A Novel Cold-Adapted Lipase from Sorangium cellulosum Strain So0157-2: Gene Cloning, Expression, and Enzymatic Characterization
Abstract
:1. Introduction
2. Results and Discussion
2.1. Gene Cloning and Sequence Analysis
2.2. Expression and Purification of the r-LipA
2.3. Effects of Temperature on the r-LipA Avtivity and Stability
2.4. Effects of pH on the r-LipA Avtivity and Stability
2.5. Effects of Metal Ions, Detergents and Organic Solvents on the r-LipA Activity
2.6. Substrate Specificity of the r-LipA
3. Experimental Section
3.1. Strains, Plasmids and Chemicals
3.2. Cloning and Sequence Analysis
3.3. Construction of Recombinant Plasmid
3.4. Expression and Purification of the r-LipA
3.5. Lipase Activity Assay
3.6. Effects of Temperature on the r-LipA Avtivity and Stability
3.7. Effects of pH on the r-LipA Avtivity and Stability
3.8. Effect of Metal Ions, Detergents and Organic Solvents on the r-LipA Avtivity
3.9. Substrate Specificity of the r-LipA
4. Conclusions
Acknowledgments
References
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Metal ions | Relative remaining activity (%) of r-LipA
| |
---|---|---|
1 mM | 10 mM | |
Control | 100.0 ± 3.0 | 100.0 ± 1.6 |
Li+ | 96.5 ± 1.2 | 93.3 ± 1.5 |
Na+ | 97.1 ± 3.8 | 105.9 ± 5.5 |
K+ | 94.3 ± 0.6 | 96.0 ± 4.7 |
Mg2+ | 105.1 ± 3.8 | 126.1 ± 2.5 |
Ca2+ | 112.6 ± 1.5 | 140.2 ± 0.3 |
Fe2+ | 140.8 ± 2.4 | 77.1 ± 2.4 |
Mn2+ | 120.1 ± 1.9 | 103.4 ± 0.9 |
Cr3+ | 110.2 ± 3.0 | 59.5 ± 1.9 |
Co2+ | 104.1 ± 1.8 | 99.2 ± 1.0 |
Ni2+ | 97.6 ± 1.9 | 63.3 ± 2.6 |
Cu2+ | 57.8 ± 1.5 | 4.0 ± 0.8 |
Zn2+ | 56.8 ± 0.9 | 42.9 ± 4.8 |
Hg2+ | 88.4 ± 2.2 | 40.1 ± 1.5 |
EDTA | 109.8 ± 1.5 | 99.8 ± 1.4 |
Detergents | Relative remaining activity (%) of r-LipA
| |
---|---|---|
0.1% | 1% | |
Control | 100.0 ± 2.9 | 100.0 ± 2.6 |
SDS | 10.2 ± 0.8 | 9.0 ± 0.7 |
CTAB | 203.7 ± 3.1 | 95.4 ± 1.4 |
Tween 20 | 139.2 ± 0.8 | 113.0 ± 5.5 |
Tween 80 | 132.4 ± 2.2 | 119.4 ± 2.2 |
Triton X-100 | 146.8 ± 0.4 | 196.3 ± 2.1 |
Organic solvents | log P | Relative remaining activity (%) of r-LipA
| |
---|---|---|---|
30 min incubation | 2 h incubation | ||
Control | - | 100.0 ± 1.6 | 100.0 ± 2.1 |
Dimethyl sulfoxide | −1.22 | 97.1 ± 0.3 | 88.1 ± 2.2 |
Methanol | −0.76 | 97.7 ± 2.5 | 90.2 ± 1.4 |
Ethanol | −0.24 | 75.4 ± 3.1 | 67.6 ± 1.1 |
Acetone | −0.23 | 78.8 ± 1.3 | 69.2 ± 2.7 |
Isopropanol | 0.14 | 57.2 ± 3.1 | 46.6 ± 2.8 |
n-Propanol | 0.28 | 76.3 ± 1.7 | 50.2 ± 0.7 |
n-Butanol | 0.8 | 99.2 ± 4.5 | 82.6 ± 1.6 |
Diethylether | 0.85 | 113.7 ± 2.0 | 118.5 ± 1.8 |
Chloroform | 2 | 139.0 ± 1.1 | 142.8 ± 3.5 |
Benzene | 2 | 140.5 ± 5.5 | 160.2 ± 3.3 |
Toluene | 2.5 | 150.9 ± 0.6 | 159.2 ± 0.9 |
p-Xylene | 3.1 | 153.7 ± 5.2 | 152.9 ± 2.5 |
Cyclohexane | 3.2 | 156.0 ± 3.7 | 136.9 ± 0.8 |
n-Hexane | 3.5 | 153.0±2.8 | 130.8 ± 3.5 |
n-Heptane | 4 | 159.1 ± 3.4 | 167.1 ± 1.2 |
Isooctane | 4.7 | 163.7 ± 0.5 | 132.8 ± 0.7 |
Substrates | Relative remaining activity (%)
| ||||||||
---|---|---|---|---|---|---|---|---|---|
pH 7.0, 20 °C | pH 7.0, 30 °C | pH 7.0, 40 °C | pH 8.0, 20 °C | pH 8.0, 30 °C | pH 8.0, 40 °C | pH 9.0, 20 °C | pH 9.0, 30 °C | pH 9.0, 40 °C | |
pNP acetate | 16.6 ± 0.3 | 32.6 ± 1.5 | 43.2 ± 4.5 | 71.6 ± 2.2 | 100.0 ± 2.6 | 60.5 ± 1.6 | 72.5 ± 1.4 | 86.1 ± 1.6 | 60.8 ± 0.8 |
pNP butyrate | 7.9 ± 3.0 | 18.3 ± 2.3 | 26.2 ± 3.9 | 38.4 ± 1.3 | 63.4 ± 2.3 | 53.3 ± 2.1 | 54.1 ± 3.6 | 83.3 ± 1.0 | 64.6 ± 1.4 |
pNP octanoate | 8.1 ± 1.2 | 12.8 ± 1.8 | 15.7 ± 2.1 | 33.3 ± 0.5 | 52.5 ± 0.3 | 52.1 ± 2.9 | 41.5 ± 2.5 | 68.4 ± 2.3 | 74.2 ± 2.6 |
pNP decanoate | 1.4 ± 0.2 | 4.9 ± 3.0 | 5.9 ± 0.5 | 22.2 ± 0.7 | 32.1 ± 0.9 | 24.7 ± 3.0 | 24.0 ± 0.2 | 40.4 ± 2.0 | 44.7 ± 2.8 |
pNP dodecanoate | 1.6 ± 0.3 | 2.5 ± 0.1 | 5.8 ± 1.4 | 13.2 ± 5.2 | 17.0 ± 1.0 | 20.5 ± 1.8 | 23.3 ± 1.8 | 43.9 ± 1.5 | 40.0 ± 1.0 |
pNP myristate | 1.6 ± 1.0 | 2.2 ± 1.4 | 5.7 ± 2.9 | 5.7 ± 1.3 | 3.9 ± 2.4 | 5.6 ± 2.2 | 5.4 ± 2.7 | 5.2 ± 3.0 | 4.8 ± 3.4 |
pNP palmitate | 1.6 ± 0.3 | 1.9 ± 1.6 | 4.1 ± 0.6 | 2.0 ± 1.2 | 2.3 ± 0.8 | 1.3 ± 0.8 | 2.6 ± 2.3 | 5.2 ± 0.1 | 1.3 ± 1.3 |
pNP stearate | 1.3 ± 1.0 | 1.0 ± 0.1 | 2.5 ± 0.6 | 3.4 ± 0.1 | 1.9 ± 1.5 | 0.7 ± 0.8 | 2.5 ± 0.1 | 5.3 ± 2.5 | 1.9 ± 0.4 |
Substrates | Temperature (°C) | Km (mM) | kcat (s−1) | kcat/Km (s−1·mM−1) |
---|---|---|---|---|
pNP acetate (C2) | 4 | 0.037 ± 0.001 | 7.008 ± 0.100 | 186.643 ± 3.201 |
pNP acetate (C2) | 30 | 0.174 ± 0.006 | 29.225 ± 0.977 | 168.114 ± 2.097 |
pNP butyrate (C4) | 30 | 0.366 ± 0.211 | 13.617 ± 0.735 | 37.173 ± 0.128 |
pNP octanoate (C8) | 30 | 0.704 ± 0.026 | 3.294 ± 0.114 | 4.677 ± 0.013 |
pNP decanoate (C10) | 30 | 1.664 ± 0.047 | 1.565 ± 0.036 | 0.940 ± 0.005 |
pNP dodecanoate (C12) | 30 | 1.946 ± 0.059 | 1.205 ± 0.032 | 0.619 ± 0.002 |
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Cheng, Y.-Y.; Qian, Y.-K.; Li, Z.-F.; Wu, Z.-H.; Liu, H.; Li, Y.-Z. A Novel Cold-Adapted Lipase from Sorangium cellulosum Strain So0157-2: Gene Cloning, Expression, and Enzymatic Characterization. Int. J. Mol. Sci. 2011, 12, 6765-6780. https://doi.org/10.3390/ijms12106765
Cheng Y-Y, Qian Y-K, Li Z-F, Wu Z-H, Liu H, Li Y-Z. A Novel Cold-Adapted Lipase from Sorangium cellulosum Strain So0157-2: Gene Cloning, Expression, and Enzymatic Characterization. International Journal of Molecular Sciences. 2011; 12(10):6765-6780. https://doi.org/10.3390/ijms12106765
Chicago/Turabian StyleCheng, Yuan-Yuan, Yun-Kai Qian, Zhi-Feng Li, Zhi-Hong Wu, Hong Liu, and Yue-Zhong Li. 2011. "A Novel Cold-Adapted Lipase from Sorangium cellulosum Strain So0157-2: Gene Cloning, Expression, and Enzymatic Characterization" International Journal of Molecular Sciences 12, no. 10: 6765-6780. https://doi.org/10.3390/ijms12106765