‘Heat-Treatment Aqueous Two Phase System’ for Purification of Serine Protease from Kesinai (Streblus asper) Leaves
Abstract
:1. Introduction
2. Results and Discussions
2.1. Effect of Temperature on Purity of Serine Protease from Kesinai Leaves
2.2. SDS-PAGE Analysis of the Purified Serine Protease
2.3. Effect of PEG on the Partitioning of Serine Protease from Kesinai Leaves
Phase Composition (%, w/w) | KP | KE | PF | Yield (%) |
---|---|---|---|---|
8% PEG4000-15% MgSO4 | ns | ns | ns | ns |
16% PEG4000-15% MgSO4 | 0.24 ± 0.03 | 1.4 ± 0.09 | 4.4 ± 0.13 | 41.3 ± 0.22 |
21% PEG4000-15% MgSO4 | 0.50 ± 0.10 | 0.03 ± 0.01 | 3.2 ± 0.10 | 33.8 ± 0.31 |
8% PEG6000-15% MgSO4 | 0.22 ± 0.74 | 2.12 ± 0.02 | 6.2 ± 0.8 | 53.4 ± 0.13 |
16% PEG6000-15% MgSO4 | 0.001 ± 0.23 | 4.52 ± 0.04 | 8.9 ± 0.02 | 83.1 ± 0.08 |
21% PEG6000-15% MgSO4 | 0.04 ± 0.18 | 3.51 ± 0.14 | 7.8 ± 0.2 | 61 ± 0.06 |
8% PEG8000-15% MgSO4 | 0.31 ± 0.15 | 1.02 ± 0.12 | 5.3 ± 0.03 | 50.2 ± 0.12 |
16% PEG8000-15% MgSO4 | 0.07 ± 0.07 | 0.23 ± 0.01 | 3.9 ± 0.01 | 31.9 ± 0.03 |
21% PEG8000-15% MgSO4 | 0.10 ± 0.02 | 0.31 ± 0.21 | 4.2 ± 0.05 | 40.2 ± 0.05 |
2.4. Effect of Salts on the Partitioning of Serine Protease from Kesinai Leaves
Phase Composition (%,w/w) | KP | KE | PF | Yield (%) |
---|---|---|---|---|
16% PEG6000-12% Na-citrate | ns | ns | ns | ns |
16% PEG6000-15% Na-citrate | 0.12 ± 0.02 | 1.8 ± 0.15 | 3.8 ± 0.80 | 34 ± 0.09 |
16% PEG6000-18% Na-citrate | 0.24 ± 0.17 | 0.8 ± 0.40 | 1.6 ± 0.17 | 21 ± 0.07 |
16% PEG6000-12% MgSO4 | 0.12 ± 0.23 | 4.1 ± 0.31 | 7.4 ± 0.14 | 74 ± 0.07 |
16% PEG6000-15% MgSO4 | 0.02 ± 0.01 | 5.38 ± 0.06 | 11.3 ± 0.21 | 87.2 ± 0.4 |
16% PEG6000-18% MgSO4 | 0.08 ± 0.11 | 3.4 ± 0.21 | 18.5 ± 0.31 | 62 ± 0.82 |
16% PEG6000-12% K2HPO4 | 0.31 ± 0.02 | 2.9 ± 0.21 | 5.1 ± 0.04 | 52 ± 0.10 |
16% PEG6000-15% K2HPO4 | 0.23 ± 0.18 | 3.8 ± 0.32 | 6.8 ± 0.22 | 63 ± 0.02 |
16% PEG6000-18% K2HPO4 | 0.18 ± 0.04 | 3.1 ± 0.11 | 4.3 ± 0.02 | 48 ± 0.12 |
2.5. Effect of pH on the Partitioning of Serine Protease from Kesinai Leaves
2.6. Effect of NaCl on the Partitioning of Serine Protease from Kesinai Leaves
3. Experimental
3.1. Plant Materials
3.2. Chemicals
3.3. Enzyme Extraction
3.4. Optimization of Heat-Treatment on Crude Enzyme
3.5. Preparation of ‘Heat Treatment Aqueous Two Phase System’
3.6. Effect of PEG on the Partitioning of Serine Protease
3.7. Effects of pH and NaCl on Partitioning of Serine Protease
3.8. Analytical Methods
3.8.1. Serine Protease Activity Assay
3.8.2. Protein Concentration Determination
3.8.3. Determination of Partition Coefficient, Specific Activity, Purification Factor and Yield
3.8.4. Sodium Dodecyl Sulfate-polyacrylamide Gel Electrophoresis (SDS-PAGE)
4. Conclusions
References and Notes
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Mehrnoush, A.; Mustafa, S.; Yazid, A.M.M. ‘Heat-Treatment Aqueous Two Phase System’ for Purification of Serine Protease from Kesinai (Streblus asper) Leaves. Molecules 2011, 16, 10202-10213. https://doi.org/10.3390/molecules161210202
Mehrnoush A, Mustafa S, Yazid AMM. ‘Heat-Treatment Aqueous Two Phase System’ for Purification of Serine Protease from Kesinai (Streblus asper) Leaves. Molecules. 2011; 16(12):10202-10213. https://doi.org/10.3390/molecules161210202
Chicago/Turabian StyleMehrnoush, Amid, Shuhaimi Mustafa, and Abdul Manap Mohd Yazid. 2011. "‘Heat-Treatment Aqueous Two Phase System’ for Purification of Serine Protease from Kesinai (Streblus asper) Leaves" Molecules 16, no. 12: 10202-10213. https://doi.org/10.3390/molecules161210202