Optimisation of Recombinant Myrosinase Production in Pichia pastoris
Abstract
:1. Introduction
2. Results and Discussion
2.1. Recombinant Strain Selection and Myrosinase Activity Assay
2.2. High-Cell Density Cultivation and the Effect of Temperature on Myrosinase Expression
2.3. Cultivation Upscale
2.4. Effect of pH on Myrosinase Expression
2.5. Enzyme Parameters
2.5.1. Temperature and pH Optimum
2.5.2. Myrosinase Activation by Ascorbic Acid
2.6. Storage of Myrosinase
3. Materials and Methods
3.1. Microorganism and Media
3.2. Recombinant Strain Preparation
3.3. Cultivation in Deep Well Plates
3.4. Microplate Assay
3.5. Flask Cultivation
3.6. High-Cell Density Cultivation and Scale-Up
3.7. Myrosinase Activity Assay
3.8. Analysis
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Gene Donor | Gene | Expression Vector | Expression Host | Reference |
---|---|---|---|---|
Arabidopsis thaliana | TGG1, TGG2 | pHIL-S1 (extracellular) | P. pastoris GS115 (Mut+) | [12] |
Arabidopsis thaliana | TGG1, TGG2 TGG4, TGG5 | pPIC3.5K (intracellular) | P. pastoris GS115 (Mut+) | [13] |
Armoracia rusticana | ArMY2 | pPIC3.5K (intracellular) | P. pastoris GS115 (Mut+) | [14] |
Brassica napus | MYR1 | pHIL-S1 (extracellular) | P. pastoris GS115 (Mut+) | [8] |
Carica papaya | CpTGG2 | pPIC3.5 (intracellular) | P. pastoris GS115 (Mut+) | [15] |
Carica papaya | CpTGG1 | pPIC3.5 (intracellular) | P. pastoris GS115 (Mut+) | [16] |
Conditions | Total Protein (mg/L) | Maximal DCW (g/L) | Maximal Activity (U/mL) | Activity after 163 h (U/mL) | Maximal Productivity (U/Lmedium/h) | Productivity after 163 h (U/Lmedium/h) | |
---|---|---|---|---|---|---|---|
1 | 30 °C, pH 5 | 267.3 | 64.9 | 0.16 | 0.15 | 1.35 | 0.89 |
2 | 20 °C, pH 5 | 256.1 | 92.05 | 0.53 | 0.47 | 3.02 | 2.83 |
3 | 20 °C, pH 4 | 383.0 | 92.35 | 0.045 | 0 | 0.39 | 0 |
4 | 20 °C, pH 6 | 286.5 | 100.5 | 0.67 | 0.67 | 4.10 | 4.10 |
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Rosenbergová, Z.; Kántorová, K.; Šimkovič, M.; Breier, A.; Rebroš, M. Optimisation of Recombinant Myrosinase Production in Pichia pastoris. Int. J. Mol. Sci. 2021, 22, 3677. https://doi.org/10.3390/ijms22073677
Rosenbergová Z, Kántorová K, Šimkovič M, Breier A, Rebroš M. Optimisation of Recombinant Myrosinase Production in Pichia pastoris. International Journal of Molecular Sciences. 2021; 22(7):3677. https://doi.org/10.3390/ijms22073677
Chicago/Turabian StyleRosenbergová, Zuzana, Kristína Kántorová, Martin Šimkovič, Albert Breier, and Martin Rebroš. 2021. "Optimisation of Recombinant Myrosinase Production in Pichia pastoris" International Journal of Molecular Sciences 22, no. 7: 3677. https://doi.org/10.3390/ijms22073677