Characterization of a Trametes versicolor aflatoxin B1-degrading enzyme (TV-AFB1D) and its application in the AFB1 degradation of contaminated rice in situ.

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Abstract

Aflatoxin B1 (AFB1) contaminates rice during harvest or storage and causes a considerable risk to human and animal health. In this study, Trametes versicolor AFB1-degrading enzyme (TV-AFB1D) gene recombinantly expressed in engineered E. coli BL21 (DE3) and Saccharomyces cerevisiae. The TV-AFB1D enzymatic characteristics and AFB1 degradation efficiency in contaminated rice were investigated. Results showed that the size of recombinant TV-AFB1D expressing in E. coli BL21 (DE3) and S. cerevisiae was appropriately 77 KDa. The kinetic equation of TV-AFB1D was y = 0.01671x + 1.80756 (R 2 = 0.994, Km = 9.24 mM, and Vmax = 553.23 mM/min). The Kcat and Kcat/Km values of TV-AFB1D were 0.07392 (s-1) and 8 M-1 s-1, respectively. The AFB1 concentration of contaminated rice decreased from 100 μg/ml to 32.6 μg/ml after treatment at 32°C for 5 h under the catabolism of TV-AFB1D. S. cerevisiae engineered strains carrying aldehyde oxidase 1 (AOX1) and Cauliflower mosaic virus 35 S (CaMV 35 S) promoters caused the residual AFB1 contents, respectively, decreased to 3.4 and 2.9 μg/g from the initial AFB1 content of 7.4 μg/g after 24 h of fermentation using AFB1-contaminated rice as substrate. The AFB1 degradation rates of S. cerevisiae engineered strains carrying AOX1 and CaMV promoters were 54 and 61%, respectively. Engineered S. cerevisiae strains integrated with TV-AFB1D expression cassettes were developed to simultaneously degrade AFB1 and produce ethanol using AFB1-contaminated rice as substrate. Thus, TV-AFB1D has significant application potential in the AFB1 decomposition from contaminated agricultural products.