Biochemical Characterization of An Arginine-Specific Alkaline Trypsin from Bacillus licheniformis
Abstract
:1. Introduction
2. Results and Discussion
2.1. Isolation of Trypsin-Producing Strains
Strain Number | Transparent Circle Diameter/mm | Diameter Ratio | Protease Activity (U/mL) | Trypsin Activity (U/mL) |
---|---|---|---|---|
ZM4 | 30 | 3.46 | 38 | 8 |
ZM5 | 32 | 5.65 | 73 | 7.5 |
CD2 | 30.33 | 5.69 | 67 | 4.5 |
CD7 | 29.67 | 9.89 | 91 | 6.5 |
DMN1 | 31.33 | 4.09 | 23 | 4 |
DMN3 | 28.67 | 8.6 | 13 | 10 |
DMN6 | 15.55 | 1.35 | 14 | 20.5 |
WS6 | 31.33 | 3.36 | 76 | 5.5 |
2.2. Strain Identification
Characteristics | DMN6 | Bacillus licheniformis a | Characteristics | DMN6 | Bacillus licheniformis |
---|---|---|---|---|---|
Oxidase | − | + b | 7% NaCl growth | + | + |
Anaerobic growth | + | + | Methyl red | + | + |
Voges-Proskauer reaction | + | + | Hippurate hydrolyzation | − | − |
Glucose acid production | − | + | 5 °C growth | − | − |
Glucose gas production | − | W c/− | 40 °C growth | + | + |
Citrate utilization | + | + | 44 °C growth | + | + |
Gelatin hydrolyzation | + | + | 50 °C growth | + | + |
Amylolysis | + | + | 55 °C growth | − | + |
Indole production | − | − |
2.3. Optimization of Fermentation Conditions
2.4. Enzyme Purification
Purification Step | Total Protein (mg) | Total Activity (U) | Specific Activity (U/mg) | Yield (%) | Purification |
---|---|---|---|---|---|
Culture filtrate | 73.1 | 3000 | 41.04 | 100 | 1 |
DEAE | 4.05 | 1200 | 296.3 | 5.54 | 7.2 |
Superdex G75 | 2.10 | 735.0 | 350.0 | 2.87 | 8.5 |
2.5. Effect of Temperature and pH on Enzyme Activity and Stability
2.6. Effect of Metal Ions, Inhibitors and Surfactants on Trypsin Activity
Metal Ions | Concentration (mM) | Relative Activity (%) | Concentration (mM) | Relative Activity (%) |
---|---|---|---|---|
Control | 0 | 100 | – | – |
K+ | 1 | 106.67 ± 1.36 | 5 | 44.44 ± 2.59 |
Zn2+ | 1 | ND | 5 | ND |
Mg2+ | 1 | 65.00 ± 6.24 | 5 | 23.02 ± 1.50 |
Na+ | 1 | 95.00 ± 4.08 | 5 | 30.95 ± 3.68 |
Fe3+ | 1 | ND | 5 | ND |
Ba2+ | 1 | 182.46 ± 2.96 | 5 | 60.32 ± 1.30 |
Al3+ | 1 | ND | 5 | ND |
Co2+ | 1 | 34.49 ± 2.61 | 5 | 21.43 ± 3.57 |
Ca2+ | 1 | 67.14 ± 3.59 | 5 | 38.10 ± 0.99 |
Sr2+ | 1 | 59.24 ± 0.00 | 5 | 36.51 ± 1.98 |
Mn2+ | 1 | 69.64 ± 7.99 | 5 | 46.83 ± 3.89 |
Ag+ | 1 | ND | 5 | ND |
Surfactants & Inhibitors | Concentration | Relative Activity (%) |
---|---|---|
Control | 0 | 100 |
DMSO | 1% | 89.71 ± 1.20 |
Triton100 | 1% | 86.27 ± 1.39 |
Tween80 | 1% | 93.63 ± 0.69 |
SDS | 1% | 39.77 ± 0.83 |
PMSF | 5 mM | 38.73 ± 0.69 |
DTT | 5 mM | 110.29 ± 1.2 |
EDTA | 5 mM | 136.27 ± 1.39 |
Benzamidine | 5 mM | 47.92 ± 1.28 |
Aprotinin | 5 mM | 40.79 ± 1.04 |
2.7. Kinetic Parameters of Enzyme
2.8. Cloning of Trypsin Gene from B. licheniformis
3. Materials and Methods
3.1. Materials
3.2. Strain Screening
3.3. Taxonomic Identification
3.4. Culture Optimization
3.5. Enzyme Purification
3.6. Effect of Temperature, pH and Stability on Trypsin Activity
3.7. Effect of Metals, Inhibitors and Surfactants on Trypsin Activity
3.8. Kinetic Parameter Determination
3.9. Cloning of B. licheniformis Trypsin Gene
3.10. Enzyme Assay
3.11. Statistical Analysis
4. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Gong, J.-S.; Li, W.; Zhang, D.-D.; Xie, M.-F.; Yang, B.; Zhang, R.-X.; Li, H.; Lu, Z.-M.; Xu, Z.-H.; Shi, J.-S. Biochemical Characterization of An Arginine-Specific Alkaline Trypsin from Bacillus licheniformis. Int. J. Mol. Sci. 2015, 16, 30061-30074. https://doi.org/10.3390/ijms161226200
Gong J-S, Li W, Zhang D-D, Xie M-F, Yang B, Zhang R-X, Li H, Lu Z-M, Xu Z-H, Shi J-S. Biochemical Characterization of An Arginine-Specific Alkaline Trypsin from Bacillus licheniformis. International Journal of Molecular Sciences. 2015; 16(12):30061-30074. https://doi.org/10.3390/ijms161226200
Chicago/Turabian StyleGong, Jin-Song, Wei Li, Dan-Dan Zhang, Min-Feng Xie, Biao Yang, Rong-Xian Zhang, Heng Li, Zhen-Ming Lu, Zheng-Hong Xu, and Jin-Song Shi. 2015. "Biochemical Characterization of An Arginine-Specific Alkaline Trypsin from Bacillus licheniformis" International Journal of Molecular Sciences 16, no. 12: 30061-30074. https://doi.org/10.3390/ijms161226200