Isolation, purification and characterization of enzyme(s) responsible for conversion of sterigmatocystin to aflatoxin B1.
The cell-free extract prepared from Aspergillus flavus ATCC 5517/A 228 showed activity in converting sterigmatocystin to aflatoxin B1. The extract was purified on Ultrogel AcA-54 and resulted in ten protein peaks, one of which (peak VI) showed activity in sterigmatocystin conversion. The protein in this peak gave one protein band using polyacrylamide gel (PAG)-disc electrophoresis. For further purification, protein(s) in peak VI were applied on DEAE-Sephadex A-50 and two protein peaks were detected. Only one peak showed enzyme activity which showed homogeneity as one band on PAGE and sodium dodecyl sulphate (SDS)-PAGE. The optimum temperature for the enzyme activity was 28 degrees C and the optimum pH was 8. The maximum conversion resulted from the action of 0.6 mg enzyme protein on 48 X 10(-8) mol sterigmatocystin. Zn2+, Co2+ and Mn2+ enhanced the enzyme activity, while ethylenediaminetetraacetic acid, parahydroxymercuric benzoate and phenylmethylsulphonic fluoride inhibited the enzyme activity in a dose-dependent manner. Amino-acid analysis showed the presence of 22 amino acids, three of which are unknown. The enzyme has a molecular weight of 64,000 daltons (by gel filtration) and 70,000 daltons (by SDS-PAGE).
D016604:Aflatoxin B1; D000348:Aflatoxins / Q000096:biosynthesis*; D000596:Amino Acids / Q000032:analysis; D001231:Aspergillus flavus / Q000201:enzymology*; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D004798:Enzymes / Q000032:analysis / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D012967:Sodium Dodecyl Sulfate; D013241:Sterigmatocystin / Q000378:metabolism*; D014966:Xanthenes / Q000378:metabolism*
D000348:Aflatoxins; D000596:Amino Acids; D004798:Enzymes; D014966:Xanthenes; D013241:Sterigmatocystin; D012967:Sodium Dodecyl Sulfate; D016604:Aflatoxin B1