Optimization of the Conditions for Extraction of Serine Protease from Kesinai Plant (Streblus asper) Leaves Using Response Surface Methodology
Abstract
:1. Introduction
2. Results and Discussions
2.1. Response Surface Analysis
- [a] Specific activity = 162.13+ 44.97 (mixing time)+ 9.67 (buffer content) + 34.79 (pH of buffer)+41.27 (temperature)2+ 7.21 (mixing time)2+ 22.13 (buffer content)2 + 8.31(pH of buffer)2 + 53.15 (mixing time *pH of buffer)
- [b] Storage stability= 86.38 + 2.50 (mixing time) + 3.25 (buffer content) + 2.57 (pH of buffer) +13 (temperature)2 + 4.65 (mixing time)2+ 7.27 (buffer content)2 +23.27 (pH of buffer)2 +8.12 (mixing time *buffer content) +2.72 (buffer content*pH of buffer)
- [c] Thermal stability = 82.36 + 3 (mixing time) +16.5 (buffer content) +2.5 (pH of buffer) +11.61 (mixing time)2 + 12.36 (buffer content)2 + 38.5 (pH of buffer)2 + 13.87 (temperature *mixing time) +14.25 (temperature*pH of buffer)
- [d] Oxidizing agent stability = 79.15+ 5.50 (mixing time) +11.50 (buffer content) +2.57 (pH of buffer) +2.25 (temperature)2 + 14.25 (mixing time) 2 + 14.02 (buffer content)2+ 7.90 (pH of buffer)2 +8.72 (mixing time *buffer content).
Model term | p-value of each independent variable | |||
---|---|---|---|---|
Specific activity | Storage stability | Thermal stability | Oxidizing agent stability | |
X1 | 0.153 | 0.411 | 0.169 | 0.131 |
X2 | 0.002 | 0.002 | 0.001 | 0.000 |
X3 | 0.001 | 0.001 | 0.000 | 0.006 |
X4 | 0.000 | 0.001 | 0.000 | 0.001 |
X12 | 0.014 | 0.020 | 0.327 | 0.044 |
X22 | 0.001 | 0.001 | 0.000 | 0.000 |
X32 | 0.008 | 0.000 | 0.002 | 0.000 |
X42 | 0.000 | 0.001 | 0.000 | 0.017 |
X1X2 | 0.202 | 0.098 | 0.004 | 0.502 |
X1X3 | 0.138 | 0.351 | 0.223 | 0.308 |
X1X4 | 0.081 | 0.123 | 0.000 | 0.056 |
X2X3 | 0.102 | 0.001 | 0.134 | 0.000 |
X2X4 | 0.000 | 0.182 | 0.073 | 0.128 |
X3X4 | 0.092 | 0.002 | 0.188 | 0.231 |
Regression coefficient | Specific activity (Y1, U/mL) | Storage stability (Y2, %) | Thermal stability (Y3, %) | Oxidizing agent stability (Y4, %) |
---|---|---|---|---|
b0 | 162.13 | 86.38 | 82.36 | 79.15 |
b1 | 9.12 | 0.57 | 1.00 | 6.25 |
b2 | 44.97 | 2.50 | 3.00 | 5.50 |
b3 | 9.67 | 3.25 | 16.50 | 11.50 |
b4 | 34.79 | 2.75 | 2.50 | 2.75 |
b12 | 41.27 | 13.00 | 1.91 | 2.25 |
b22 | 7.21 | 4.65 | 11.61 | 14.25 |
b32 | 22.13 | 7.27 | 12.36 | 14.02 |
b42 | 8.31 | 23.27 | 3.86 | 7.90 |
b12 | 6.71 | 1.83 | 13.87 | 10.23 |
b13 | 3.27 | 1.01 | 2.52 | 7.89 |
b14 | 9.12 | 11.48 | 14.25 | 13.26 |
b23 | 13.26 | 8.12 | 10.80 | 8.72 |
b24 | 53.15 | 2.68 | 0.97 | 15.61 |
b34 | 1.12 | 9.27 | 1.60 | 12.03 |
R2 | 0.989 | 0.999 | 0.999 | 0.994 |
R2 (adj.) | 0.955 | 0.995 | 0.989 | 0.988 |
Regression (p-value) | 0.001a | 0.000a | 0.001a | 0.002a |
Variables | Main effects | Quadratic effects | Interaction effects | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
X1 | X2 | X3 | X4 | X12 | X22 | X32 | X42 | X1X2 | X1X3 | X1X4 | X2X3 | X2X4 | X3X4 | ||
Specific activity (Y1, U/mg) | p-value | 0.153 | 0.002 a | 0.001 a | 0.000 a | 0.014 a | 0.001 a | 0.008 a | 0.000 a | 0.202 | 0.138 | 0.081 | 0.102 | 0.000 a | 0.092 |
F-ratio | 2.82 | 36.72 | 57.76 | 148.10 | 13.83 | 62.25 | 18.49 | 131.10 | 0.66 | 2.56 | 2.07 | 0.033 | 125.44 | 4.04 | |
Storage stability (Y2, %) | p-value | 0.411 | 0.002 a | 0.001 a | 0.001 a | 0.020 a | 0.001 a | 0.000 a | 0.001 a | 0.098 | 0.351 | 0.123 | 0.001a | 0.182 | 0.002 a |
F-ratio | 0.82 | 14.06 | 62.09 | 60.84 | 53.58 | 97.41 | 81.90 | 30.25 | 2.56 | 0.08 | 1.96 | 107.32 | 1.69 | 52.85 | |
Thermal stability (Y3, %) | p-value | 0.169 | 0.001 a | 0.000 a | 0.000 a | 0.327 | 0.000 a | 0.002 a | 0.000 a | 0.004 a | 0.223 | 0.000 a | 0.134 | 0.073 | 0.188 |
F-ratio | 3.25 | 441.00 | 384.16 | 144.96 | 1.36 | 364.04 | 310.81 | 538.24 | 234.09 | 1.44 | 265.69 | 0.64 | 4.41 | 1.44 | |
Oxidizing agent stability (Y4, %) | p-value | 0.131 a | 0.000 a | 0.006 a | 0.001 a | 0.044 a | 0.000 a | 0.000 a | 0.017 | 0.502 | 0.308 | 0.056 | 0.000 a | 0.128 | 0.231 |
F-ratio | 1.44 | 189.00 | 21.16 | 43.03 | 11.28 | 148.59 | 154.00 | 12.39 | 0.04 | 1.08 | 4.96 | 197.96 | 2.40 | 0.022 |
2.2. Specific Activity of Seine Protease
2.3. Storage Stability of Serine Protease
2.4. Thermal Stability of Serine Protease
2.5. Oxidizing Agent Stability of Serine Protease
2.6. Optimization Procedure
2.7. Model Validation
3. Experimental
3.1. Materials
3.2. Serine Protease Extraction
Independent variables | Independent variables level | ||||
---|---|---|---|---|---|
Low | Center | High | Axial (−α) | Axial (+α) | |
Temperature (°C) | −20 | 2.5 | +25 | −42.5 | 47.5 |
Time (min) | 3 | 4 | 5 | 2 | 6 |
Buffer content (mL) | 20 | 40 | 60 | 0 | 80 |
pH of buffer | 6 | 7.5 | 9 | 4.5 | 10.5 |
3.3. Determination of Serine Protease Activity
3.4. Determination of Protein Concentration
3.5. Determination of Serine Protease Specific Activity
3.6. Determination of Serine Protease Storage Stability
3.7. Determination of Serine Protease Thermal Stability
3.8. Determination of Oxidizing Agent Stability
3.9. Statistical Design
4. Conclusions
Treatments | Blocks | Temperature (X1, °C) | Mixing time (X2, min) | Buffer content (X3, mL) | pH of buffer (X4) | Specific activity (U/mL) | Storage stability (%) | Thermal stability (%) | Oxidizing agent stability |
---|---|---|---|---|---|---|---|---|---|
1 C | 1 | 2.5 | 4 | 40 | 7.5 | 162.4 | 86.8 | 82.6 | 79.3 |
2 | 1 | 25 | 3 | 20 | 6.0 | 161.8 | 80.4 | 73.4 | 62.7 |
3 | 1 | 25 | 5 | 60 | 6.0 | 150.0 | 20.0 | 30.0 | 30.6 |
4 | 1 | 25 | 3 | 60 | 9.0 | 130.7 | 52.0 | 62.0 | 43.2 |
5 | 1 | −20 | 3 | 60 | 6.0 | 120.4 | 56.7 | 53.3 | 61.1 |
6 | 1 | 25 | 5 | 20 | 9.0 | 140.5 | 15.3 | 21.2 | 29.3 |
7 | 1 | −20 | 3 | 20 | 9.0 | 10.0 | 73.4 | 66.1 | 52.2 |
8 C | 1 | 2.5 | 4 | 40 | 7.5 | 158.9 | 80.1 | 81.1 | 77.7 |
9 | 1 | −20 | 5 | 60 | 9.0 | 110.0 | 58.8 | 38.8 | 33.1 |
10 | 1 | −20 | 5 | 20 | 6.0 | 62.4 | 86.4 | 42.0 | 32.0 |
11 C | 2 | 2.5 | 4 | 40 | 7.5 | 162.4 | 85.4 | 80.6 | 79.4 |
12 | 2 | 25 | 3 | 60 | 6.0 | 105.3 | 42.7 | 68.0 | 42.1 |
13 C | 2 | 2.5 | 4 | 40 | 7.5 | 160.0 | 86.0 | 82.3 | 71.3 |
14 | 2 | 25 | 5 | 60 | 9.0 | 67.2 | 32.4 | 48.0 | 42.2 |
15 | 2 | 25 | 3 | 20 | 9.0 | 88.1 | 44.2 | 78.2 | 61.3 |
16 | 2 | −20 | 5 | 60 | 6.0 | 93.8 | 85.8 | 32.2 | 30.3 |
17 | 2 | −20 | 5 | 20 | 9.0 | 54.9 | 34.8 | 42.2 | 44.7 |
18 | 2 | 25 | 5 | 20 | 6.0 | 90.1 | 14.2 | 18.8 | 21.3 |
19 | 2 | −20 | 3 | 60 | 9.0 | 123.0 | 51.0 | 63.2 | 55.8 |
20 | 2 | −20 | 3 | 20 | 6.0 | 132.1 | 42.8 | 34.1 | 23.2 |
21 | 3 | 2.5 | 6 | 40 | 7.5 | 111.9 | 68.0 | 71.2 | 70.2 |
22 | 3 | 2.5 | 4 | 0 | 7.5 | 74.3 | 73.4 | 57.1 | 68.8 |
23 | 3 | 2.5 | 2 | 40 | 7.5 | 121.2 | 54.5 | 73.6 | 40.2 |
24 | 3 | −42.5 | 4 | 40 | 7.5 | 81.4 | 86.1 | 29.2 | 33.4 |
25 | 3 | 2.5 | 4 | 40 | 10.5 | 122.2 | 77.0 | 66.6 | 51.7 |
26 C | 3 | 2.5 | 4 | 40 | 7.5 | 161.9 | 72.3 | 80.8 | 78.7 |
27 | 3 | 2.5 | 4 | 40 | 4.5 | 101.1 | 42.2 | 52.4 | 54.3 |
28 C | 3 | 2.5 | 4 | 40 | 7.5 | 162.1 | 86.5 | 81.2 | 79.1 |
29 | 3 | 2.5 | 4 | 80 | 7.5 | 115.0 | 69.4 | 53.8 | 56.5 |
30 | 3 | 47.5 | 4 | 40 | 7.5 | 51.3 | 23.4 | 38.1 | 22.0 |
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Mehrnoush, A.; Mustafa, S.; Sarker, M.Z.I.; Yazid, A.M.M. Optimization of the Conditions for Extraction of Serine Protease from Kesinai Plant (Streblus asper) Leaves Using Response Surface Methodology. Molecules 2011, 16, 9245-9260. https://doi.org/10.3390/molecules16119245
Mehrnoush A, Mustafa S, Sarker MZI, Yazid AMM. Optimization of the Conditions for Extraction of Serine Protease from Kesinai Plant (Streblus asper) Leaves Using Response Surface Methodology. Molecules. 2011; 16(11):9245-9260. https://doi.org/10.3390/molecules16119245
Chicago/Turabian StyleMehrnoush, Amid, Shuhaimi Mustafa, Md. Zaidul Islam Sarker, and Abdul Manap Mohd Yazid. 2011. "Optimization of the Conditions for Extraction of Serine Protease from Kesinai Plant (Streblus asper) Leaves Using Response Surface Methodology" Molecules 16, no. 11: 9245-9260. https://doi.org/10.3390/molecules16119245