An affinity-column procedure for the purification of veratrate O-demethylase from fungi.
An affinity column procedure is reported for purifying veratrate O-demethylase from higher fungi. The procedure is based on the affinity of the fungal demethylases for veratrate, which was coupled to AH-Sepharose 4B. An over 300-fold purification of the enzyme from an Ascomycete (Chaetomium piluliferum), and a lower degree of purification (20-fold) from a Basidiomycete (Xerocomus badius), were obtained. The O-demethylases from higher fungi require NADH and oxygen. The enzyme activity is sensitive to exposure to oxygen. The pH optima are 5 for enzyme from Chaetomium, and 7 for demethylase from Xerocomus, respectively. The enzymes are not specific for veratrate. They also demethylate p- and m-anisate and 3,4-dimethoxycinnamate, but to a lower degree.
D001203:Ascomycota / Q000201:enzymology*; D001487:Basidiomycota / Q000201:enzymology*; D001565:Benzoates; D002597:Chaetomium / Q000201:enzymology*; D002846:Chromatography, Affinity; D004229:Dithiothreitol / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008722:Methods; D008738:Methyl Ethers; D010088:Oxidoreductases / Q000302:isolation & purification*; D010090:Oxidoreductases, O-Demethylating / Q000302:isolation & purification* / Q000378:metabolism
D001565:Benzoates; D008738:Methyl Ethers; D010088:Oxidoreductases; D010090:Oxidoreductases, O-Demethylating; D004229:Dithiothreitol