Purification and characterization of an extracellular phenol oxidase from culture filtrates of Pyricularia oryzae

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Purification and characterization of an extracellular phenol oxidase from culture filtrates of Pyricularia oryzae

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Title: Purification and characterization of an extracellular phenol oxidase from culture filtrates of Pyricularia oryzae
Author: Alsubaey, A.; Sisler, H.D.; Matthews, B.F.
Abstract: Extracellular phenol oxidase activity was characterized and compared in Pyricularia oryzae wild-type and albino cell types to determine if this phenol oxidase was responsible for lack of melanization in the albino culture. Filtrates of the albino mutant Alb-5 showed activity similar to those of the wild type, while those of a buff mutant (Cp62) showed weak phenol oxidase activity. This indicated that the lack of melanization in the albino mutant was not due to an absence of phenol oxidase activity. The phenol oxidase isoform patterns from the wild type and two mutants were similar when analyzed by polyacrylamide gel electrophoresis. The slowest migrating isoform of phenol oxidase from wild-type Pyricularia oryzae was the major form and had a molecular mass of 380 kDa. The molecular masses of two of the minor forms were 220 and 130 kDa. The isoforms oxidized 1,8-dihydroxynaphthalene, the terminal metabolite in the polyketide pathway to melanin. The major phenol oxidase isoform was also present in extracts from albino mutants and the buff mutant. The major form was enriched by a combination of ammonium sulfate precipitation, DEAE-Sepharose column chromatography, and elution from preparative polyacrylamide gels. The enriched isoform of phenol oxidase separated into two forms after a second electrophoresis, indicating that these two isoforms interconvert. Analysis of both forms by sodium dodecyl sulfate polacrylamide gel electrophoresis indicated that both were composed of a single subunit with a molecular mass of 70 kDa. The enriched isoform preferred phloroglucinol as a substrate and had a Michaelis constant (K(m)) of 19.3 mM for phloroglucinol and a pH optimum between 6 and 7.5.
URI: http://hdl.handle.net/10576/10532
Date: 1996-05

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