Cloning, Expression, and Characterization of a Highly Stable Heparinase I from Bacteroides xylanisolvens
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals and Reagents
2.2. Cloning of BxHep-I Gene from B. xylanisolvens
2.3. Expression and Purification of BxHep-I
2.4. Enzyme Assay
2.5. Optimization of BxHep-I Expression
2.6. Biochemical Characterization of BxHep-I
2.6.1. Effects of Temperature on the Activity and Stability of BxHep-I
2.6.2. Effect of pH on the Activity and Stability of BxHep-I
2.6.3. Effects of Metal Ions and EDTA on the Activity of BxHep-I
2.7. Enzyme Kinetics
2.8. Homology Modeling and Substrate Docking of BxHep-I
3. Results and Discussion
3.1. Sequence Analysis of BxHep-I
3.2. Cloning, Expression, and Purification of BxHep-I
3.3. Biochemical Characterization of BxHep-I
3.4. Thermostability Analysis of BxHep-I
Organisms | Heparinase I | Specific Activity (U/mg) | Kinetic Parameters | t1/2 (min) | Residual Activity (%) * | Reference | ||
---|---|---|---|---|---|---|---|---|
Km (mg/mL) | Vmax (U/mg) | 30 °C | 37 °C | |||||
F. heparinum | Native | 90.33 | 17.8 | 219.48 | - | - | <30 | [45] |
F. heparinum | Recombinant MBP-fused | 149 | - | - | 10 | - | 11.3 | [26] |
F. heparinum | Recombinant MBP-fused mutant | 56.31 | - | - | 130 | - | 85.1 | [46] |
B. stercoris | Recombinant | 46.02 | 0.023 | 51.81 | - | 30 | - | [19] |
B. eggerthii | Recombinant | 480 | 3.6 | 647.93 | 350 | 60 | 89.1 | [17] |
B. cellulosilyticus | Recombinant | 738.8 | 0.17 | 750.58 | 300 | 59 | 58.6 | [18] |
B. xylanisolvens | Recombinant | 57.6 | 0.79 | 124.58 | 597 | 158 | 73.1 | This work |
3.5. Homology Modeling and Substrate Docking
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Pei, J.-L.; Wei, W.; Wang, D.-R.; Liu, C.-Y.; Zhou, H.-P.; Xu, C.-L.; Zhang, Y.-W. Cloning, Expression, and Characterization of a Highly Stable Heparinase I from Bacteroides xylanisolvens. Polymers 2023, 15, 1776. https://doi.org/10.3390/polym15071776
Pei J-L, Wei W, Wang D-R, Liu C-Y, Zhou H-P, Xu C-L, Zhang Y-W. Cloning, Expression, and Characterization of a Highly Stable Heparinase I from Bacteroides xylanisolvens. Polymers. 2023; 15(7):1776. https://doi.org/10.3390/polym15071776
Chicago/Turabian StylePei, Jia-Lu, Wei Wei, Ding-Ran Wang, Cai-Yun Liu, Hua-Ping Zhou, Chen-Lu Xu, and Ye-Wang Zhang. 2023. "Cloning, Expression, and Characterization of a Highly Stable Heparinase I from Bacteroides xylanisolvens" Polymers 15, no. 7: 1776. https://doi.org/10.3390/polym15071776