Response Surface Methodology Modelling of an Aqueous Two-Phase System for Purification of Protease from Penicillium candidum (PCA 1/TT031) under Solid State Fermentation and Its Biochemical Characterization
Abstract
:1. Introduction
2. Results
2.1. Optimizing the Protease Purification Using an Aqueous Two-Phase System
2.1.1. Fitting of the RSM Models
2.1.2. Partition Coefficient (Y1)
2.1.3. Purification Factor (Y2)
2.1.4. Yields (Y3)
2.1.5. Experimental Validation of the Models
2.2. Characterization of Protease from P. candidum (PCA 1/TT031) in Aqueous Solutions
2.2.1. Influence of Temperature on the Activity and Stability of P. candidum (PCA 1/TT031) Protease
2.2.2. Influence of pH on the Activity and Stability of P. candidum (PCA 1/TT031) Protease
2.2.3. Influence of Inhibitors and Metal Ions on the Activity of P. candidum (PCA 1/TT031) Protease
2.2.4. SDS-PAGE Assessment of Purified Protease and ATPS
3. Discussion
3.1. Protease Purification Using the Aqueous Two-Phase System
3.1.1. Partition Coefficient (Y1)
3.1.2. Purification Factor (Y2)
3.1.3. Yields (Y3)
3.1.4. Experimental Validation of the Models
3.2. Characterization of Protease from P. candidum (PCA 1/TT031) in Aqueous Solutions
3.2.1. Influence of Temperature on the Activity and Stability of P. candidum (PCA 1/TT031) Protease
3.2.2. Influence of pH on the Activity and Stability of P. candidum (PCA 1/TT031) Protease
3.2.3. Influence of Metal Ions and Inhibitors on the Activity of P. candidum (PCA 1/TT031) Protease
3.2.4. SDS-PAGE Assessment of Purified Protease and ATPS
4. Methods and Materials
4.1. Materials
4.2. Production of Protease by Solid-State Fermentation
4.3. Enzyme Extraction
4.4. Proteolytic Assay
4.5. Protein Determination
4.6. Optimizing the Protease Purification Using an Aqueous Two-Phase System
4.6.1. ATPS Preparation
4.6.2. Calculations
4.6.3. Experimental Design
4.6.4. Variance Analysis
4.6.5. Optimization and Validation of the Experimental Process
4.7. Protease Characterization
4.7.1. Influence of Temperature on Activity and Stability of P. candidum (PCA 1/TT031) Protease
4.7.2. Influence of pH on Activity and Stability of P. candidum (PCA 1/TT031) Protease
4.7.3. Influence of Metal Ions and Inhibitors on Activity of P. candidum (PCA 1/TT031) Protease
4.7.4. SDS–PAGE Analysis
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Regression Coefficient | Partition Coefficient (Y1) | Purification Factor (Y2) | Yield (Y3) |
---|---|---|---|
b0 | 4.25371 | 8.00997 | 122.231 |
b1 | −0.00019 | 0.000124 | 0.007 |
b2 | −0.18962 | −0.72459 | −4.359 |
b3 | −0.20842 | −0.61406 | −10.700 |
b4 | −0.14749 | 0.99890 | −2.721 |
b12 | 0.00000 | −0.00000 | −0.000 |
b22 | 0.00426 | 0.01864 | 0.099 |
b32 | 0.01023 | 0.00242 | 0.281 |
b42 | 0.01415 | −0.07165 | −0.140 |
b12 | 0.00001 | −0.00004 | 0.000 |
b13 | −0.00001 | 0.00001 | 0.000 |
b14 | 0.00000 | −0.00003 | 0.000 |
b23 | 0.00170 | 0.03580 | 0.023 |
b24 | −0.00082 | −0.02791 | −0.127 |
b34 | 0.00063 | 0.02063 | 0.350 |
R2 | 0.96 | 0.94 | 0.98 |
R2 (adj.) | 0.92 | 0.87 | 0.97 |
Regression (p-value) | 0.000 a | 0.000 a | 0.000 a |
Ariables | Main Effects | Quadratic Effects | Interaction Effects | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Independent Variable | X1 | X2 | X3 | X4 | X12 | X22 | X32 | X42 | X1X2 | X1X3 | X1X4 | X2X3 | X2X4 | X3X4 | |
Partition coefficient (Y1) | p-value | 0.010 a | 0.005 a | 0.024 a | 0.015 a | 0.000 a | 0.013 a | 0.003 a | 0.000 a | 0.016 a | 0.002 a | 0.432 | 0.533 | 0.707 | 0.834 |
F-ratio | 9.05 | 11.24 | 6.55 | 7.82 | 26.16 | 8.29 | 13.40 | 62.56 | 7.70 | 14.44 | 0.66 | 0.41 | 0.15 | 0.05 | |
Purification factor (Y2) | p-value | 0.002 a | 0.028 a | 0.170 | 0.003 a | 0.005 a | 0.030 a | 0.870 | 0.000 a | 0.016 a | 0.670 | 0.040 a | 0.022 a | 0.025 a | 0.197 |
F-ratio | 14.00 | 6.11 | 2.12 | 13.35 | 11.65 | 5.91 | 0.03 | 59.96 | 7.61 | 0.19 | 5.20 | 6.73 | 6.39 | 1.85 | |
Yield %(Y3) | p-value | 0.037 a | 0.141 | 0.019 a | 0.313 | 0.000 a | 0.196 | 0.062 | 0.136 | 0.016 a | 0.022 a | 0.011 a | 0.865 | 0.246 | 0.030 a |
F-ratio | 5.39 | 2.45 | 7.13 | 1.10 | 66.90 | 1.86 | 4.18 | 2.53 | 7.71 | 6.78 | 8.70 | 0.03 | 1.48 | 5.90 |
Reagent | Relative Activity (% ± SD) a | |
---|---|---|
Concentration 5 mM | Concentration 10 mM | |
Without component | 100 ± 0.00 d | 100 ± 0.00 b |
NaCl | 136 ± 1.7 a | 112 ± 2.5 a |
ZnCl2 | 122 ± 2.5 b | 92 ± 2.0 c |
KCl | 112 ± 2.0 c | 85 ± 3.0 d |
MnCl2 | 110 ± 2.0 c | 98 ± 2.0 b |
MgCl2 | 44 ± 2.6 f | 31 ± 1.5 e |
CaCl2 | 34 ± 2.0 g | 23 ± 1.5 f |
FeCl3 | 25 ± 3.00 h | 0.00 ± 0.00 g |
Sodium dodecyl sulfate (SDS) | 54 ± 3.5 e | 33 ± 2.5 e |
Ethylenediaminetetraacetate (EDTA) | 15 ± 2.0 i | 7 ± 2.0 h |
PMSF (Phenylmethanesulfonylflouride) | 100 ± 1.3 d | 100 ± 1.7 b |
Run Order | Independent Variable | ||||
---|---|---|---|---|---|
Block | PEG Molecular Mass (g/mol,X1) | PEG Concentration (w/w, X2) | Citrate Concentration (w/w, X3) | NaCl Concentration (w/w, X4) | |
1 | 3 | 6000 | 20 | 12 | 5 |
2c | 3 | 6000 | 14.5 | 12 | 5 |
3 | 3 | 1500 | 14.5 | 12 | 5 |
4 | 3 | 6000 | 14.5 | 16 | 5 |
5 | 3 | 10,000 | 14.5 | 12 | 5 |
6 | 3 | 6000 | 9 | 12 | 5 |
7 | 3 | 6000 | 14.5 | 12 | 10 |
8 | 3 | 6000 | 14.5 | 12 | 0 |
9 | 3 | 6000 | 14.5 | 8 | 5 |
10 c | 3 | 6000 | 14.5 | 12 | 5 |
11 c | 1 | 6000 | 14.5 | 12 | 5 |
12 c | 1 | 6000 | 14.5 | 12 | 5 |
13 | 1 | 4000 | 17.25 | 14 | 7.5 |
14 | 1 | 8000 | 17.25 | 10 | 7.5 |
15 | 1 | 8000 | 11.75 | 10 | 2.5 |
16 | 1 | 8000 | 11.75 | 14 | 7.5 |
17 | 1 | 8000 | 17.25 | 14 | 2.5 |
18 | 1 | 4000 | 11.75 | 10 | 7.5 |
19 | 1 | 4000 | 11.75 | 14 | 2.5 |
20 | 1 | 4000 | 17.25 | 10 | 2.5 |
21 c | 2 | 6000 | 14.5 | 12 | 5 |
22 | 2 | 4000 | 17.25 | 10 | 7.5 |
23 | 2 | 8000 | 17.25 | 10 | 2.5 |
24 | 2 | 8000 | 11.75 | 14 | 2.5 |
25 | 2 | 8000 | 17.25 | 14 | 7.5 |
26 | 2 | 4000 | 11.75 | 14 | 7.5 |
27 | 2 | 4000 | 11.75 | 10 | 2.5 |
28 | 2 | 8000 | 11.75 | 10 | 7.5 |
29 | 2 | 4000 | 17.25 | 14 | 2.5 |
30 c | 2 | 6000 | 14.5 | 12 | 5 |
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Alhelli, A.M.; Abdul Manap, M.Y.; Mohammed, A.S.; Mirhosseini, H.; Suliman, E.; Shad, Z.; Mohammed, N.K.; Meor Hussin, A.S. Response Surface Methodology Modelling of an Aqueous Two-Phase System for Purification of Protease from Penicillium candidum (PCA 1/TT031) under Solid State Fermentation and Its Biochemical Characterization. Int. J. Mol. Sci. 2016, 17, 1872. https://doi.org/10.3390/ijms17111872
Alhelli AM, Abdul Manap MY, Mohammed AS, Mirhosseini H, Suliman E, Shad Z, Mohammed NK, Meor Hussin AS. Response Surface Methodology Modelling of an Aqueous Two-Phase System for Purification of Protease from Penicillium candidum (PCA 1/TT031) under Solid State Fermentation and Its Biochemical Characterization. International Journal of Molecular Sciences. 2016; 17(11):1872. https://doi.org/10.3390/ijms17111872
Chicago/Turabian StyleAlhelli, Amaal M., Mohd Yazid Abdul Manap, Abdulkarim Sabo Mohammed, Hamed Mirhosseini, Eilaf Suliman, Zahra Shad, Nameer Khairulla Mohammed, and Anis Shobirin Meor Hussin. 2016. "Response Surface Methodology Modelling of an Aqueous Two-Phase System for Purification of Protease from Penicillium candidum (PCA 1/TT031) under Solid State Fermentation and Its Biochemical Characterization" International Journal of Molecular Sciences 17, no. 11: 1872. https://doi.org/10.3390/ijms17111872