Department of Chemical Engineering and Food Technology, Campus Universitario de Puerto Real, University of Cadiz, 11510 Puerto Real, Spain.
Institute of Viticulture and Agri-Food Research (IVAGRO)-International Campus of Excellence (ceiA3), University of Cadiz, 11510 Puerto Real, Spain.
Int J Mol Sci. 2020 Nov 12;21(22):8523. doi: 10.3390/ijms21228523.
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 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 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.
D-DIBOA（4-羟基-（2H）-1,4-苯并恶嗪-3-（4H）-酮）是一种具有除草、杀菌和杀虫特性的化感衍生化合物，其生产已成功通过使用基因工程菌株的生物催化来实现。然而，由于目前 D-DIBOA 定量的方法，该方法基于高效液相色谱（HPLC），这是一种耗时的技术，需要昂贵的设备和使用对环境不安全的溶剂，因此该过程的改进和扩大受到了阻碍。在这项工作中，我们建立并验证了一种快速、简单和灵敏的分光光度法，用于定量全细胞生物催化产生的 D-DIBOA，检测限和定量限分别为 0.0165 和 0.0501 µmol·mL。该分析仅需几秒钟即可完成，并且可以使用微量滴定板读数器中的 100 µL 样品进行分析。我们进行了几种全细胞生物催化策略，通过每小时监测 D-DIBOA 的产生来优化该过程，以控制生物反应器中前体和 D-DIBOA 的浓度。这些实验将 D-DIBOA 的产量从之前报道的 5.01 mM 提高到 7.17 mM（增加了 43%）。这种方法将有助于优化生物催化剂、扩大规模和下游纯化等过程。
