Characterization of Protocatechuate 4,5-Dioxygenase from Pseudarthrobacter phenanthrenivorans Sphe3 and In Situ Reaction Monitoring in the NMR Tube
Abstract
:1. Introduction
2. Results
2.1. PCA 4,5-Dioxygenase Activity and Transcriptional Analysis with RT-PCR and RT-qPCR
2.2. Heterologous Expression and Catalytic Properties of the Recombinant PcaA
2.3. Substrate Specificity
2.4. In Situ Monitoring of the PcaA Enzymatic Reaction Using PCA or Gallate as Substrates in the NMR Tube Bioreactor
3. Discussion
4. Materials and Methods
4.1. Bacterial Strains and Growth Conditions
4.2. Preparation of Sphe3 Cell Extracts
4.3. Enzyme Assays
4.4. Substrate Specificity Assessment
4.5. Cloning and Heterologous Expression of pcaA Gene
4.6. Purification of Recombinant PCA 4,5-Dioxygenase
4.7. In situ Biotransfomation Monitoring in the NMR Tube
4.8. Transcriptional Analysis with RT-PCR and Quantitative Real-Time PCR (RT-qPCR)
4.9. 3D Model Construction of PcaA
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Tsagogiannis, E.; Vandera, E.; Primikyri, A.; Asimakoula, S.; Tzakos, A.G.; Gerothanassis, I.P.; Koukkou, A.-I. Characterization of Protocatechuate 4,5-Dioxygenase from Pseudarthrobacter phenanthrenivorans Sphe3 and In Situ Reaction Monitoring in the NMR Tube. Int. J. Mol. Sci. 2021, 22, 9647. https://doi.org/10.3390/ijms22179647
Tsagogiannis E, Vandera E, Primikyri A, Asimakoula S, Tzakos AG, Gerothanassis IP, Koukkou A-I. Characterization of Protocatechuate 4,5-Dioxygenase from Pseudarthrobacter phenanthrenivorans Sphe3 and In Situ Reaction Monitoring in the NMR Tube. International Journal of Molecular Sciences. 2021; 22(17):9647. https://doi.org/10.3390/ijms22179647
Chicago/Turabian StyleTsagogiannis, Epameinondas, Elpiniki Vandera, Alexandra Primikyri, Stamatia Asimakoula, Andreas G. Tzakos, Ioannis P. Gerothanassis, and Anna-Irini Koukkou. 2021. "Characterization of Protocatechuate 4,5-Dioxygenase from Pseudarthrobacter phenanthrenivorans Sphe3 and In Situ Reaction Monitoring in the NMR Tube" International Journal of Molecular Sciences 22, no. 17: 9647. https://doi.org/10.3390/ijms22179647