Cryptococcus diffluens D44 tarafından üretilen lipaz(lar)ın saflaştırılması ve karakterizasyonuna ait ön çalışma

Öz

Bu çalışmada, petrol çamurundan izole edilmiş olan Cryptococcus diffluens D44'ten lipazların saflaştırılması ve karakterizasyonuna ait ön çalışmalar gerçekleştirilmiştir. Saflaştırma çalışmalarında, aseton çöktürmesinin ardından, C. diffluens D44’ye ait lipazlar, DEAE Sepharose ile saflaştırılarak iki farklı tepe noktası elde edilmiş, Lip1 ve Lip4 olarak isimlendirilmiştir. Sephadex G-100 boyut ayırma kromatografisi kullanılarak, Lip1 ve Lip4'ün saflaştırılması sonucunda da, Lip1-1 (%2,4 geri kazanım ile 1,0 saflaştırma katında), Lip1-2 (%7,2 geri kazanım ile 0,8 saflaştırma katında) ve Lip4-1 (%4,5 geri kazanımla 1,2 saflaştırma katında) olmak üzere üç farklı lipaz elde edilmiştir. Farklı piklerden elde edilen bu üç lipazın karakterizasyon çalışmaları sonucunda, Lip1-1, Lip1-2 ve Lip4-1 için belirlenen optimum sıcaklıklar sırasıyla 60 °C, 65 °C ve 65 °C olarak bulunmuştur. Ayrıca, termal stabilite çalışmaları 50 °C, 60 °C ve 70 °C'de yürütülmüş, C. diffluens D44'ten elde edilen lipazlara ait enzim aktivitesi, 50 ºC'de, başlangıç aktivitesinin %70'inin üzerinde korunabilmiştir. Optimum pH değerlerine bakıldığında, bu değerler, Lip1-1 ve Lip1-2 için pH 9.0 olarak bulunurken, Lip4-1 için pH 5.0 olarak belirlenmiştir. Ayrıca organik çözücülerin lipaz aktivitesi üzerindeki etkileri dikkate alındığında %10 metanol, Lip1-1 ve Lip4-1'in nispi aktivitesini arttırırken, %10 etanol ise Lip1-2 dışındaki lipazların nispi aktivitesini azaltmıştır. Çalışmadaki belirlenen özelliklere göre, C. diffluens D44'ten elde edilen bu farklı lipazların, endüstriyel ve biyoteknolojik uygulamalar için umut verici adaylar olabileceği düşünülmüştür. Bildiğimiz kadarıyla bu çalışma, C. diffluens D44'ten lipazların saflaştırılmasına ilişkin ilk çalışma niteliğidedir.

Anahtar Kelimeler

• Lipaz
• Cryptococcus diffluens D44
• saflaştırma
• karakterizasyon

A Preliminary Study on Purification and Characterization of Lipase(s) Produced by Cryptococcus Diffluens D44

Öz

In the present work, preliminary purification, and characterization of lipases from Cryptococcus diffluens D44, which was isolated from petroleum sludge, were performed. In the purification steps, subsequential to acetone precipitation, lipases from C. diffluens D44 were purified by DEAE Sepharose resulting in two different peaks, named Lip1 and Lip4. Sephadex G-100 size-exclusion chromatography was also performed for further purification of Lip1 and Lip4 and resulted in three different lipases as Lip1-1 (1.0 purification fold with 2.4% recovery), Lip1-2 (0.8 purification fold with 7.2% recovery), and Lip4-1 (1.2 purification fold with 4.5% recovery). As a result of characterization studies of these three lipases resulting from different peaks, optimum temperatures were found as 60 °C, 65 °C, and 65 °C for Lip1-1, Lip1-2, and Lip4-1, respectively. Furthermore, thermal stability studies were conducted at 50 °C, 60 °C, and 70 °C, and lipases of C. diffluens D44 maintained over 70% of their initial activity at 50 °C. The optimum pH for Lip1-1 and Lip1-2 was pH 9.0 although pH 5.0 was for Lip4-1. Considering the organic solvent effect on lipase activity, 10% methanol enhanced the relative activity of Lip1-1 and Lip4-1 while 10% ethanol caused a decrease in the relative activity of lipases except for Lip1-2. According to the indicated features based on the results, these different lipases from C. diffluens D44 could be promising candidates for industrial and biotechnological applications. To the best of our knowledge, this is the first study on the purification of lipases from C. diffluens D44.

Anahtar Kelimeler

• Lipase
• Cryptococcus diffluens D44
• purification
• characterization

Kaynakça

1. Saraswat, R., Bhushan, I., Gupta, P., Kumar, V., and Verma, V. (2018). Production and purification of an alkaline lipase from Bacillus sp. for enantioselective resolution of (±)-Ketoprofen butyl ester. 3 Biotech, 8(12), 1-12. https://doi.org/10.1007/s13205-018-1506-6
2. Priyanka, P., Kinsella, G., Henehan, G.T., and Ryan, B.J. (2019). Isolation, purification and characterization of a novel solvent stable lipase from Pseudomonas reinekei. Protein Expr. Purif., 153, 121-130. https://doi.org/10.1016/j.pep.2018.08.007
3. Hasan, F., Shah, A.A., and Hameed, A. (2006). Industrial applications of microbial lipases, Enzyme Microb. Technol., 39(2), 235–251. https://doi.org/10.1016/j.enzmictec.2005.10.016
4. Del Hierro, J.N., Gutiérrez-Docio, A., Otero, P., Reglero, G., and Martin, D. (2020). Characterization, antioxidant activity, and inhibitory effect on pancreatic lipase of extracts from the edible insects Acheta domesticus and Tenebrio molitor. Food Chem., 309, 125742. https://doi.org/10.1016/j.foodchem.2019.125742
5. Singh, R.S., Singh, T., and Singh A.K. (2019). Biomass, Enzymes as diagnostic tools. In: Biofuels, Biochemicals: Advances in Enzyme Technology, R.S. Singh, R.R. Singhania, A. Pandey, C. Larroche (ed.), 1st edition, Elsevier, Netherlands, p. 225–271.
6. Al-Zuhair, S. (2011). Biochemical catalytic production of biodiesel. In: Handbook of Biofuels Production: Processes and Technologies, R. Luque, J. Campelo, J. Clark (ed.), 1st edition, Woodhead Publishing, Elsevier Inc., Cambridge, p. 134–159. https://doi.org/10.1533/9780857090492.2.134
7. Anbu, P. (2013). Characterization of an Extracellular Lipase by Pseudomonas koreensis BK-L07 Isolated from Soil. Prep. Biochem. Biotechnol., 44(3), 266-280. https://doi.org/10.1080/10826068.2013.812564
8. Priji, P., Unni, K.N., Sajith, S., Binod, P., and Benjamin, S. (2015). Production, optimization, and partial purification of lipase from Pseudomonas sp. strain BUP6, a novel rumen bacterium characterized from Malabari goat. Biotechnol. Appl. Biochem., 62(1), 71-78. https://doi.org/10.1002/bab.1237

9. Das, A., Shivakumar, S., Bhattacharya, S., Shakya, S., and Swathi, S.S. (2016). Purification and characterization of a surfactant-compatible lipase from Aspergillus tamarii JGIF06 exhibiting energy-efficient removal of oil stains from polycotton fabric. 3 Biotech, 6(131), 1-8. https://doi.org/10.1007/s13205-016-0449-z
10. Edupuganti, S., Parcha, L., and Mangamoori, L.N. (2017). Purification and Characterization of Extracellular Lipase from Staphylococcus epidermidis (MTCC 10656). J. Appl. Pharm. Sci., 7(1), 57-63. https://doi.org/10.7324/JAPS.2017.70108
11. Rios, N.S., Pinheiro, B.B., Pinheiro, M.P., Bezerra, R.M., dos Santos, J.C., Gonçalves, L.R. (2018). Biotechnological potential of lipases from Pseudomonas: Sources, properties and applications, Process Biochem., 75, 99-120. https://doi.org/10.1016/j.procbio.2018.09.003
12. Kademi, A., Lee, B., and Houde, A. (2003). Production of heterologous microbial lipases by yeasts. Indian J. Biotechnol., 2, 346-355.
13. Vakhlu, J., and Kour, A. (2006). Yeast lipases: enzyme purification, biochemical properties and gene cloning. Electron J., 9(1), 717–3458. https://doi.org/10.2225/vol9-issue1-fulltext-9
14. Cesário, L.M., Pires, G.P., Pereira, R.F., Fantuzzi, E., da Silva Xavier, A., Cassini, S.T., and de Oliveira, J.P. (2021). Optimization of lipase production using fungal isolates from oily residues. BMC Biotechnol., 21, 1-13. https://doi.org/10.1186/s12896-021-00724-4
15. Szymczak, T., Cybulska, J., Podleśny, M., and Frąc, M. (2021). Various perspectives on microbial lipase production using agri-food waste and renewable products. Agriculture, 11 (6), (2021), 540. https://doi.org/10.3390/agriculture11060540
16. Patel, R.N. (2008). Synthesis of chiral pharmaceutical intermediates by biocatalysis. Coord. Chem. Rev., 252(5), 659-701. https://doi.org/10.1016/j.ccr.2007.10.031
17. Singh, A.K., and Mukhopadhyay, M. (2012). Overview of fungal lipase: a review. Appl. Biochem., 166(2), 486-520. https://doi.org/10.1007/s12010-011-9444-3
18. Navvabi, A., Razzaghi, M., Fernandes, P., Karami, L., and Homaei, A. (2018). Novel lipases discovery specifically from marine organisms for industrial production and practical applications. Process Biochem., 70, 61-70. https://doi.org/10.1016/j.procbio.2018.04.018
19. Mishra, S., and Baranwal, R. (2009). Yeast genetics and biotechnological applications. In: T. Satyanarayana, G. Kunze (ed.), Yeast biotechnology: diversity and applications, Springer, Dordrecht, p. 323-355.
20. Silveira, E.A., Tardioli, P.W., and Farinas, C.S. (2016). Valorization of palm oil industrial waste as feedstock for lipase production. Appl. Biochem. Biotechnol., 179(4), 558-571. https://doi.org/10.1007/s12010-016-2013-z
21. Guerrand, D. (2017). Lipases industrial applications: Focus on food and agro industries. OCL - Oilseeds fats Crops Lipids, 24(4), D403.
22. Yalçın, H.T., Çorbacı, C., and Ucar, F.B. (2014). Molecular characterization and lipase profiling of the yeasts isolated from environments contaminated with petroleum. J. Basic. Microbiol., 54, S85-S92. https://doi.org/10.1002/jobm.201300029
23. Yılmaz, D.E., and Sayar, N.A. (2015). Organic solvent stable lipase from Cryptococcus diffluens D44 isolated from petroleum sludge. J. Mol. Catal., 122, 72-79. https://doi.org/10.1016/j.molcatb.2015.08.021
24. Thermo Fisher Scientific, Acetone Precipitation of Proteins, https://tools.thermofisher.com/content/sfs/brochures/TR0049-Acetone-precipitation.pdf (2009).
25. Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72(1–2), 248-254. https://doi.org/10.1016/0003-2697(76)90527-3
26. Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680–685. https://doi.org/10.1038/227680a0
27. Palekar, A.A., Vasudevan, P.T., and Yan, S. (2000). Purification of Lipase: A Review. Biocatal. and Biotransfor., 18:3, 177-200. https://doi.org/10.3109/10242420009015244
28. Ezema, B.O., Omeje, K.O., Bill, R.M., Goddard, A.D., Eze, S.O.O., and Fernandez-Castane, A. (2023). Bioinformatic characterization of a triacylglycerol lipase produced by Aspergillus flavus isolated from the decaying seed of Cucumeropsis manniii. J. Biomol. Struct. Dyn., 41(6), 2587-2601. https://doi.org/10.1080/07391102.2022.2035821
29. Syihab, S.F., Madayanti, F., Akhmaloka, A., and Widhiastuty, M.P. (2017). Purification and characterization of thermostable and alcohol tolerant lipase from Pseudoxanthomonas sp. Afr. J. Biotechnol., 16(31), 1670–1677. https://doi.org/10.5897/AJB2017.16044
30. Jermsuntiea, W., Aki, T., Toyoura, R., Iwashita, K., Kawamoto, S., and Ono, K. (2011). Purification and characterization of intracellular lipase from the polyunsaturated fatty acid-producing fungus Mortierella alliacea. New Biotechnol., 28, 158–164. https://doi.org/10.1016/j.nbt.2010.09.007
31. Ai, L., Huang, Y., and Wang, C. (2018). Purification and characterization of halophilic lipase of Chromohalobacter sp. from ancient salt well. J. Basic Microbiol., 58(8), 647–657. https://doi.org/10.1002/jobm.201800116
32. Hambarliiska, A.P., Dobreva, V.T., H.N. Strinska, B.Y. Zhekova, and G.T. Dobrev, Isolation and purification of lipase produced from Rhizopus arrhizus in solid state fermentation by fractional precipitation. Bulg. Chem. Commun., 51, 184-188.
33. Rade, L.L., Da Silva, M.N., Vieira, P.S., Milan, N., De Souza, C.M., De Melo, R.R., Klein, B.C., Bonomi, A., de Castro, H.F., Murakami, M.T., and Zanphorlin, L.M. (2020). A novel fungal lipase with methanol tolerance and preference for macaw palm oil. Front. Bioeng. Biotechnol., 8, 304. https://doi.org/10.3389/fbioe.2020.00304
34. Paitaid, P., Buatong, J., Phongpaichit, S., and Aran, H. (2021). Purification and characterization of an extracellular lipase produced by Aspergillus oryzae ST11 as a potential catalyst for an organic synthesis. Trends in Sci., 18(21), 45. https://doi.org/10.48048/tis.2021.45
35. Ayinla, Z.A., Ademakinwa, A.N., Gross, R.A., and Agboola, F.K. (2022). Biochemical and biophysical characterisation of a small purified lipase from Rhizopus oryzae ZAC3, Biocatal. Biotransfor., 40(3), 195-208. https://doi.org/10.1080/10242422.2021.1883006
36. Hernández-Rodríguez, B., Córdova, J., Bárzana, E., and Favela-Torres, E. (2009). Effects of organic solvents on activity and stability of lipases produced by thermotolerant fungi in solid-state fermentation. J. Mol. Catal., 61(3-4), 136-142. https://doi.org/10.1016/j.molcatb.2009.06.004