Activity enhancement of Trametes versicolor aflatoxin B1-degrading enzyme (TV-AFB1D) by molecular docking and site-directed mutagenesis techniques
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.
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