzymes were stable over a wide range of pH from 6 to 11. AO int oxidized short straight-chain alcohols (K(m) for methanol, 13.5 mM and K(m) for ethanol, 15.8 mM). On the other hand, AO ext could oxidize secondary alcohols and aromatic alcohols (veratryl alcohol and benzyl alcohol) in addition to straight-chain alcohols (K(m) for methanol, 0.5 mM and K(m) for ethanol, 10.2 mM).
Ko
Hee-Sun
HS
Department of Bioresources Science, Graduate School of Science and Technology, Chiba University, 648 Matsudo, Matsudo-city, Chiba 271-8510, Japan.
Yokoyama
Yuichi
Y
Ohno
Nobuko
N
Okadome
Miho
M
Amachi
Seigo
S
Shinoyama
Hirofumi
H
Fujii
Takaaki
T
D000429:Alcohol Oxidoreductases / Q000032:analysis* / Q000737:chemistry* / Q000145:classification; D000468:Alkalies / Q000737:chemistry*; D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology*; D004789:Enzyme Activation; D004795:Enzyme Stability; D045604:Extracellular Fluid / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007424:Intracellular Fluid / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013696:Temperature
D000468:Alkalies; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase