Characterization of immobilized aflatoxin-detoxizyme / 生物工程学报

ABSTRACT

Aflatoxins, found in contaminated food, are potent hepatocarcinogen. The aflatoxin-detoxiczyme (ADTZ) isolated from the edible fungus Armillariella sp., detoxifies aflatoxin B1 (AFB1). This paper reports on the characterization of immobilized ADTZ using a hydrophobic adsorption method. The ADTZ was isolated from cryo-homogenated fungus, previously cultivated at 24 - 28 degrees C for 20 - 30 days, using n-alkyl amino-agar beads. Various adsorption conditions of the enzyme to n-alkyl or n-octyl amino-agar beads were carried out. The effects of enzyme immobilization on different alkyl amino-agar beads, at different pH values (5.5 - 7.5), at different temperature (20 - 40 degrees C) and at different salt concentrations were investigated. The enzyme activity was measured at OD360 by reacting 133.3 ng/mL of AFB1 at 30 degrees C for 30 min with the immobilized ADTZ. The Km value of the immobilized enzyme, determined using Schematic Linewearver-Burk plot, is 3.308 x 10(-3) mol/L, lower than that of free enzyme, which is 2.16 x 10(-6) mol/L. This indicated the affinity of the detoxiczyme to AFB1 decreased after immobilization. The immobilized enzyme activity in oil-phase (n-hexane) was also studied with different concentration of water. After the treatment of the immobilized ADTZ, the toxin no longer causes liver toxicity in the rat toxicity test, no longer causes mutagenicity in Ames test and is no longer toxic in the chicken embryo test. Results also indicated that the pH stability, the thermostability and the freezing stability of ADTZ were improved after the immobilization.