Optimization of the Biocatalysis for D-DIBOA Synthesis Using a Quick and Sensitive New Spectrophotometric Quantification Method
D-DIBOA (4-hydroxy-(2H)-1,4-benzoxazin-3-(4H)-one) is an allelopathic-derived compound with interesting herbicidal, fungicidal, and insecticide properties whose production has been successfully achieved by biocatalysis using a genetically engineered <i>Escherichia coli</i> strain. Howeve...
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| Format: | Article |
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MDPI AG
2020-11-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/21/22/8523 |
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| author | Gema Cabrera Teresa Linares Maria Elena de la Calle Domingo Cantero Antonio Valle Jorge Bolivar |
| author_facet | Gema Cabrera Teresa Linares Maria Elena de la Calle Domingo Cantero Antonio Valle Jorge Bolivar |
| author_sort | Gema Cabrera |
| collection | DOAJ |
| description | D-DIBOA (4-hydroxy-(2H)-1,4-benzoxazin-3-(4H)-one) is an allelopathic-derived compound with interesting herbicidal, fungicidal, and insecticide properties whose production has been successfully achieved by biocatalysis using a genetically engineered <i>Escherichia coli</i> strain. However, improvement and scaling-up of this process are hampered by the current methodology for D-DIBOA quantification, which is based on high-performance liquid chromatographic (HPLC), a time-consuming technique that requires expensive equipment and the use of environmentally unsafe solvents. In this work, we established and validated a rapid, simple, and sensitive spectrophotometric method for the quantification of the D-DIBOA produced by whole-cell biocatalysis, with limits of detection and quantification of 0.0165 and 0.0501 µmol·mL<sup>−1</sup> respectively. This analysis takes place in only a few seconds and can be carried out using 100 µL of the sample in a microtiter plate reader. We performed several whole-cell biocatalysis strategies to optimize the process by monitoring D-DIBOA production every hour to keep control of both precursor and D-DIBOA concentrations in the bioreactor. These experiments allowed increasing the D-DIBOA production from the previously reported 5.01 mM up to 7.17 mM (43% increase). This methodology will facilitate processes such as the optimization of the biocatalyst, the scaling up, and the downstream purification. |
| first_indexed | 2024-03-10T14:53:30Z |
| format | Article |
| id | doaj.art-0d726266f5774255805c711c5f2ad796 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T14:53:30Z |
| publishDate | 2020-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-0d726266f5774255805c711c5f2ad7962023-11-20T20:44:06ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672020-11-012122852310.3390/ijms21228523Optimization of the Biocatalysis for D-DIBOA Synthesis Using a Quick and Sensitive New Spectrophotometric Quantification MethodGema Cabrera0Teresa Linares1Maria Elena de la Calle2Domingo Cantero3Antonio Valle4Jorge Bolivar5Department of Chemical Engineering and Food Technology, Campus Universitario de Puerto Real, University of Cadiz, 11510 Puerto Real, SpainDepartment of Biomedicine, Biotechnology and Public Health-Biochemistry and Molecular Biology, Campus Universitario de Puerto Real, University of Cadiz, 11510 Puerto Real, SpainDepartment of Chemical Engineering and Food Technology, Campus Universitario de Puerto Real, University of Cadiz, 11510 Puerto Real, SpainDepartment of Chemical Engineering and Food Technology, Campus Universitario de Puerto Real, University of Cadiz, 11510 Puerto Real, SpainInstitute of Viticulture and Agri-Food Research (IVAGRO)—International Campus of Excellence (ceiA3), University of Cadiz, 11510 Puerto Real, SpainDepartment of Biomedicine, Biotechnology and Public Health-Biochemistry and Molecular Biology, Campus Universitario de Puerto Real, University of Cadiz, 11510 Puerto Real, SpainD-DIBOA (4-hydroxy-(2H)-1,4-benzoxazin-3-(4H)-one) is an allelopathic-derived compound with interesting herbicidal, fungicidal, and insecticide properties whose production has been successfully achieved by biocatalysis using a genetically engineered <i>Escherichia coli</i> strain. However, improvement and scaling-up of this process are hampered by the current methodology for D-DIBOA quantification, which is based on high-performance liquid chromatographic (HPLC), a time-consuming technique that requires expensive equipment and the use of environmentally unsafe solvents. In this work, we established and validated a rapid, simple, and sensitive spectrophotometric method for the quantification of the D-DIBOA produced by whole-cell biocatalysis, with limits of detection and quantification of 0.0165 and 0.0501 µmol·mL<sup>−1</sup> respectively. This analysis takes place in only a few seconds and can be carried out using 100 µL of the sample in a microtiter plate reader. We performed several whole-cell biocatalysis strategies to optimize the process by monitoring D-DIBOA production every hour to keep control of both precursor and D-DIBOA concentrations in the bioreactor. These experiments allowed increasing the D-DIBOA production from the previously reported 5.01 mM up to 7.17 mM (43% increase). This methodology will facilitate processes such as the optimization of the biocatalyst, the scaling up, and the downstream purification.https://www.mdpi.com/1422-0067/21/22/8523whole-cell biocatalysisD-DIBOAspectrophotometric methodnitroreductase NfsB |
| spellingShingle | Gema Cabrera Teresa Linares Maria Elena de la Calle Domingo Cantero Antonio Valle Jorge Bolivar Optimization of the Biocatalysis for D-DIBOA Synthesis Using a Quick and Sensitive New Spectrophotometric Quantification Method International Journal of Molecular Sciences whole-cell biocatalysis D-DIBOA spectrophotometric method nitroreductase NfsB |
| title | Optimization of the Biocatalysis for D-DIBOA Synthesis Using a Quick and Sensitive New Spectrophotometric Quantification Method |
| title_full | Optimization of the Biocatalysis for D-DIBOA Synthesis Using a Quick and Sensitive New Spectrophotometric Quantification Method |
| title_fullStr | Optimization of the Biocatalysis for D-DIBOA Synthesis Using a Quick and Sensitive New Spectrophotometric Quantification Method |
| title_full_unstemmed | Optimization of the Biocatalysis for D-DIBOA Synthesis Using a Quick and Sensitive New Spectrophotometric Quantification Method |
| title_short | Optimization of the Biocatalysis for D-DIBOA Synthesis Using a Quick and Sensitive New Spectrophotometric Quantification Method |
| title_sort | optimization of the biocatalysis for d diboa synthesis using a quick and sensitive new spectrophotometric quantification method |
| topic | whole-cell biocatalysis D-DIBOA spectrophotometric method nitroreductase NfsB |
| url | https://www.mdpi.com/1422-0067/21/22/8523 |
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