Enzymatic studies of riboflavin oversynthesis in Eremothecium ashbyii

Mechanisms of riboflavin oversynthesis in a high flavinogenic mold, Eremothecium ashbyii, were examined in relation to growth, riboflavin formation and related synthases, and medium pH with increasing culture periods. Growth reached maximum at 1 d and then decreased, riboflavin formation proceeded rapidly up to 5 d and approached almost a plateau region. The medium pH reached minimum at 1 d and thereafter fairly rapidly increased until 3 d, then gradually increased to 7 d after cultivation. The crude enzyme solution from the mycelia at specified culture periods was run through a column of Sephadex G-200, indicating two riboflavin synthase activities on the chromatogram. The fluctuation of the growth and the specific activities of the two enzymes were examined with increasing culture periods, which showed that the heavy enzyme may be a constitutive one and that the light enzyme may be concerned with the oversynthesis of riboflavin in E. ashbyii. The heavy enzyme was then purified by 49-fold after dialysis of the ammonium sulfate precipitate by a series of column chromatographies with Sephadex G-200, hydroxyapatite, DEAE-Sepharose A-50 and DEAE-cellulose. The purified enzyme which was treated with weak alkaline solution was broken into the light enzyme, showing two bands on an acrylamide disc gel electrophoresis. The relation of the heavy and the light enzymes to the oversynthesis of riboflavin in E. ashbyii was discussed.