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 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.

Expression and application of aflatoxin degrading enzyme gene in Pichia pastoris

“…However, most of the enzymes found so far are natural degradation enzymes, which are difficult to be widely applied and developed because of their low content, poor activity and instability. [ 8,9 ] Using molecular biology and genetic engineering techniques to clone and express the target degradation enzyme gene in different hosts can improve the production and activity of AFB 1 degradation enzyme, which has great development and application value to mitigate aflatoxin contamination. [ 10–12 ] The expression system of Pichia pastoris has a strict regulatory mechanism of one of the promoters, alcohol oxidase gene AOX1 promoter, which is very conducive to the regulation of exogenous gene expression [ 13 ] ; can be expressed both intracellularly and extracellularly; has high genetic stability; has post‐translational protein processing modification functions such as phosphorylation, glycosylation, and low culture cost, which is a eukaryotic expression system that gathers many advantages.…”

Mechanisms by which microbial enzymes degrade four mycotoxins and application in animal production: A review

“…17-2-E-8 which is responsible for the oxidation of the C3 hydroxyl group in DON to a keto group. On this basis, Yang et al. (2022) further revealed the interactions between dehydrogenase, cofactor, and substrate DON.…”

Research Progress on Electrochemical Sensors based on Metal-Organic Framework Materials for the Detection of Mycotoxins in Food

“…Therefore, aflatoxin contamination represents a major risk to food safety and the well-being of humans and animals, leading to considerable economic impacts. Consequently, swiftly and sensitively identifying aflatoxins in agricultural products like grains or dairy items, including corn, peanut oil, and milk, is crucial [29].…”