Publicação
Innovative Ochratoxin A (OTA) extraction platforms using OTA-binding proteins
| Resumo: | Wine is a widely consumed product that is often associated with contaminations of toxic metabolites called mycotoxins. The most important mycotoxin associated to wine is ochratoxin A (OTA), and its detection usually involves the clean-up of samples with immunoaffinity columns (IAC) and quantification by HPLC coupled with fluorescence detection (FL). However, the several drawbacks associated with the use of the IAC led to the search for alternative clean-up methods. Thus, in this work, new platforms for OTA clean-up from wine were developed, based on proteins whose affinity towards OTA makes them suitable candidates to mimic the binding properties of the antibodies used in the IAC method. In a first approach, a protein with high affinity towards OTA, was used to develop a new solid phase extraction (SPE) method for the extraction of the mycotoxin from wine and subsequent quantification by HPLC-FL. The capture of OTA by the columns constructed with agarose-immobilized OTA binding protein was optimized to allow the full recovery of OTA in wine, and the method was further validated by the evaluation of various parameters such as recovery rates, selectivity and limits of detection (LOD) and quantification (LOQ). The developed method was selective enough for a reliable determination of OTA in wine, presenting recovery rates superior to 98% and LOD and LOQ of 0.02 and 0.05 μg L-1, respectively. Furthermore, the performance of the developed method revealed no significant differences in relation to the IAC method in concentrations up to 2 μg L-1 of OTA. In comparison with other conventional SPE methods reported in the literature, the developed method has proved to be suitable to be employed in the determination of OTA in wine. In a second approach, the domain where lies the primary binding site of OTA in the OTA-binding protein used in the first approach was evaluated as OTA ligand for developing OTA extraction platforms based on this domain. For that, the domain was recombinantly produced, fused to a 6xHis tag, with and without the thioredoxin (TrxA) solubility partner, in two E. coli strains, BL21 (DE3) and Origami 2 (DE3). Soluble proteins, with and without TrxA, were produced by both strains, but the Origami strain provided higher yields of production (18.7 and 23.4 mg per litre of culture, respectively). In addition, differences observed in the affinity of the proteins for the nickel resin in the purification suggested that the structures acquired by the recombinant proteins produced in each strain were different. Furthermore, the fact that fusion proteins were less prone to degradation suggested that TrxA contributed for their stability. Studies of fluorescence spectroscopy revealed that only the recombinant proteins produced by the Origami strain were capable of interacting with OTA, thus indicating that these proteins were functional. The ability of the proteins produced from this strain, with and without TrxA, immobilized in nickel via the 6xHis tag, to capture the mycotoxin in buffer solutions was evaluated. SPE columns constructed with the nickel-immobilized recombinant proteins did not show the ability to effectively capture OTA. On the other hand, incubation assays performed in eppendorfs allowed decreasing OTA in solution up to 54 and 63%, respectively for immobilized proteins, with and without TrxA. These results open perspectives for the development of OTA extraction platforms based on the recombinant domain of this OTA-binding protein with matrixes less expensive than the agarose used in the first approach by means of specific purification tags. |
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| Autores principais: | Leal, Tânia Vanessa da Silva |
| Assunto: | Ciências Agrárias Ciências Médicas Engenharia e Tecnologia Engenharia e Tecnologia |
| Ano: | 2018 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Wine is a widely consumed product that is often associated with contaminations of toxic metabolites called mycotoxins. The most important mycotoxin associated to wine is ochratoxin A (OTA), and its detection usually involves the clean-up of samples with immunoaffinity columns (IAC) and quantification by HPLC coupled with fluorescence detection (FL). However, the several drawbacks associated with the use of the IAC led to the search for alternative clean-up methods. Thus, in this work, new platforms for OTA clean-up from wine were developed, based on proteins whose affinity towards OTA makes them suitable candidates to mimic the binding properties of the antibodies used in the IAC method. In a first approach, a protein with high affinity towards OTA, was used to develop a new solid phase extraction (SPE) method for the extraction of the mycotoxin from wine and subsequent quantification by HPLC-FL. The capture of OTA by the columns constructed with agarose-immobilized OTA binding protein was optimized to allow the full recovery of OTA in wine, and the method was further validated by the evaluation of various parameters such as recovery rates, selectivity and limits of detection (LOD) and quantification (LOQ). The developed method was selective enough for a reliable determination of OTA in wine, presenting recovery rates superior to 98% and LOD and LOQ of 0.02 and 0.05 μg L-1, respectively. Furthermore, the performance of the developed method revealed no significant differences in relation to the IAC method in concentrations up to 2 μg L-1 of OTA. In comparison with other conventional SPE methods reported in the literature, the developed method has proved to be suitable to be employed in the determination of OTA in wine. In a second approach, the domain where lies the primary binding site of OTA in the OTA-binding protein used in the first approach was evaluated as OTA ligand for developing OTA extraction platforms based on this domain. For that, the domain was recombinantly produced, fused to a 6xHis tag, with and without the thioredoxin (TrxA) solubility partner, in two E. coli strains, BL21 (DE3) and Origami 2 (DE3). Soluble proteins, with and without TrxA, were produced by both strains, but the Origami strain provided higher yields of production (18.7 and 23.4 mg per litre of culture, respectively). In addition, differences observed in the affinity of the proteins for the nickel resin in the purification suggested that the structures acquired by the recombinant proteins produced in each strain were different. Furthermore, the fact that fusion proteins were less prone to degradation suggested that TrxA contributed for their stability. Studies of fluorescence spectroscopy revealed that only the recombinant proteins produced by the Origami strain were capable of interacting with OTA, thus indicating that these proteins were functional. The ability of the proteins produced from this strain, with and without TrxA, immobilized in nickel via the 6xHis tag, to capture the mycotoxin in buffer solutions was evaluated. SPE columns constructed with the nickel-immobilized recombinant proteins did not show the ability to effectively capture OTA. On the other hand, incubation assays performed in eppendorfs allowed decreasing OTA in solution up to 54 and 63%, respectively for immobilized proteins, with and without TrxA. These results open perspectives for the development of OTA extraction platforms based on the recombinant domain of this OTA-binding protein with matrixes less expensive than the agarose used in the first approach by means of specific purification tags. |
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