Acinetobacter psychrotolerans Suşlarından Elde Edilen Lipazların Biyokimyasal Karakterizasyonu

Bu çalışmada, Acinetobacter psychrotolerans suşlarından (Xg1 ve Xg2) elde edilen ekstrasellülar lipazlar karakterize edilmiştir. Farklı pH değerlerinin (3.0- 10.0) ve çeşitli sıcaklıkların (10-90 °C) lipaz aktiviteleri üzerindeki etkileri incelenmiştir. Ayrıca farklı metal iyonlarının, organik çözücülerin ve deterjanların lipazlar üzerindeki etkileri de araştırılmıştır. Ekstrasellülar ham lipazlar ultrafiltrasyon ile konsantre edilmiştir. Bu lipazların zymogram analizi yapılmıştır. Lipazlar pH 8 ve 30 °C’de maksimum aktivite gösterdiği bulundu. Xg1 suşundan elde edilen lipaz; hekzan, etil asetat, kloroform ve N,N dietil formamid gibi organik çözücülerinin varlığında yüksek stabilite gösterirken, Xg2 suşundan elde edilen lipaz izopropanol, asetonitril ve butan 1-ol varlığında hassas olduğu bulundu. Xg1 ve Xg2 suşlarından elde edilen lipazlar Cu2+ ve Zn2+ varlığında inhibe edilmiştir. Xg1 suşundan elde edilen enzim aynı zamanda Fe3+ varlığında da inhibe edilmiştir. Xg1 ve Xg2 suşlarının lipaz aktiviteleri EDTA varlığında kısmen inhibe edilmiştir. SDS varlığında, lipazlar tamamen inhibe oldular. Zymogram analiz sonuçlarına göre Acinetobacter psychrotolerans Xg1 ve Xg2 suşlarından elde edilen lipazların molekül ağırlıkları yaklaşık olarak sırasıyla 37 ve 30 kDa’dır.

Biochemical Characterization of Lipases Obtained from Acinetobacter psychrotolerans Strains

In this study, extracellular lipases obtained from Acinetobacterpsychrotolerans strains (Xg1 and Xg2) were characterized. The effects of varyingpH values (3.0-10.0) and various temperatures (10-90 °C) on lipase activities wereexamined. Also the effects of different metal ions, organic solvents and detergentson lipases were studied. The extracellular crude lipases were concentrated usingultrafiltration. Zymogram analysis of these lipases was performed. Lipasesexhibited maximum activity at pH 8 and 30 °C. While lipase obtained from the Xg1strain exhibited the highest stability in the presence of various organic solvents,including hexane, ethyl acetate, chloroform and N,N dietil formamide, lipaseobtained from the Xg2 strain was sensitive in the presence of isopropanol,acetonitrile, and butan-1-ol. The lipases of the Xg1 and Xg2 strains were inhibitedin the presence of Cu2+ and Zn2+. Also, the lipase of the Xg1 strain was inhibited inthe presence of Fe3+. In the presence of EDTA, the lipase activities of the Xg1 andXg2 strains were partially inhibited. In presence of SDS, they were exactlyinhibited. According to the zymogram results, the molecular weights of the lipasesobtained from the Acinetobacter psychrotolerans Xg1 and Xg2 strains have beenfound approximately 37 and 30 kDa, respectively.

PDF

___

[1] Thakur, S. 2012. Lipases, its sources, properties and applications: A review. International Journal of Scientific and Engineering Research, 3(7), 1- 29.
[2] Gupta, R., Gupta, N., Rathi, P. 2004. Bacterial lipases: An overview of production, purification and biochemical properties. Applied Microbiology and Biotechnology, 64(6), 763- 781.
[3] Rubin, B., Dennis, E.A. 1997. Lipases: Part A. Biotechnology Methods Enzymology (Vol. 284). Academic Press, New York, 1-408p.
[4] Rubin, B., Dennis, E.A. 1997. Lipases: Part B. Enzyme characterization and utilization (Vol. 286). Academic Press, New York, 1-563p.
[5] Kazlauskas, R.J., Bornscheuer, U.T. 1998. Biotransformations with lipases. In: Rehm, H.J., Pihler, G., Stadler, A., Kelly, P.J.W. (Eds.), Biotechnology, (Vol. 8), VCH, New York, 37-192 p.
[6] Kamini, N.R., Fujii, T., Kurosu, T., Iefuji, H. 2000. Production, purification and characterization of an extracellular lipase from yeast, Cryptococcus sp. S-2. Process Biochemistry, 36(4), 317-324.
[7] Ruchi, G., Anshu, G., Khare, S.K. 2008. Lipase from solvent tolerant Pseudomonas aeruginosa strain: Production optimization by response surface methodology and application. Bioresource Technology, 99(11), 4796-4802.
[8] Shariff, F.M., Leow, T.C., Mukred, A.D., Salleh, A.B., Basri, M., Rahman, R.N.Z.R.A. Production of L2 lipase by Bacillus sp. strain L2: Nutritional and physical factors. Journal of Microbiology and Biotechnology, 47(5)(2007), 406-412.
[9] Breuil, C., Kushner, D.J. 1975. Partial purification and characterization of the lipase of a facultatively psychrophilic bacterium (Acinetobacter 016). Canadian Journal of Microbiology, 21(4), 434-441.
[10] Kok, R.G., van Thor, J.J., Nugteron-Roodzant, I.M., Brouwer, M.B.W., Egmond, M.R., Nudel, C.B., Vosman, B., Hellingwerf, K.J. 1995. Characterization of the extracellular lipase, LipA, of Acinetobacter calcoaceticus BD413 and sequence analysis of the cloned structural gene. Molecular Microbiology, 15(5), 803-818.
[11] Kasana, R.C., Kaur, B., Yadav, S.K. 2008. Isolation and identification of a psychrotrophic Acinetobacter sp. CR9 and characterization of its alkaline lipase. Journal of Basic Microbiology, 48(3), 207-212.
[12] Kati, A., Kati, H. 2013. Isolation and identification of bacteria from Xylosandrus germanus (Blandford) (Coleoptera: Curculionidae). African Journal of Microbiology Research, 7(47), 5288- 5299.
[13] Kugiyama, W., Otani, Y., Hashimoto, Y., Tagagi, Y. 1986. Molecular cloning and nucleotide sequence of lipase gene from Pseudomonas fragi. Biochemical and Biophysical Research Communications, 141(1), 185-190.
[14] Smibert, R.M., Krieg, N.R. 1981. General characterization. In: Gerhardt, P., Murray, R.G.E., Costilow, R.N., Neste,r E.W., Wood, W.A., Krieg, N.R., Phillips, G.B. (Eds.), Manual of methods for general bacteriology, American Society for Microbiology, Washington, D.C., 409-43p.
[15] Kouker, G., Jaeger, K.E. 1987. Specific and sensitive plate assay for bacterial lipase. Applied Microbiology and Biotechnology, 53(1), 211- 213.
[16] Winkler, U.K., Stuckmann, M. 1979. Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens. Journal of Bacteriology, 138(3), 663-670.
[17] Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680-685.
[18] Wang, H.K., Shao, J., Wei, Y.J., Zhang, J., Qi, W. 2011. A Novel Low-Temperature Alkaline Lipase from Acinetobacter johnsonii LP28 Suitable for Detergent Formulation. Food Technology and Biotechnology, 49(1), 96-102.
[19] Lee, K.W., Bae, H.A., Shin, G.S., Lee, Y.H. 2006. Purification and catalytic properties of novel enantioselective lipase from Acinetobacter sp. ES-1 for hydrolysis of (S)-ketoprofen ethyl ester. Enzyme and Microbial Technology, 38(3), 443- 448.
[20] Saisubramanian, N., Sivasubramanian, S., Nandakumar, N., Indirakumar, B., Chaudhary, N.A., Puvanakrishnan, R. 2008. Two step purification of Acinetobacter sp. lipase and its evaluation as a detergent additive at low temperatures. Applied Biochemistry and Biotechnology, 150(2), 139-156.
[21] Uttatree, S., Winayanuwattikun, P., Charoenpanich, J. 2010. Isolation and Characterization of a Novel Thermophilic- Organic Solvent Stable Lipase from Acinetobacter baylyi. Applied Biochemistry and Biotechnology, 162(5), 1362-1376.
[22] Hun, C.J., Rahman, R.N.Z.A., Salleh, A.B., Basri, M. 2003. A newly isolated organic solvent tolerant Bacillus sphaericus 205y producing organic solvent-stable lipase. Biochemical Engineering Journal, 15(2), 147-151.
[23] Wang, H., Zhong, S., Ma, H., Zhang, J., Qi, W. 2012. Screening and characterization of a novel alkaline lipase from Acinetobacter calcoaceticus 1–7 Isolated from bohai bay in china for detergent formulation. Brazilian Journal of Microbiology, 43(1), 148-56.
[24] Cherif, S., Mnif, S., Hadrich, F., Abdelkafi, S., Sayadi, S. 2011. Newly high alkaline lipase: an ideal choice for application in detergent formulations. Lipids in Health and Disease, 10(221),1-8.
[25] Yoon, M.Y., Shin, P.K., Han, Y.S., Lee, S.H., Park, J.K., Cheong, C.S. 2004. Isolation of an Acinetobacter junii SY-01 strain producing an extracellular lipase enantioselectively hydrolyzing itraconazole precursor and some properties of the lipase. Journal of Microbiology and Biotechnology, 14(1), 97-104.
[26] Son, S.H., Park, K.R. 2004. Purification and characterization of the lipase from Acinetobacter sp. B2 isolated from oil-contaminated soil. Korean Journal of Microbiology, 40(4), 320-327.
[27] Pratuangdejkul, J., Dharmsthiti, S. 2000. Purification and characterization of lipase from psychrophilic Acinetobacter calcoaceticus LP009. Microbiology Research,155(2), 95-100.
[28] Hong, M.C., Chang, M.C. 1998. Purification and characterization of an alkaline lipase from a newly isolated Acinetobacter radioresistens CMC- 1, Biotechnology Letters, 20(11), 1027-1029.
[29] Mitsuhashi, K., Yamashita, M., Hwan, Y.S., Ihara, F., Nihira, T., Yamada, Y. 1999. Purification and characterization of a novel extracellular lipase catalyzing hydrolysis of oleyl benzoate from Acinetobacter nov sp. strain KM109. Bioscience, Biotechnology, and Biochemistry, 63(11), 1959- 1964.
[30] Ng, I.S., Tsai, S.W., Chen, S.J. 1999. Purification and characterization of extracellular lipase from Acinetobacter radioresistens CMC-2. Journal of the Chinese Institute of Chemical Engineers, 30(5), 355-362.