Purification and Characterization of Organic Solvent and Detergent Tolerant Lipase from Thermotolerant Bacillus sp. RN2
Abstract
:1. Introduction
2. Results and Discussion
2.1. Isolation and Characterization of Bacillus sp. RN 2 Lipase
2.2. Expression and Purification of Lip-SBRN2
2.3. Optimum pH and Temperature for Lip-SBRN2 Activity
2.4. Effect of Metal Ions and Chemical Reagent on Lip-SBRN2 Activity
2.5. Substrate Specificity
2.6. Effect of Organic Solvents and Detergent on Lip-SBRN2 Activity
3. Experimental Section
3.1. Bacterial Strains Collection
3.2. PCR Amplification of Thermotolerant Bacillus sp. RN2 Lipase Gene (Lip-SBRN2 Gene)
3.3. Over-Expression of the Lip-SBRN2 Gene
3.4. Purification of Lip-SBRN2
3.5. Lipase Assay
4. Conclusion
Acknowledgements
References
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Steps | Volume (mL) | Total activity (Units) | Total activity/mL (Units/mL) | Total protein (mg) | Specific activity (Units/mg) | Purification factor (fold) | Recovery yield (%) |
---|---|---|---|---|---|---|---|
Crude enzyme | 5 | 281 | 56.2 | 174 | 1.6 | 1 | 100 |
Ni-NTA column | 2 | 144 | 72 | 1.3 | 118 | 74 | 51 |
Substrates | Relative activity (%) |
---|---|
p-nitrophenylester | |
pNP-butyrate (C4) | 40 ± 6 |
pNP-caprylate (C8) | 92 ± 8 |
pNP-caprate (C10) | 68 ± 9 |
pNP-laurate (C12) | 100 ± 15 |
pNP-myristate (C14) | 59 ± 7 |
pNP-palmitate (C16) | 54 ± 10 |
pNP-stearate (C18) | 35 ± 4 |
Natural oils | |
Coconut oil (12:0) | 727.3 ± 16 |
Palm oil (16:0) | 32.0 ± 13 |
Olive oil (18:1) | 100.0 ± 9 |
Grape seed oil (18:2) | 32.4 ± 7 |
Soybean oil (18:3) | 42.2 ± 7 |
Organic solvents/detergents (25%) | Relative activity of Bacillus sp. RN2 lipase | |
---|---|---|
30 min incubation Relative activity (%) | 2 h incubation Relative activity (%) | |
None | 100.0 ± 7 | 100.0 ± 4 |
Organic solvents | ||
Benzene | 108.0 ± 11 | 104.4 ± 3.4 |
Diethylether | 102.4 ± 5.2 | 103.6 ± 0.5 |
Acetone | 100.8 ± 0.8 | 82.2 ± 11.3 |
n-hexane | 94.0 ± 4 | 94.5 ± 3.5 |
Dimethylsulfoxide | 85.2 ±.8.4 | 96.4 ± 11.3 |
Cyclohexane | 84.4 ± 11.2 | 85.1 ± 5.6 |
N,N-dimethylformamide | 80.4 ± 5.5 | 86.9 ± 3.4 |
Chloroform | 80.0 ± 11 | 84.4 ± 10.5 |
Acetonitrile | 69.6 ± 9.5 | 82.5 ± 11 |
Toluene | 68.4 ± 12.3 | 80.4 ± 13.4 |
2-propanol | 60.8 ± 8.7 | 66.5 ± 6.6 |
Detergents | ||
25%Tween-20 | 60.8 ± 11.5 | 96.4 ± 7.3 |
25%Triton X-100 | 41.6 ± 10.6 | 86.5 ± 8.7 |
1% SDS | 112.5 ± 11.3 | 485.7 ± 11.5 |
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Kanjanavas, P.; Khuchareontaworn, S.; Khawsak, P.; Pakpitcharoen, A.; Pothivejkul, K.; Santiwatanakul, S.; Matsui, K.; Kajiwara, T.; Chansiri, K. Purification and Characterization of Organic Solvent and Detergent Tolerant Lipase from Thermotolerant Bacillus sp. RN2. Int. J. Mol. Sci. 2010, 11, 3783-3792. https://doi.org/10.3390/ijms11103783
Kanjanavas P, Khuchareontaworn S, Khawsak P, Pakpitcharoen A, Pothivejkul K, Santiwatanakul S, Matsui K, Kajiwara T, Chansiri K. Purification and Characterization of Organic Solvent and Detergent Tolerant Lipase from Thermotolerant Bacillus sp. RN2. International Journal of Molecular Sciences. 2010; 11(10):3783-3792. https://doi.org/10.3390/ijms11103783
Chicago/Turabian StyleKanjanavas, Pornpimon, Sintawee Khuchareontaworn, Paisarn Khawsak, Arda Pakpitcharoen, Khajeenart Pothivejkul, Somchai Santiwatanakul, Kenji Matsui, Tadahiko Kajiwara, and Kosum Chansiri. 2010. "Purification and Characterization of Organic Solvent and Detergent Tolerant Lipase from Thermotolerant Bacillus sp. RN2" International Journal of Molecular Sciences 11, no. 10: 3783-3792. https://doi.org/10.3390/ijms11103783