Biochemical Characterization of a Novel Alkaline-Tolerant Xaa-Pro Dipeptidase from Aspergillus phoenicis
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals and Reagents
2.2. Plasmids, Strains and Growth Conditions
2.3. Gene Cloning and Sequence Analysis
2.4. Expression of ApXPD in P. pastoris
2.5. Enzyme Purification
2.6. Enzyme and Protein Assays
2.7. Biochemical Characterization of Recombinant ApXPD
3. Results
3.1. Molecular Cloning and Sequence Analysis of the xpd Gene from A. phoenicis ATCC 14332
3.2. Reclassification of XPDs Based on Phylogenetic Analysis and MEROPS Database
3.3. Three-Dimensional Structure Analysis of ApXPD
3.4. Expression and Purification of Recombinant ApXPD
3.5. Effects of Temperature and pH on the Activity and Stability of Recombinant ApXPD
3.6. Metal Dependency of Recombinant ApXPD
3.7. Substrate Specificity of Recombinant ApXPD
3.8. Enzyme Kinetics
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Substrate | Relative Activity (%) |
---|---|
Lys-Pro † | 100 ± 2.1 |
Gly-Pro † | 86.3 ± 1.7 |
Ala-Pro † | 32.7 ± 1.3 |
Pro-Pro † | ND |
Cys-Gly ∗ | – |
Lys-Ala ∗ | – |
Lys-Ser ∗ | – |
Lys-Gly ∗ | – |
Leu-Ala-Pro † | ND |
Lys-Pro-Ala ∗ | – |
Gly-Pro-Ala ∗ | – |
Lys-Trp-Ala-Pro † | ND |
Lys-Pro-Ala-Ala ∗ | – |
Substrate | Vmax (μmol min−1 mg−1) | Km (mM) | kcat (s−1) | kcat/Km (mM−1 s−1) | h |
---|---|---|---|---|---|
Lys-Pro | 85.7 | 1.0 | 123.5 | 123.5 | 1.03 |
Gly-Pro | 82.8 | 1.2 | 105.9 | 88.3 | 1.05 |
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Dong, Z.; Yang, S.; Zhang, K.; Tang, C.; Kan, Y.; Yao, L. Biochemical Characterization of a Novel Alkaline-Tolerant Xaa-Pro Dipeptidase from Aspergillus phoenicis. Fermentation 2023, 9, 978. https://doi.org/10.3390/fermentation9110978
Dong Z, Yang S, Zhang K, Tang C, Kan Y, Yao L. Biochemical Characterization of a Novel Alkaline-Tolerant Xaa-Pro Dipeptidase from Aspergillus phoenicis. Fermentation. 2023; 9(11):978. https://doi.org/10.3390/fermentation9110978
Chicago/Turabian StyleDong, Zixing, Shuangshuang Yang, Kun Zhang, Cunduo Tang, Yunchao Kan, and Lunguang Yao. 2023. "Biochemical Characterization of a Novel Alkaline-Tolerant Xaa-Pro Dipeptidase from Aspergillus phoenicis" Fermentation 9, no. 11: 978. https://doi.org/10.3390/fermentation9110978