Purification and characterization of the esterases involved in aflatoxin biosynthesis in Aspergillus parasiticus.
The esterases from the cell-free extracts (CFEs) of Aspergillus parasiticus ATCC15517, an aflatoxin-producing strain, catalyzing the hydrolytic conversion of versiconal hemiacetal acetate (VHA) to versiconal was biochemically studied. The specific activity of the enzymes increased 2.5-fold during incubation of mycelia through 40-55 h. No metal ions were required for enzyme stability, but EDTA at 1 mM and dithiothreitol at 0.5-5 mM increased its stability. Three peaks of VHA esterase activity were resolved when the proteins in the CFEs prepared from the mycelia of different ages were separated by anion-exchange column chromatography, suggesting that at least three VHA esterases were present in the eluate of this purification step. One of these esterases extracted from the mycelia of a 55-h culture was partially purified in five steps by means of preparative chromatography and fast protein liquid chromatography. The partially purified enzyme when reacted with [14C]diisopropylfluorophosphate followed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis gave a single radiolabelled band, which corresponded to a protein of 32 kDa. The molecular mass of the partially purified VHA esterase determined with gel filtration was around 60 kDa. The results suggested that the enzyme consists of two isomeric subunits.
Kusumoto
K
K
Department of Environmental Toxicology, University of California, Davis 95616, USA. kusumoto@cgk.affrc.go.jp
Hsieh
D P
DP
D000348:Aflatoxins / Q000096:biosynthesis*; D000880:Anthraquinones / Q000378:metabolism*; D001230:Aspergillus / Q000201:enzymology*; D002265:Carboxylic Ester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D002273:Carcinogens / Q000378:metabolism*; D004795:Enzyme Stability; D008970:Molecular Weight
D000348:Aflatoxins; D000880:Anthraquinones; D002273:Carcinogens; C027121:versiconal hemiacetal acetate; D002265:Carboxylic Ester Hydrolases; C104588:versiconal hemiacetal acetate esterase