Molecular Cloning of Novel-Type Phosphoenolpyruvate Carboxylase Isoforms in Pitaya (Hylocereus undatus)
Abstract
:1. Introduction
2. Results
2.1. Amino Acid Sequence Alignment and Phylogenetic Analysis of PEPC Isoforms
2.2. Partial Purification and Characterization of HuPPC Isoforms
3. Discussion
4. Materials and Methods
4.1. Plant Material
4.2. Extraction of RNA
4.3. cDNA Cloning of PEPC
4.4. Phylogenetic Analysis
4.5. Partial Purification of HuPPC
4.5.1. Extraction and Concentration Using Anion Exchange Resin
4.5.2. Phenyl-Sepharose Column Chromatography
4.5.3. Mono Q Column Chromatography
4.5.4. Hydroxyapatite (HA) Column Chromatography
4.5.5. Mono S Column Chromatography
4.6. Enzyme Assay
4.7. MS Analysis to Identify the Amino Acid Sequence
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Total Activity (U) | Recovery (%) | Specific Activity (U/mg) | Total Protein (mg) | Purification Fold | |
---|---|---|---|---|---|
Extract | 33.3 | 100 | 0.0318 | 1050 | 1 |
Q-Sepharose | 24.3 | 73.0 | 0.122 | 199 | 3.84 |
Phenyl-Sepharose-a | 9.10 | 27.4 | 0.315 | 28.9 | 9.91 |
Phenyl-Sepharose-b | 11.3 | 34.1 | 0.486 | 23.3 | 15.3 |
Mono Q-a | 7.18 | 21.6 | 2.19 | 3.28 | 68.9 |
Mono Q-b | 8.17 | 24.6 | 0.836 | 9.78 | 26.3 |
Hydroxyapatite-a | 4.37 | 13.1 | 3.87 | 1.13 | 122 |
Hydroxyapatite-b | 7.43 | 22.3 | 1.49 | 5.00 | 46.9 |
Mono S-a | 4.24 | 12.7 | 7.45 | 0.256 | 234 |
Mono S-b | 1.91 | 5.73 | 1.07 | 2.38 | 33.6 |
HuPPC1 | HuPPC2 | HuPPC3 | |
---|---|---|---|
HuPPCa | 12% | 18% | 75% |
HuPPCb | 10% | 67% | 13% |
HuPPC2 | HuPPC3 | |
---|---|---|
pH stability | 6.5–7.0 | 5.5–6.5 |
Heat stability | Up to 35 °C | Up to 50 °C |
Optimal pH | 6.5–7.2 | 7.0–7.2 |
Optimal temperature | 28 °C | 55 °C |
Km,PEP (μM) | 44.3 ± 7.0 | 48.5 ± 4.0 |
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Nomura, K.; Sakurai, Y.; Dozono, M. Molecular Cloning of Novel-Type Phosphoenolpyruvate Carboxylase Isoforms in Pitaya (Hylocereus undatus). Plants 2020, 9, 1241. https://doi.org/10.3390/plants9091241
Nomura K, Sakurai Y, Dozono M. Molecular Cloning of Novel-Type Phosphoenolpyruvate Carboxylase Isoforms in Pitaya (Hylocereus undatus). Plants. 2020; 9(9):1241. https://doi.org/10.3390/plants9091241
Chicago/Turabian StyleNomura, Keiichi, Yuho Sakurai, and Mayu Dozono. 2020. "Molecular Cloning of Novel-Type Phosphoenolpyruvate Carboxylase Isoforms in Pitaya (Hylocereus undatus)" Plants 9, no. 9: 1241. https://doi.org/10.3390/plants9091241