Optimization of Culture Conditions for Oxygen-Tolerant Regulatory [NiFe]-Hydrogenase Production from Ralstonia eutropha H16 in Escherichia coli
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strains and Growth Conditions
2.2. Plasmid Design and Construction
2.3. Deepwell Plate Cultivation
2.4. Shake-Flask Cultivation
2.5. Purification of Strep-Tagged Hox Proteins
2.6. Western Blot Analysis
2.7. Spectroscopic Analysis and Activity Assays of RH
3. Results
3.1. Construction of Expression Vectors for Heterologous Hydrogenase Production in E. coli
3.2. Screening of Inducer Concentrations for HoxBC Overproduction in E. coli
3.3. Low Inducer Concentrations and Low Temperatures Support Production of Soluble RH
3.4. Optimization of Expression Time
3.5. Fed-Batch Like Cultivation for Heterologous RH Production in E. coli
3.6. Upscalable RH Production in Shake Flask in Batch and Fed-Batch Cultivations
3.7. Biochemical and Spectroscopic Characterization of the Recombinant HoxBC and HoxC Proteins
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
References
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Fan, Q.; Caserta, G.; Lorent, C.; Lenz, O.; Neubauer, P.; Gimpel, M. Optimization of Culture Conditions for Oxygen-Tolerant Regulatory [NiFe]-Hydrogenase Production from Ralstonia eutropha H16 in Escherichia coli. Microorganisms 2021, 9, 1195. https://doi.org/10.3390/microorganisms9061195
Fan Q, Caserta G, Lorent C, Lenz O, Neubauer P, Gimpel M. Optimization of Culture Conditions for Oxygen-Tolerant Regulatory [NiFe]-Hydrogenase Production from Ralstonia eutropha H16 in Escherichia coli. Microorganisms. 2021; 9(6):1195. https://doi.org/10.3390/microorganisms9061195
Chicago/Turabian StyleFan, Qin, Giorgio Caserta, Christian Lorent, Oliver Lenz, Peter Neubauer, and Matthias Gimpel. 2021. "Optimization of Culture Conditions for Oxygen-Tolerant Regulatory [NiFe]-Hydrogenase Production from Ralstonia eutropha H16 in Escherichia coli" Microorganisms 9, no. 6: 1195. https://doi.org/10.3390/microorganisms9061195
APA StyleFan, Q., Caserta, G., Lorent, C., Lenz, O., Neubauer, P., & Gimpel, M. (2021). Optimization of Culture Conditions for Oxygen-Tolerant Regulatory [NiFe]-Hydrogenase Production from Ralstonia eutropha H16 in Escherichia coli. Microorganisms, 9(6), 1195. https://doi.org/10.3390/microorganisms9061195