Lipase Production and Characterization by Pseudomonas aeruginosa Isolated from Oil Mill Effluents
Keywords:
Lipase, Pseudomonas Aeruginosa, Dialysis, Column Chromatography, Oil Mill EffluentsAbstract
Lipases have numerous biotechnological uses. This study aimed to isolate lipase-producing bacteria from oil mill effluents and to optimize, purify, and characterize the lipase produced. Fifty bacteria isolates were obtained from the various samples screened in this study. Eighteen (18) isolates showed zones of hydrolysis on tributyrin agar, while the isolate with the highest lipase activity was selected for lipase production. The selected isolate was identified as Pseudomonas aeruginosa based on its physiological attributes. Optimal lipase activity (3.72U/ml) was observed at a 36-hour incubation period and total protein content was 13.5mg/ml. The optimization of various production parameters was evaluated. The optimal conditions were an incubation period of 36 hours, yeast extract as nitrogen source, palm oil as carbon source, temperature of 50ºC and pH 8.0. The lipase produced was purified by ammonium sulphate precipitation, dialysis, and column chromatography. The protein determination at each purification step was highest for gel chromatographic step (4.814) and lowest for the crude enzyme (0.999). A determination of the molecular weight using Sodium dodecyl sulphate polyacrylamide gel electrophoresis SDS-PAGE analysis of the lipase showed a molecular weight of 30.68 kDa. Characterization showed the lipase was stable at a temperature of 50ºC (98.6%), pH of 7.0 (97.3%) in the presence of Mg2+ (107.7%). This result showed that Pseudomonas aeruginosa is a good producer of lipase, which can be further researched for their biotechnological applications.
References
Benattouche, Z., & Abbouni, B. (2012). Production, optimization and characterization of the lipase from Pseudomonas aeruginosa. Romanian Biotechnological Letters, 17(2), 7192.
Borkar, P. S., Ragini, J., Bodade, G., Srinivasa, R., Rao, R., & Khobragade, T. (2009). Purification and characterization of extracellular lipase from a new strain: Pseudomonas aeruginosa SRT 9. Brazilian Journal of Microbiology, 40(2), 358. https://doi.org/10.1590/S1517-838220090002000028
Bornscheuer, U.T., Bessler, C., Srivinas, R., & Krishna, S. H. (2002). Bornscheuer, U. T., Bessler, C., Srivinas, R., & Krishna, S. H. Issues in biochemistry. Trends in Biotechnology, 20, 433-437.
Câmara Ribas, R. K., dos Santos Carboni, D., Cazarolli, J. C., Flôres, S. H., Ramirez-Castrillon, M., & Valente, P. (2019). Nitrogen source and pH interact and modulate lipase secretion in a non-clinical strain of Candida parapsilosis. Acta Scientiarum. Biological Sciences, 41(1), e45481. https://doi.org/10.4025/actascibiolsci.v41i1.45481
Cardenas J., Alvrez, E., Castro-Alvarez, M. S., Sanchwe-Monero, J. M., Valmsda, M., Elson, S.W. & Sinisterra, J. V. (2001). Screening and catalytic activity in organic synthesis of novel fugal and yeast lipases. Journal of Molecular Catalysis B: Enzymatic, 14, 111-123.
Chaturvedi, S., & Khare, A. (2016). Isolation and optimization for extracellular lipase using ground nut shell under submerged fermentation. Indonesian American Journal of Pharmaceutical Research, 6(3), 4727–4732.
Fatima, H., Khan, N., Rehman, A. U., & Hussain, Z. (2014). Production and partial characterization of lipase from pseudomonas putida. Fermentation Technology, 4, 112.
Hasanuzzaman, M., Umadhay-Briones, K. M., Zsiros, S. M., Morita, N., Nodasaka, Y., & Yumoto, I. O. H. (2004). Isolation, identification, and characterization of a novel, oil-degrading bacterium, and pseudomonas aeruginosa T1. Current Microbiology, 49, 108-114.
Javed, S., Azeem, F., Hussain, S., Rasul, I., Siddique, M. H., Riaz, M., Afzal, M., Kouser, A., & Nadeem, H. (2018a). Bacterial lipases: A review on purification and characterization. Progress in Biophysics and Molecular Biology, 132, 23-34. https://doi.org/10.1016/J.PBIOMOLBIO.2017.07.014
Javed, S., Azeem, F., Hussain, S., Rasul, I., Siddique, M. H., Riaz, M., Afzal, M., Kouser, A., & Nadeem, H. (2018b). Bacterial lipases: A review on purification and characterization. Progress in Biophysics and Molecular Biology, 132, 23-34. https://doi.org/10.1016/j.pbiomolbio.2017.07.014
Jia, J., Yang, X., Wu, Z., Zhang, Q., Lin, Z., Guo, H., Lin, C. S. K., Wang, J., & Wang, Y. (2015). Optimization of fermentation medium for extracellular lipase production from aspergillus Niger using response surface methodology. BioMed Research International, 2015(1), 497462. https://doi.org/10.1155/2015/497462
Kalapatapu, V. R., & Bhaskar, R. I. (2015). Lipase activity when palm oil was used as carbon source. International Journal of Scientific & Engineering Research, 6(1), 2168.
Khemika Lomthaisong, A. B. (2012). Investigation of isolated lipase producing bacteria from oil-contaminated soil with proteomic analysis of its proteins responsive to lipase inducer. Journal of Biological Sciences, 12, 161-167.
Kunamnein, A., Poluri, E., & Davuluri, S. P. (2003). Purification and partial characterization of thermostable serine alkaline protease from a newly isolated Bacillus subtilis PE-11. American Association of Pharmaceutical Sciences, 4(4), 564-573.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680-685.
Lotrakul, P and Dharmsthiti, S. (1997). Lipase production by Aeromonas sobria LP004 in a medium containing whey and soybean meal. World Journal of Microbiology and Biotechnology, 13(3), 163-166.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with folin phenol reagent. Journal of Biological Chemistry, 193(1), 265-275.
Mahmoud, G. A., Koutb, M. M., Morsy, F. M., & Bagy, M. M. K. (2015). Characterization of lipase enzyme produced by hydrocarbons utilizing fungus Aspergillus terreus. European Journal of Biological Research, 5(3), 70-77.
Mobarak-Qamsari, E., Kasra-Kermanshahi, R., & Moosavi-Nejad, Z. (2011). Isolation and identification of a novel, lipase-producing bacterium, Pseudomnas aeruginosa KM110. Iranian Journal of Microbiology, 3(2), 92-98.
Muthumari, G. M., Thilagavathi, S., & Hariram, N. (2016). Industrial enzymes: Lipase producing microbes from waste volatile substances. International Journal Pharmaceutical Science Resources, 7(5), 2201-2208.
Nagarajan, S. (2012). New tools for exploring old friends-microbial lipases. Applied Biochemistry and Biotechnology, 168(5), 1163-1196.
Norris, J.R., & Ribbons, D. W. (1971). Methods in Microbiology. Academic Press.
Femi-Ola T. O., Odeyemi A. T., Olaiya B. S., & Ojo O. O. (2018). Characterization of lipase from bacillus subtilis isolated from oil contaminated soil. Journal of Applied & Environmental Microbiology, 6(1), 10-17. https://doi.org/10.12691/jaem-6-1-2
Okino-Delgado, C. H., & Fleuri, L. F. (2014). Obtaining lipases from byproducts of orange juice processing. Food Chemistry, 163(April), 103–107. https://doi.org/10.1016/j.foodchem.2014.04.090
Peng, R., Lin, J. and Wei, D. (2010). Purification and Characterization of an Organic Solvent-Tolerant Lipase from Pseudomonas aeruginosa CS-2. Applied Biochemistry and Biotechnology, 162, 733–743.
Saxena, R. K., Misra, S., Rawat, I., Gupta, P., Dutt, K., & Parmar, V. S. (2011). Production of 1, 3 regiospecific lipase from Bacillus sp. RK-3: Its potential to synthesize Cocoa Butter Substitute. Malaysian Journal of Microbiology, March. https://doi.org/10.21161/mjm.25010
Shanthi, K. and. (2002). Isolation and Characterization of Cold active lipase producing Pseudomonas sp. 4 from Marine samples of Tamilnadu Coast. Research Journal of Biotechnology, 8(4), 57–62.
T., M. H. & S. (1994). Matsumae H.,& Shibatani T. Journal of Fermentation Bioengineering, 77, 152–158.
Veerapagu, M., Jeya, K.R., & Sankaranarayananan, A. (2016). Screening and production of lipase producing bacteria from vegetable oil industry. International Journal of Scientific Research in Science, Engineering and Technology, 2(6), 726.
Wakil, S. M., & Osesusi, A. O. (2017). Production, Characterization And Purification Of Lipase By Bacteria Isolated From Palm Oil Mill Effluent And Its Dumpsites Soil. Nigerian Journal of Microbiology, 31(1), 3691–3703. http://nsmjournal.org.ng/2017-31-1/07.pdf
Zhang, H.Y.,& Wang X, C. C. (2005). Experimental optimization of enzymic kinetic resolution of racemic flurbiprofen. Biotechnology and Applied Biochemistry, 46, 67–71.
