Recombinant Thaumatin-Like Protein (rTLP) and Chitinase (rCHI) from Vitis vinifera as Models for Wine Haze Formation
Abstract
:1. Introduction
2. Results
2.1. Validation of Transformants
2.2. Heterologous Expression of rTLP and rCHI
2.3. Purification of rTLP and rCHI and Characterization by MS-Based Bottom-Up Proteomics
2.4. TLP and CHI: Recombinant Versus Native Proteins
2.4.1. Glycosylation Analysis
2.4.2. Chitinolytic Activity
2.4.3. Thermostability of rTLP and rCHI
2.4.4. Adsorption of the Proteins to Bentonite (Bentonite Fining)
2.4.5. Influence of Polyphenols and Sulfite Ions on the Haze Potential of rTLP and rCHI
3. Discussion
3.1. Molecular Characterization and Comparison with Native Proteins
3.2. Influence of Polyphenols and Sulfite Ion on the Haze Potential of TLP and CHI
3.3. Heterologous rTLP and rCHI as Haze-Forming Protein Models for Research and Applications
4. Materials and Methods
4.1. Plasmid Amplification and Isolation
4.2. Transformation into K. phaffii, Selection of Transformed Cells and Phenotype Determination
4.3. Recombinant Expression, Purification and Identification of rTLP and rCHI
4.3.1. Recombinant Expression
4.3.2. Protein Purification
4.3.3. MS-Based Proteomics Analysis
4.4. Protein Glycosylation
4.5. Chitinolytic Activity
4.6. Analysis of Protein Thermostability by Differential Scanning Fluorimetry (DSF)
4.7. Bentonite Fining
4.8. Influence of Haze-Inducing Agents on Protein Aggregation
4.8.1. Haze Test
4.8.2. Extraction and Analysis of Polyphenols and Monosaccharides from Grape (V. vinifera) Juices
4.8.3. Aggregation Assays
5. 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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Albuquerque, W.; Sturm, P.; Schneider, Q.; Ghezellou, P.; Seidel, L.; Bakonyi, D.; Will, F.; Spengler, B.; Zorn, H.; Gand, M. Recombinant Thaumatin-Like Protein (rTLP) and Chitinase (rCHI) from Vitis vinifera as Models for Wine Haze Formation. Molecules 2022, 27, 6409. https://doi.org/10.3390/molecules27196409
Albuquerque W, Sturm P, Schneider Q, Ghezellou P, Seidel L, Bakonyi D, Will F, Spengler B, Zorn H, Gand M. Recombinant Thaumatin-Like Protein (rTLP) and Chitinase (rCHI) from Vitis vinifera as Models for Wine Haze Formation. Molecules. 2022; 27(19):6409. https://doi.org/10.3390/molecules27196409
Chicago/Turabian StyleAlbuquerque, Wendell, Pia Sturm, Quintus Schneider, Parviz Ghezellou, Leif Seidel, Daniel Bakonyi, Frank Will, Bernhard Spengler, Holger Zorn, and Martin Gand. 2022. "Recombinant Thaumatin-Like Protein (rTLP) and Chitinase (rCHI) from Vitis vinifera as Models for Wine Haze Formation" Molecules 27, no. 19: 6409. https://doi.org/10.3390/molecules27196409