Date of Award


Degree Type


Degree Name

Doctor of Philosophy


A black yeast isolated from oak bark was identified as a species of Phaeococcomyces. The yeast produced black pigment constitutively which was identified as a pentaketide melanin, since its production was inhibited by the systemic fungicide tricyclazole. The black yeast melanin was produced as granules with a diameter of ca. 30 nm. Three types of pigmentation mutant were produced; albino mutants, which did not produce melanin, diffusion mutants which were not melanized, and excreted 2-hydroxy-juglone and flaviolin in culture media, and cross feeder mutants which were not melanized and excreted scytalone, a component of the main pentaketide pathway, into culture media. Oxidation products of the pentaketide pathway, and main pathway phenolic intermediates, produced by treatment with tricyclazole, or which were excreted by mutants, were identified by High Performance Liquid Chromatography and Thin Layer Chromatography. The melanin of the black yeast protected against enzymatic cell wall lysis. Albino mutants were susceptible to enzymatic cell wall lysis. The melanin of the black yeast did not protect against toxic phenolic compounds, ultraviolet light irradiation, or desiccation. An azure A dye binding assay was developed to quantify cell wall melanin. A defined low pH ascorbate medium was developed which completely inhibited melanin production by Phaeococcomyces sp., while allowing normal growth. Polyacrylamide gel electrophoresis was used to identify a number of copper containing phenoloxidases in cell wall enzyme and cytoplasmic enzyme extracts of the black yeast, which acted as melanin polymerases. An enzyme (referred to as convertase) which acted against scytalone, was found in cytoplasmic extracts of all but cross feeder mutants. The enzyme had a molecular mass of ca. 65000, a K{dollar}\sb{lcub}\rm m{rcub}{dollar} of 1.7 mM for scytalone, an isoelectric point of 7.9., a pH optimum of 7.5, and did not appear to be glycosylated. The convertase enzyme had activity against 1,3,8-trihydroxynaphthalene, the presumed product of scytalone conversion. The latter activity was inhibited by tricyclazole.



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