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

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 = 0.01671x + 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 mu g/ml to 32.6 mu g/ml after treatment at 32 degrees 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 mu g/g from the initial AFB(1) content of 7.4 mu g/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.