Expression, Purification and Characterization of Chondroitinase AC II from Marine Bacterium Arthrobacter sp. CS01
Abstract
:1. Introduction
2. Results and Discussion
2.1. Isolation and Identification of Strain CS01
2.2. Cloning and Expression of Recombinant ChSase AC II
2.3. Purification of Recombinant ChSase AC II
2.4. Substrate Specificity and Kinetic Parameters of Recombinant ChSase AC II
2.5. Biochemical Properties of Recombinant ChSase AC II
2.6. Analysis of Reaction Mode and Products
3. Materials and Methods
3.1. Strains, Plasmids, Reagents and Medium
3.2. Isolation and Identification of Strain CS01
3.3. Cloning and Expression of Recombinant ChSase AC II
3.4. Purification of Recombinant ChSase AC II
3.5. Assay of Enzyme Activity
3.6. Kinetic Parameters of the Recombinant ChSase AC II
3.7. Biochemical Properties of ChSase AC II
3.8. Analysis of Reaction Mode and Products
4. Conclusions
Author Contributions
Funding
Conflicts of Interest
References
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Step | Total | Total | Specific Activity | Fold | Yield |
---|---|---|---|---|---|
Protein (mg) | Activity (U) | (U/mg Protein) | Purification | (%) | |
Fermentation media | 889.9 | 2723 | 3.06 | 1.0 | 100.0 |
Ultrafiltration | 480.2 | 1969 | 4.10 | 1.34 | 72.3 |
HiTrap QFF column | 77.8 | 1454 | 18.7 | 6.11 | 53.4 |
Substrate | Relative Activity | Vmax | Km | Kcat | Kcat/Km |
---|---|---|---|---|---|
(%) | (µM/min) | (µM) | (min−1) | (min−1/µM) | |
CS-A | 115.64 | 186 | 2.44 | 232 | 95.1 |
CS-C | 100 | 168 | 1.68 | 210 | 125 |
HA | 294.93 | 472 | 0.165 | 590 | 3575 |
Media | Composition |
---|---|
Selective | 0.5% CS, 0.5% yeast extract (YE), 0.5% NaCl, 0.2% MgSO4, 0.05% KH2PO4, pH 7.0 |
Luria-Bertani (LB) | 0.5% YE, 1% NaCl, 1% peptone, pH 7.0 |
Low Salt LB (LLB) | 0.5% YE, 0.5% NaCl, 1% peptone, pH 7.0 |
Yeast extract peptone dextrose medium (YPD) | 1% YE, 2% peptone, 2% glucose, pH 7.0 |
YPD+sorbitol (YPDS) | 1% YE, 2% peptone, 2% glucose, 1 M sorbitol, pH 7.0 |
Minimal dextrase medium (MD) | 2% glucose, 1.34% yeast nitrogen base without amino acids and ammonium sulfate (YNB), 4 × 10−5% biotin, pH 7.0 |
Buffered glycerol-complex medium (BMGY) | 1% Glycerol, 1% YE, 2% peptone, 0.34% YNB, 1% (NH4)2SO4 (w/v) and 4 × 10−5% biotin in pH 6.0 100 mM potassium phosphate buffer |
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Fang, Y.; Yang, S.; Fu, X.; Xie, W.; Li, L.; Liu, Z.; Mou, H.; Zhu, C. Expression, Purification and Characterization of Chondroitinase AC II from Marine Bacterium Arthrobacter sp. CS01. Mar. Drugs 2019, 17, 185. https://doi.org/10.3390/md17030185
Fang Y, Yang S, Fu X, Xie W, Li L, Liu Z, Mou H, Zhu C. Expression, Purification and Characterization of Chondroitinase AC II from Marine Bacterium Arthrobacter sp. CS01. Marine Drugs. 2019; 17(3):185. https://doi.org/10.3390/md17030185
Chicago/Turabian StyleFang, Yangtao, Suxiao Yang, Xiaodan Fu, Wancui Xie, Li Li, Zhemin Liu, Haijin Mou, and Changliang Zhu. 2019. "Expression, Purification and Characterization of Chondroitinase AC II from Marine Bacterium Arthrobacter sp. CS01" Marine Drugs 17, no. 3: 185. https://doi.org/10.3390/md17030185