The affinity purification and characterization of a dehydrogenase from Aspergillus parasiticus involved in aflatoxin B1 biosynthesis.
A two step scheme has been developed for the purification of a dehydrogenase from mycelia of 84 hours old Aspergillus parasiticus (1-11-105 Wh 1), which catalyzes the conversion of norsolorinic acid (NA) to averantin (AVN). The dehydrogenase was purified from cell-free extracts using reactive green 19-agarose and norsolorinic acid-agarose affinity chromatography. The latter affinity matrix was synthesised by attaching norsolorinic acid to omega-aminohexylagarose. The purified protein was shown to be homogenous on non-denaturing polyacrylamide gel electrophoresis. A final purification of 215-fold was achieved. Results of gel filtration chromatography indicated the approximate molecular mass of the native protein to be 140,000 daltons. The isoelectric point of the protein was about 5.5 as determined by chromatofocusing. The reaction catalyzed by the dehydrogenase was optimum at pH 8.5 and between 25 degrees to 35 degrees C. The Km of the enzyme for NA and NADPH was determined to be 3.45 microM and 103 microM respectively.
Chuturgoon
A A
AA
Department of Biochemistry, University of Natal, Pietermaritzburg, South Africa.
Dutton
M F
MF
D016604:Aflatoxin B1 / Q000096:biosynthesis* / Q000737:chemistry; D000429:Alcohol Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D000880:Anthraquinones / Q000302:isolation & purification / Q000378:metabolism; D001230:Aspergillus / Q000201:enzymology*; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D064430:NAD (+) and NADP (+) Dependent Alcohol Oxidoreductases
D000880:Anthraquinones; D005656:Fungal Proteins; C024820:averantin; D016604:Aflatoxin B1; D000429:Alcohol Oxidoreductases; D064430:NAD (+) and NADP (+) Dependent Alcohol Oxidoreductases; C062609:norsolorinic acid ketoreductase; C002754:norsolorinic acid