Gene Expression and Enzyme Kinetics of Polyphenol Oxidases in Strawberry and Their Possible Involvement in Enzymatic Browning Reactions in Strawberry Nectar
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals
2.2. PPO Genes and Plasmid Construct
2.3. Protein Expression and Purification
2.4. SDS-PAGE and Western Blotting
2.5. Enzyme Assays
2.6. Kinetic Studies
2.7. Plant Material
2.8. Nectar Preparation
2.9. Color Measurement and Acceptance Factor Calculation
2.10. RT-qPCR Analysis
3. Results and Discussion
3.1. Selection of PPO Gene Candidates
3.2. Protein Expression and Purification
3.3. Enzyme Characterization
3.3.1. Activation of the Latent Enzyme and pH Optimum
3.3.2. Substrate Specificity of the Recombinant PPOs
3.4. RT-qPCR Analysis
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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FaPPO | Tyramine (470 nm) | Dopamine (465 nm) | L-DOPA (475 nm) | Cathechol (400 nm) | Catechin (440 nm) | Chlorogenic Acid (400 nm) | Phloretin (455 nm) |
---|---|---|---|---|---|---|---|
1 | 1 | 0.2 | 0.1 | 0.2 | 0.2 | 0.5 | 1 |
2 | 1 | 0.2 | 0.1 | 0.2 | 0.2 | 0.5 | 1 |
4 | 3 | 1 | 1 | 1 | 1 | 2.5 | 3 |
Stage | Code | Height (mm) | Width (mm) | Weight (g) |
---|---|---|---|---|
Green | 0 | 20.3 | 10.8 | 2.9 |
White | 0.25 | 20.6 | 20.3 | 7.7 |
Turning | 0.50 | 20.9 | 20.9 | 12.2 |
Ripe | 0.75 | 30.9 | 20.9 | 13.6 |
Overripe | 1.00 | 30.0 | 40.0 | 22.2 |
Substrate | Structure | PPO | kcat (s−1) | KM (mM) | kcat/KM (s−1 mM−1) |
---|---|---|---|---|---|
Tyramine | 1 2 4 | 42 ± 1 32.0 ± 0.4 142 ± 20 | 0.5 ± 0.1 0.6 ± 0.1 1.3 ± 0.1 | 85 ± 7 54 ± 4 244 ± 45 | |
Dopamine | 1 2 4 | 502 ± 18 384 ± 14 125 ± 0.4 | 0.6 ± 0.1 0.95 ± 0.12 0.99 ± 0.11 | 827 ± 94 406 ± 39 125 ± 31 | |
L-DOPA | 1 2 4 | 468 ± 8 460 ± 7 30 ± 0.2 | 2 ± 0.4 5.7 ± 1.9 3.2 ± 1.6 | 187 ± 47 97 ± 42 17.4 ± 1.5 | |
Catechol | 1 2 4 | 1086 ± 7 827 ± 27 681 ± 10 | 27 ± 2 7.5 ± 0.6 39 ± 12 | 292 ± 10 111 ± 7 25 ± 10 | |
Catechin | 1 2 4 | 176 ± 11 146 ± 4 142 ± 20 | 0.12 ± 0.03 0.19 ± 0.04 0.87 ± 0.08 | 1510 ± 367 772 ± 59 165 ± 11 | |
Chloro-genic acid | 1 2 4 | 352 ± 20 198 ± 10 82 ± 1 | 2.7 ± 0.4 1.3 ± 0.2 1.3 ± 0.2 | 130 ± 67 153 ± 59 64 ± 5 | |
Phloretin | 1 2 4 | 13.5 ± 0.4 1 35 ± 8 2 - 3 | 0.082 ± 0.004 1 0.6 ± 0.4 2 - 3 | 165 ± 5 1 55 ± 18 2 - 3 |
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Zavarise, A.; Rabeeah, I.; Molitor, C.; Davoudi Pahnekolayi, M.; Gruber-Schmidt, V.; Winter, A.; Olbricht, K.; Haselmair-Gosch, C.; Stich, K.; Goessinger, M.; et al. Gene Expression and Enzyme Kinetics of Polyphenol Oxidases in Strawberry and Their Possible Involvement in Enzymatic Browning Reactions in Strawberry Nectar. Foods 2025, 14, 2064. https://doi.org/10.3390/foods14122064
Zavarise A, Rabeeah I, Molitor C, Davoudi Pahnekolayi M, Gruber-Schmidt V, Winter A, Olbricht K, Haselmair-Gosch C, Stich K, Goessinger M, et al. Gene Expression and Enzyme Kinetics of Polyphenol Oxidases in Strawberry and Their Possible Involvement in Enzymatic Browning Reactions in Strawberry Nectar. Foods. 2025; 14(12):2064. https://doi.org/10.3390/foods14122064
Chicago/Turabian StyleZavarise, Alberto, Ibrahim Rabeeah, Christian Molitor, Mahboubeh Davoudi Pahnekolayi, Viktoria Gruber-Schmidt, Andrea Winter, Klaus Olbricht, Christian Haselmair-Gosch, Karl Stich, Manfred Goessinger, and et al. 2025. "Gene Expression and Enzyme Kinetics of Polyphenol Oxidases in Strawberry and Their Possible Involvement in Enzymatic Browning Reactions in Strawberry Nectar" Foods 14, no. 12: 2064. https://doi.org/10.3390/foods14122064
APA StyleZavarise, A., Rabeeah, I., Molitor, C., Davoudi Pahnekolayi, M., Gruber-Schmidt, V., Winter, A., Olbricht, K., Haselmair-Gosch, C., Stich, K., Goessinger, M., & Halbwirth, H. (2025). Gene Expression and Enzyme Kinetics of Polyphenol Oxidases in Strawberry and Their Possible Involvement in Enzymatic Browning Reactions in Strawberry Nectar. Foods, 14(12), 2064. https://doi.org/10.3390/foods14122064