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

Abstract

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