Enzymatic conversion of sterigmatocystin to aflatoxin B1.
The age of Aspergillus parasiticus (1-11-105Wh1) mycelium was found to have an influence on the level of enzymes, responsible for the conversion of sterigmatocystin to aflatoxin B and O-methylsterigmatocystin, present. These enzymes were active over a wide range of temperature and pH. Production of a cell free system by lyophiliization yielded the highest aflatoxin B synthesising activity. Three other methods of preparing the cell free system capable of synthesising aflatoxin B were also studied, ie,: french press, protoplast, and grinding, but with limited success. The lyophilized preparation had narrower temperature and pH optima for the conversion than whole mycelia. Initial purification of the aflatoxin B synthesising enzyme was achieved by separating the crude cell free extract by gel filtration. The enzyme activity was located in a membrane fraction. The involvement of endoplasmic reticulum was indirectly concluded by the use of marker enzyme and chelating agents. This membrane fraction was ultracentrifuged and the released extrinsic proteins were separated by gel filtration. A fraction containing two proteins which were capable of converting sterigmatocystin to aflatoxin B was isolated and characterised by isoelectric focusing and gel electrophoresis. The temperature and pH optima together with the cofactor requirements were studied. The Michaelis-Menten constant (Km) and the stoichiometry for the conversion of sterigmatocystin to aflatoxin B was determined.