Recombinant Expression of Trametes versicolor Aflatoxin B1-Degrading Enzyme (TV-AFB1D) in Engineering Pichia pastoris GS115 and Application in AFB1 Degradation in AFB1-Contaminated Peanuts
Abstract
:1. Introduction
2. Results
2.1. TV-AFB1D Cloning and Evolutionary Tree Analysis
2.2. Expression Vector Construction of TV-AFB1D
2.3. Transformation of TV-AFB1D into P. pastoris GS115
2.4. Recombinant TV-AFB1D Expression in P. pastoris GS115
2.5. Effect of Methanol Concentration and Time on the TV-AFB1D Expression
2.6. Effect of Temperatures and Treatment Time on the Expression of TV-AFB1D
2.7. Product Formation of AFB1-Contaminated Peanuts Catalyzed by TV-AFB1D
2.8. Effect of Time on the Residue of AFB1 Standard Sample
2.9. Effect of Time on the Proportion of AFB1 Residue in Peanuts
2.10. Effect of Temperature on the Proportion of AFB1 Residue in Peanuts
3. Discussion
4. Conclusions
5. Materials and Methods
5.1. Materials and Reagents
5.2. Construction of pPIC9K-His-TV-AFB1D Vector
5.3. Transformation of pPIC9K-His-TV-AFB1D into P. pastoris GS115
5.4. Inducible Expression and Condition Optimizations of TV-AFB1D
5.5. Purification of Recombinant TV-AFB1D and Definition of Activity
5.6. Measurement of AFB1 Content and Degradation Products
5.7. Application in the AFB1 Degradation of AFB1-Contaminated Peanuts
5.8. Data Analysis
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Microbial Sources and Enzymes | Conditions | Catalytic Efficiency and Time |
---|---|---|
Mycobacterium smegmatis, F420H2-dependent reductase [25] | 20 mM Tris-HCl, pH 7.5 | 63%, 4 h |
Myxococcus fulvus, MADE [30] | pH 6 | 96% |
Armillariella tabescens, ADTZ [28] | 0.02 M PBS, pH 6 | 80% |
Phanerochaete sordida, peroxidase [33] | 50 mM PBS, pH 6 | 86%, 48 h |
Pleurotus ostreatus, peroxidase [34] | pH 4.5 | 90%, 48 h |
Bacillus subtilis, BsCotA laccase [35] | 50 mM Tris–HCl, pH 7.0 | 98%, 10 h |
Pleurotus pulmonarius, laccase [36] | 1 mM sodium acetate, pH 5 | 90%, 72 h |
Aspergillus flavus, HRP [29] | 1mM sodium acetate, pH 5 | 42%, 1 h |
Bacillus shackletonii, BADE [31] | 50 mM PBS, pH 7.0 | 47.5%, 72 h |
Pseudomonas aeruginosa, PADE [32] | 0.02 M PBS, pH 7 | 72.5%, 12h |
T. versicolor, TV-AFB1D, in this study | AFB1 standard sample | 75.9%, 12 h |
T. versicolor, TV-AFB1D, in this study | AFB1-contaminated peanuts | 48.5%, 18 h |
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Yang, P.; Xiao, W.; Lu, S.; Jiang, S.; Zheng, Z.; Zhang, D.; Zhang, M.; Jiang, S.; Jiang, S. Recombinant Expression of Trametes versicolor Aflatoxin B1-Degrading Enzyme (TV-AFB1D) in Engineering Pichia pastoris GS115 and Application in AFB1 Degradation in AFB1-Contaminated Peanuts. Toxins 2021, 13, 349. https://doi.org/10.3390/toxins13050349
Yang P, Xiao W, Lu S, Jiang S, Zheng Z, Zhang D, Zhang M, Jiang S, Jiang S. Recombinant Expression of Trametes versicolor Aflatoxin B1-Degrading Enzyme (TV-AFB1D) in Engineering Pichia pastoris GS115 and Application in AFB1 Degradation in AFB1-Contaminated Peanuts. Toxins. 2021; 13(5):349. https://doi.org/10.3390/toxins13050349
Chicago/Turabian StyleYang, Peizhou, Wei Xiao, Shuhua Lu, Suwei Jiang, Zhi Zheng, Danfeng Zhang, Min Zhang, Shaotong Jiang, and Shuying Jiang. 2021. "Recombinant Expression of Trametes versicolor Aflatoxin B1-Degrading Enzyme (TV-AFB1D) in Engineering Pichia pastoris GS115 and Application in AFB1 Degradation in AFB1-Contaminated Peanuts" Toxins 13, no. 5: 349. https://doi.org/10.3390/toxins13050349