Aflatoxin B 1 (AFB 1 ) is highly carcinogenic and teratogenic as a secondary metabolite of Aspergillus flavus and Aspergillus parasiticus . Trametes versicolor aflatoxin B 1 -degrading enzyme (TV-AFB 1 D) has capability of AFB 1 degradation. In this study, in order to improve the catalytic performance of TV-AFB 1 D, the molecular docking and site-directed mutagenesis techniques were applied to modify the key amino acid sites. Three mutation sites of Y305A, E436A, and H554A were selected for the directed mutation of TV-AFB 1 D. Four TV-AFB 1 D combinations of mutant H554A, Y305A/H554A, E436A/H554A, Y305A/E436A/H554A had potential to modify the catalytic performance according to the value of de-folding free energy. The TV-AFB 1 D mutations were expressed in engineering E. coli BL21 (DE3) with the size of approximately 77 kDa. The optimal temperature and pH value of TV-AFB 1 D were 32 °C and pH 7, respectively. E436A/H554A mutant represented the highest enzyme activities among these four mutants. The specific activity of E436A/H554A mutant (36 U/mg) was 1.84-fold in comparison with wild-type TV-AFB 1 D (19.6 U/mg) under the conditions of pH 7 and 32 °C. This study would contribute to the activity increase of TV-AFB 1 D by the performance improvement of AFB 1 degradation by site-directed mutagenesis approach.
Yang Peizhou,Xiao Wei,Lu Shuhua,Jiang Shuying,Jiang Suwei,Chen Jianchao,Wu Wenjing,Zheng Zhi,Jiang Shaotong
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 (R2 = 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.
危险品化学品经营许可证（带存储）