Substrate specificity and properties of the aryl-alcohol oxidase from the ligninolytic fungus Pleurotus eryngii.
The production in a 5-1 fermenter of the extracellular enzymes laccase and aryl-alcohol oxidase by the fungus Pleurotus eryngii was studied. The latter enzyme has been purified 50-fold by Sephacryl S-200 and Mono Q chromatography. Purified aryl-alcohol oxidase is a unique flavoprotein with 15% carbohydrate content, a molecular mass of 72.6 kDa (SDS/PAGE) and a pI of 3.9. The enzyme presents wide specificity, showing activity on benzyl, cinnamyl, naphthyl and aliphatic unsaturated alcohols. Neither activity nor inhibition of veratryl alcohol oxidation was found with saturated alcohols, but competitive inhibition was produced by aromatic compounds which were not aryl-alcohol oxidase substrates, such as phenol or 3-phenyl-1-propanol. From these results, it was apparent that a double bond conjugated with a primary alcohol is necessary for substrate recognition by aryl-alcohol oxidase, and that activity is increased by the presence of additional conjugated double bonds and electron donor groups. Both affinity and maximal velocity during enzymic oxidation of methoxybenzyl alcohols were affected in a similar way by ring substituents, increasing from benzyl alcohol (Km = 0.84 mM, Vmax = 52 U/mg) to 4-methoxybenzyl alcohol (Km = 0.04 mM, Vmax = 208 U/mg). Aryl-alcohol oxidase presents also a low oxidase activity with aromatic aldehydes, but the highest activity was found in the presence of electron-withdrawing groups.
Guillén
F
F
Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
Martínez
A T
AT
Martínez
M J
MJ
D000429:Alcohol Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011124:Polyporaceae / Q000201:enzymology* / Q000254:growth & development; D013379:Substrate Specificity
D000429:Alcohol Oxidoreductases; C077759:aryl-alcohol oxidase