The l-amino-acid oxidase of Neurospora

Abstract

17 strains of N. crassa and 3 of N. sltophila were examined for amino-acid oxidases. Under suitable conditions, which were described, both an L-alpha-amino-acid and a D-alpha-amino-acid oxidase were detected in all strains. The activity of both enzymes varied considerably from strain to strain. An L-oxidase was also found in Melanospora destruens; it was absent from several other species. The highest yields of L-amino-acid oxidase were obtained with N. crassa CMI 3411 in stationary culture on a liquid medium containing sucrose, ammonium tartrate, 0.2% casein hydrolysate, inorganic salts, and 0.1-0.5 [mu]g. biotin/l. The oxidase activity in the mycelium and also in the culture fluid was highest after 12 days'' growth at 25[degree]. Concns. of biotin above 1 [mu]g./l. considerably reduced the yield of L-amino-acid oxidase. The kinetics of the L-amino-acid oxidase were studied. The pH-activity curve had a broad maximum between pH 6 and 9.5. High concns. of substrate inhibited. The specificity was not changed by 4.5-fold purification of the oxidase from the culture fluid. Bound flavin-adenine dinucleotide was present in the purified oxidase (1 g. mol./ll,000 g. non-dialysable N) as shown by the activation of the D-amino-acid oxidase apoenzyme, fluorimetry and paper chromatography. Riboflavin phosphate and riboflavin were not detected. Anaerobic addition of alanine decreased the optical density of the enzyme prepn. between 380 and 500 m[mu]; this change was that which is characteristic of the chemical reduction of flavoproteins. It may, therefore, be taken that FAD was the prosthetic group of the L-amino-acid oxidase. The turnover number was estimated to be 2100 molecules phenyla-lanine per minute at 30[degree].