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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 (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-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.

SUBMITTER: Yang P

PROVIDER: S-EPMC9515612 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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

Yang Peizhou P Xiao Wei W Lu Shuhua S Jiang Shuying S Jiang Suwei S Chen Jianchao J Wu Wenjing W Zheng Zhi Z Jiang Shaotong S

Frontiers in microbiology 20220914

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 ...[more]

PMID: 36187979

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Project description:Aflatoxin B1 (AFB1) is the most harmful mycotoxin that occurs as natural contaminant of agricultural commodities, particularly maize. Practical solutions for detoxification of contaminated staples and reduction of agricultural wastes are scarce. We investigated the capability of the white-rot and edible fungus Plerotus eryngii (king oyster mushroom) to degrade AFB1 both in vitro and in a laboratory-scale mushroom cultivation, using a substrate similar to that routinely used in mushroom farms. In malt extract broth, degradation of AFB1 (500 ng/mL) by nine isolates of P. eryngii ranged from 81 to 99% after 10 days growth, and reached 100% for all isolates after 30 days. The growth of P. eryngii on solid medium (malt extract-agar, MEA) was significantly reduced at concentrations of AFB1 500 ng/mL or higher. However, the addition of 5% wheat straw to the culture medium increased the tolerance of P. eryngii to AFB1 and no inhibition was observed at a AFB1 content of 500 ng/mL; degradation of AFB1 in MEA supplemented with 5% wheat straw and 2.5% (w/v) maize flour was 71-94% after 30 days of growth. Further, AFB1 degradation by P. eryngii strain ITEM 13681 was tested in a laboratory-scale mushroom cultivation. The mushroom growth medium contained 25% (w/w) of maize spiked with AFB1 to the final content of 128 μg/kg. Pleurotus eryngii degraded up to 86% of the AFB1 in 28 days, with no significant reduction of either biological efficiency or mushroom yield. Neither the biomass produced on the mushroom substrate nor the mature basidiocarps contained detectable levels of AFB1 or its metabolite aflatoxicol, thus ruling out the translocation of these toxins through the fungal thallus. These findings make a contribution towards the development of a novel technology for remediation of AFB1- contaminated corn through the exploitation of the degradative capability of P. eryngii and its bioconversion into high nutritional value material intended for feed production.

Project description:Aflatoxin B1 is a carcinogenic and mutagenic mycotoxin mainly produced by Aspergillus flavus and A. parasiticus, and prevalent in food and feed. Microbial degradation is a promising strategy which can be performed in mild and environmental friendly condition. This work is a step towards identifying the enzyme responsible for biodegradation of AFB1 by P. putida. Experiments were performed with P. putida lysate and compared with commercial lipase to see the degradation efficiency and the temperature stability. The cell free lysate of P. putida efficiently degraded AFB1 in a range of temperature from 20 to 90 °C. The lysate is thermostable and could retain its activity on pre-incubation up to 90 °C. Highest rate of degradation was observed at 70 °C. These observations show that the P. putida lysate is not only stable at higher temperatures but its enzymatic activity increases after incubation. Similarly, the commercial lipase degraded AFB1 efficiently. However, both, the P. putida lysate and lipase ceased degradation in presence of a lipase inhibitor, HgCl2. The Hill function accurately predicted enzyme activity at various times and temperatures. Like lipase, the lysate also hydrolyses the p-nitrophenyl palmitate to p-nitrophenol. Kinetic parameters such as Vmax, Km and n values are good measures to characterize the lysate response with respect to changing paranitro phenyl palmitate levels. The substrate specificity test of lipase showed linear correlation between the absorbance at 410 nm vs amount of product paranitro phenol. The value of Km, Vmax and n are 0.62 mM, 355.7 μmol min-1 and 1.29, respectively. The lipase gene presence in P. putida was confirmed using PCR technique. These observations indicate that the main enzyme responsible for AFB1 degradation by P. putida is lipase. Thus, lipase as a multifunctional biocatalyst provides a promising future for a variety of industries and may also help to ensure the food safety by degrading the mycotoxins.

Dataset Information

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

Publications

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

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