Cloning of Three Aflatoxin B1 Oxidases of the Dipeptidyl Peptidase III Family and Evaluation of Their Potential for Practical Applications as Decontamination Enzymes.

Abstract

Aflatoxin B1 (AFB1) produced by some Aspergillus species belongs to the most dangerous contaminants of animal feeds. Development of safe and cost efficient decontamination methods saving feed quality and nutritional value are of paramount importance. The use of recombinant AFB1-detoxifying microbial enzymes represents a promising biotechnological approach meeting the aforementioned requirements. In this study, three AFB1-degrading oxidases (AFOs) from edible basidiomycetes Cantharellus cibarius, Lentinula edodes and Pleurotus eryngii as well as AFO from Armillaria tabescens were expressed in E. coli Rosetta (DE3) and purified by immobilized metal-chelate chromatography. The stabilizing effect of the addition of glycerol and β-mercaptoethanol during protein extraction is shown. The catalytic constants of the recombinant AFOs (rAFOs) and other characteristics, which might be important for their practical application (and optimal temperature and pH, thermolability, regulation of the activity by metal ions and chelating agents, storage stability) were investigated. Among the obtained enzymes, rAFO from P. eryngii (Pe-AFO), which was characterized by the highest specific activity, thermostability and pH stability (especially at acidic pH range), the lowest K_m, and relative resistance to the inhibition by phytate, showed the best AFB1-degrading efficacy. However, Pe-AFO and all other rAFOs significantly decreased the target activity during heating above 45 °C, storage frozen or lyophilization.