Table I
Purification of thermostable protease from Streptomyces sp. CNXK100.
| Purification steps | Total protein (mg) | Total activity (U) | Specific activity (U/mg) | Activity recovery (%) | Purification level |
|---|---|---|---|---|---|
| Crude extract | 54.64 | 1.14 x 106 | 2.09 x 104 | 100.00 | 1.00 |
| Heat-treatment | 18.62 | 4.14 x 105 | 2.22 x 104 | 36.32 | 1.06 |
| Fractional precipitation with 60% (NH4)2SO4 | 0.81 | 1.95 x 105 | 2.41 x 105 | 17.11 | 11.53 |
| Gel filtration | 0.011 | 2.63 x 104 | 2.40 x 106 | 2.31 | 114.83 |
Fig. 1.
SDS-PAGE analysis of protease from Streptomyces sp. CNXK100. Lane 1 – protein marker, lane 2 – purified protease.
Fig. 2.
Effect of temperature (A) and pH (B) on CNXK100 thermostable protease activity.
Fig. 3.
Effect of metal ions on CNXK100 thermostable protease activity.
Fig. 4.
Substrate specificity of CNXK100 thermostable protease.
Fig. 5.
Michaelis-Menten plot of CNXK100 thermostable protease.
Fig. 6.
Stability of CNXK100 thermostable protease; thermostability (A); pH stability (B).
Table II
Effect of various chemicals on CNXK100 thermostable protease activity.
| Group | Chemical | Concentration | Residual activity (%) |
|---|---|---|---|
| Control | – | – | 100.00 |
| Organic solvents | Methanol | 10.0% | 89.28 ± 3.63 |
| Ethanol | 86.54 ± 2.36 | ||
| Isopropanol | 79.47 ± 1.85 | ||
| Butanol | 43.93 ± 2.32 | ||
| Chloroform | 86.54 ± 2.34 | ||
| Surfactants | Triton X-100 | 1.0% | 101.27 ± 2.58 |
| Tween 20 | 88.13 ± 1.93 | ||
| Tween 80 | 92.06 ± 3.21 | ||
| SDS | 53.42 ± 3.05 | ||
| Bleaching agents | NaClO | 0.5% | 105.86 ± 0.57 |
| 1.0% | 111.36 ± 0.86 | ||
| 1.5% | 113.17 ± 1.62 | ||
| 2.0% | 114.11 ± 0.53 | ||
| 2.5% | 121.47 ± 2.28 | ||
| 3.0% | 120.75 ± 1.34 | ||
| H2O2 | 0.5% | 89.21 ± 2.03 | |
| 1.0% | 77.85 ± 9.51 | ||
| 1.5% | 74.12 ± 10.34 | ||
| 2.0% | 71.68 ± 12.29 | ||
| 2.5% | 75.51 ± 2.12 | ||
| 3.0% | 66.96 ± 13.07 | ||
| Proteolytic enzymes | Pepsin | 0.1 mg/ml | 101.54 ± 1.43 |
| Trypsin | 68.97 ± 0.51 | ||
| Chymotrypsin | 91.34 ± 0.13 | ||
| Proteinase K | 10.80 ± 0.44 |
Fig. 7.
Compatibility of CNXK100 thermostable protease with commercial detergent (A) and (B) stain removal ability (a – unwashed control; b – distilled water + buffer A; c – CNXK100 protease + buffer A; d – IZI detergent; e – CNXK100 protease + IZI).
Fig. 8.
Blood clot lysis activity of CNXK100 thermostable protease.
Fig. 9.
Residual activity (column) and protein concentration (line) of CNXK100 thermostable protease in storage condition.