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Purification and Characterization of a Small Thermostable Protease from Streptomyces sp. CNXK100

Tan Viet Pham

Tan Viet Pham

Institute of Biotechnology and Food Technology, Industrial University of Ho Chi Minh CityHo Chi Minh City, Vietnam
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Pham, Tan Viet
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Truong Chinh Hua

Truong Chinh Hua

Faculty of Biology and Biotechnology, University of Science, Vietnam National UniversityHo Chi Minh City, Vietnam
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Hua, Truong Chinh
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Ngoc An Nguyen

Ngoc An Nguyen

Institute of Biotechnology and Food Technology, Industrial University of Ho Chi Minh CityHo Chi Minh City, Vietnam
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Nguyen, Ngoc An
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Hanh Thi Dieu Nguyen

Hanh Thi Dieu Nguyen

Institute of Biotechnology and Food Technology, Industrial University of Ho Chi Minh CityHo Chi Minh City, Vietnam
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Nguyen, Hanh Thi Dieu
  
28 apr 2024
Purification and Characterization of a Small Thermostable Protease from Streptomyces sp. CNXK100's Cover Image
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Categoria dell'articolo: Original Paper
Pubblicato online: 28 apr 2024
Pagine: 155 - 165
Ricevuto: 16 dic 2023
Accettato: 13 mar 2024
DOI: https://doi.org/10.33073/pjm-2024-014
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© 2024 Tan Viet Pham et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
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Fig. 1.

SDS-PAGE analysis of protease from Streptomyces sp. CNXK100. Lane 1 – protein marker, lane 2 – purified protease.
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.
Effect of temperature (A) and pH (B) on CNXK100 thermostable protease activity.

Fig. 3.

Effect of metal ions on CNXK100 thermostable protease activity.
Effect of metal ions on CNXK100 thermostable protease activity.

Fig. 4.

Substrate specificity of CNXK100 thermostable protease.
Substrate specificity of CNXK100 thermostable protease.

Fig. 5.

Michaelis-Menten plot of CNXK100 thermostable protease.
Michaelis-Menten plot of CNXK100 thermostable protease.

Fig. 6.

Stability of CNXK100 thermostable protease; thermostability (A); pH stability (B).
Stability of CNXK100 thermostable protease; thermostability (A); pH stability (B).

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).
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.
Blood clot lysis activity of CNXK100 thermostable protease.

Fig. 9.

Residual activity (column) and protein concentration (line) of CNXK100 thermostable protease in storage condition.
Residual activity (column) and protein concentration (line) of CNXK100 thermostable protease in storage condition.

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

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
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