ENZYMATIC TRANSFORMATION OF AFLATOXIN B 1 BY RH_DYPB PEROXIDASE AND CHARACTERIZATION OF THE REACTION PRODUCTS

Aflatoxins (AFs) are fungal secondary metabolites which contaminate several staple food and feed commodities worldwide. Aflatoxin B1 (AFB1) is highly toxic and hepatocarcinogenic in humans. AF's exposure, especially in food-insecure countries, increases the rate of liver cancer, child mortality, malnutrition, immune suppression and hepatotoxicity, also in livestock animals. Biological methods, such as enzymatic biotransformation, represent a mild and environmental friendly method to reduce AFs contamination. Different peroxidases have been shown to degrade several mycotoxins, including aflatoxin B1 (AFB1). Therefore, the aim of this study was to investigate the degrading capability of a recombinant type B dye decolorizing peroxidase (Rh_DypB) towards AFB1. In vitro assays were set up in 50 mM sodium malonate buffer, pH 6.0 containing 2 mM MnCl2 and AFB1 (1 µg/mL) using different enzyme and H2O2 concentrations. Kinetic parameters on AFB1 were assessed, its reduction was assessed by HPLC during a 96 hours bioconversion time and the reaction products were characterized by mass spectrometry analysis. AFB1 was reduced up to 96% using low enzyme and H2O2 dosages; an approximative maximal rate of 0.0021 µg/mL x min and a Km of 3.2 mM were estimated at the optimal enzyme and H2O2 concentrations. AFB1 was quantitatively converted to the hydroxylated metabolite AFQ1, which is known to possess lower acute toxicity and mutagenicity than AFB1. Further studies are currently ongoing to investigate the mechanism of action of Rh_DypB through the use of enzyme mutants and to identify other reaction products other than AFQ1. Rh_DypB application could represent an effective method to reduce AFs contamination, especially in highly contaminated feed commodities.