Production, Purification of Aspergillus niger Catalase by Three-Phase Partitioning and Its Biochemical Characterization

Abstract

In this study, it was aimed to produce, purify and characterize catalase enzyme from Aspergillus niger. For that purpose, the cells of A. niger were grown at 37°C and 155 rpm in 1 liter YpSs broth media and on the 7th day of cultivation the crude enzyme extract was obtained from the growth media. Catalase enzyme was purified by three-phase partitioning method. Accordingly, the system prepared by 80% (w/w) ammonium sulfate saturation at pH 7.0 with 1:1.5 (v/v) ratio of crude extract to t-butanol gave 7.9-fold purification with 263% activity recovery. The Km value (21.4 mM), the optimum reaction temperature (50°C) and the optimum reaction pH (6.0) were determined. Stability tests indicated that the enzyme can remain its activity in a broad pH (4.0-9.0) range. Additionally, it was observed that about 77% of initial catalase activity was maintained in the presence of 7.5% (v/v) ethanol. Besides its main function, it can also oxidase some phenolic compounds like 4-methyl catechol and pyrocatechol in a peroxide-independent way. As a consequence, it was seen that the catalase enzyme can be purified from A. niger by a three-phase partitioning system known as a time-effective and inexpensive system and very easy to use rather than a conventional chromatography. Its biochemical properties (pH and ethanol stabilities and presence of a secondary oxidase activity) can provide advantages in various industrial applications.

Keywords

• Aspergillus niger,Catalase,Three-phase partitioning,Oxidase,Peroxidase

Aspergillus niger Katalazının Üretimi, Üçlü-Faz Ayırma ile Saflaştırılması ve Biyokimyasal Karakterizasyonu

Abstract

Bu çalışmada Aspergillus niger katalazının üretimi, saflaştırılması ve karakterize edilmesi amaçlanmıştır. Buna göre 1 litrelik YpSs sıvı büyüme ortamında 37°C ve 155 rpm çalkalama hızında büyütülen A. niger’den 7. günde ham enzim ekstraktı elde edilmiştir. Katalaz enzimi üçlü-faz ayırma (TPP) tekniği ile saflaştırılmıştır. Bunun için %80 (w/w) amonyum sülfat içeren ve ham ekstrakt:t-butanol oranı 1:1.5 olacak şekilde pH 7.0’da hazırlanan sistemden enzim %263 verim ile 7.9 kat saflaştırılmıştır. Km değeri (21.4 mM), optimum reaksiyon sıcaklığı (50°C) ve optimum reaksiyon pH’sı (6.0) belirlenmiştir. Kararlılık testleri, enzimin geniş pH (4.0-9.0) aralığında dayanıklı kalabildiğini göstermiştir. Ayrıca katalaz aktivitesinin %7.5’lik (v/v) etanol varlığında yaklaşık %77’sinin korunduğu gözlenmiştir. Bununla birlikte, esas fonksiyonunun yanında 4-metil katekol ve katekol gibi fenolik bileşikleri peroksitten bağımsız olarak okside edebilmiştir. Sonuç olarak, A. niger’den katalaz enziminin geleneksel kromatografi yöntemi yerine zamandan tasarruf sağlayan, maliyeti ucuz ve kullanımı oldukça kolay olan üçlü faz sistemleri ile saflaştırılabildiği görülmektedir. Enzimin sahip olduğu biyokimyasal özellikleri (pH ve etanol kararlılığı ve ikincil oksidaz aktivite varlığı), çeşitli endüstriyel uygulama alanlarında avantaj sağlayabilir.

Keywords

• Aspergillus niger,Katalaz,Üçlü-faz ayırma sistemi,Oksidaz,Peroksidaz

References

1. [1] Switala, J., Loewen, P. C. 2002. Diversity of properties among catalases. Archives of Biochemistry and Biophysics, 401, 145–154.
2. [2] Calera, J. A., Sánchez-Weatherby, J., López-Medrano, R., Leal, F. 2000. Distinctive properties of the catalase B of Aspergillus nidulans. FEBS Letters, 475, 117–120.
3. [3] Hansberg, W., Salas-Lizana, R., Domínguez, L. 2012. Fungal catalases: function, phylogenetic origin and structure. Archives of Biochemistry and Biophysics, 525, 170–180.
4. [4] Paris, S., Wysong, D., Debeaupuis, J-P., Shibuya, K., Philippe, B., Diamond, R. D., Latge, J-P. 2003. Catalases of Aspergillus fumigatus. Infection and Immunity, 71, 3551–3562.
5. [5] Witteveen, C. F. B., Veenhuis, M., Visser, J. 1992. Localization of glucose oxidase and catalase activities in Aspergillus niger. Applied and Environmental Microbiology, 58, 1190–1194.
6. [6] Dennison, C., Lovrien, R. 1997. Three phase partitioning: concentration and purification of proteins. Protein Expression and Profication, 11, 149–161.
7. [7] Duman, Y., Kaya, E. 2014. Purification and recovery of invertase from potato tubers by three phase partitioning and determination of kinetic properties of purified enzyme. Turkish Journal of Biochemistry, 39, 443–448.
8. [8] Sharma, A., Gupta, M. N. 2001. Purification of pectinases by three-phase partitioning. Biotechnology Letters, 23, 1625–1627.

9. [9] Duman, Y., Kaya, E. 2013. Purification, recovery, and characterization of chick pea β-galactosidase in single step by three phase partitioning as a rapid and easy technique. Protein Expression and Purification, 91, 155–160.
10. [10] Saxena, L., Iyer, B. K., Ananthanarayan, L. 2007. Three phase partitioning as a novel method for purification of ragi (Eleusine coracana) bifunctional amylase/protease inhibitor. Process Biochemistry, 42, 491–495.
11. [11] Roy, I., Gupta, M. N. 2002. Three-phase affinity partitioning of proteins. Analytical Biochemistry, 300, 11–14.
12. [12] Şen, A., Eryılmaz, M., Bayraktar, H., Önal, S. 2011. Purification of α–galactosidase from pepino (Solanum muricatum) by three-phase partitioning. Separation and Purification Technology, 83, 130–136.
13. [13] Roy, I., Sharma, A., Gupta, M. N. 2004. Three-phase partitioning for simultaneous renaturation and partial purification of Aspergillus niger xylanase. Biochimica Et Biophysica Acta, 1698, 107–110.
14. [14] Sooch, B. S., Kauldhar, B. S., Puri, M. 2014. Recent insights into microbial catalases: isolation, production and purification. Biotechnology Advances, 32, 1429–1447.
15. [15] Yuzugullu, Y., Trinh, C. H., Smith, M. A., Pearson, A. R., Phillips, S. E. V., Sutay Kocabas, D., Bakir, U., Ogel, Z. B., McPherson, M. J. 2013. Structure, recombinant expression and mutagenesis studies of the catalase with oxidase activity from Scytalidium thermophilum. Acta Crystallogrographica Section D, 69, 398–408.
16. [16] Kacem-Chaouche, N., Destain, J., Meraihi, Z., Dehimat, L., Haddoum, T., Wathelet, J. P., Thonart, P. 2013. Optimization of extracellular catalase production from Aspergillus phoenicis K30 by a linear regression method using date flour as single carbon source and purification of the enzyme. African Journal of Biotechnology, 12, 2646–2653.
17. [17] Tian, Y. S., Xu, H., Peng, R. H., Yao, Q. H. 2013. Heterlogous expression and initial characterization of the peroxisomal catalase from the methylotropic yeast Hansenula polymorpha in Pichia pastoris. Applied Biochemistry and Microbiology, 49, 507–513.
18. [18] Garay-Flores, R. V., Segura-Ceniceros, E. P., De Leon-Gamez, R., Balvantin-Garcia, C., Martinez-Hernandez, J. L., Betancourt-Galindo, R., Ramirez, A. R. P., Aguilar, C. N., Ilyina, A. 2014. Production of glucose oxidase and catalase by Aspergillus niger free and immobilized in alginate-polyvinyl alcohol beads. The Journal of General and Applied Microbiology, 60, 262–269.
19. [19] Söyler, B. 2012. Characterization and analysis of the antioxidant capacity of functional phenolics oxidized by Scytalidium thermophilum catalase phenol oxidase (CATPO). Orta Doğu Teknik Üniversitesi, Fen Bilimleri Enstitüsü, Doktora Tezi, Ankara.
20. [20] Chang, Y. C., Tsai, H.-F., Karos, M., Kwon-Chung, K. J. 2004. THTA, a thermotolerance gene of Aspergillus fumigatus. Fungal Genetics and Biology, 41, 888–896.
21. [21] Kawasaki, L., Aguirre, J. 2001. Multiple catalase genes are differentially regulated in Aspergillus nidulans. Journal of Bacteriology, 183, 1434–1440.
22. [22] Ögel, Z. B., Yüzügüllü, Y., Mete, S., Bakir, U., Kaptan, Y., Sutay, D., Demir, A. S. 2006. Production, properties and application to biocatalysis of a novel extracellular alkaline phenol oxidase from the thermophilic fungus Scytalidium thermophilum. Applied Microbiology and Biotechnology, 71, 853–862.
23. [23] Bradford M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding. Analytical Biochemistry, 72, 248–254.
24. [24] Alici, E. H., Arabaci, G., 2016. Prufication of polyphenol oxidase from borage (Trachystemon orientalis L.) by using three-phase partitioning and investigation of kinetic properties. International Journal of Biological Macromolecules, 93, 1051–1056.
25. [25] Yuzugullu Karakus, Y., Isik, S. 2019. Partial characterization of Bacillus pumilus catalase partitioned in poly(ethylene glycol)/sodium sulfate aqueous two-phase systems. Preperative Biochemistry and Purification, 49, 391–399.
26. [26] Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.
27. [27] Blum, H., Beier, H., Gross, H. J. 1987. Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis, 8, 93–99.
28. [28] Kocabas, D. S., Bakir, U., Phillips, S. E. V., McPherson, M. J., Ogel, Z. B. 2008. Purification, characterization, and identification of a novel bifunctional catalase-phenol oxidase from Scytalidium thermophilum. Applied Microbiology and Biotechnology, 79, 407–415.
29. [29] Lineweaver, H., Burk, D. 1934. The determination of enzyme dissociation constants. Journal of American Chemical Society, 56, 658–66.
30. [30] Vetrano, A. M., Heck, D. E., Mariano, T. M., Mishin, V., Laskin, D. L., Laskin, J. D. 2005. Characterization of the oxidase activity in mammalian catalase. The Journal of Biological Chemistry, 280, 35372–35381.
31. [31] Carrea, G., Riva, S. 2000 Properties and synthetic applications of enzymes in organic solvents. Angewandte Chemie International ed. in English, 39, 2226–2254.
32. [32] Sangar, S., Pal, M., Moon, L. S., Jolly, R. S. 2012. A catalase-peroxidase for oxidation of β–lactams to their (R)-sulfoxides. Bioresource Technology, 115, 102–110.
33. [33] Loncar, N., Fraaije, M. W. 2015. Not so monofunctional-a case of the thermostable Thermobifida fusca catalase with peroxidase activity. Applied Microbiology and Biotechnology, 99, 2225–2232.
34. [34] Chen, N., Teng, X.-L., Xiao, X.-G. 2017. Subcellular localization of a plant catalase-phenol oxidase, AcCATPO, from Amaranthus and identification of a non-canonical peroxisome targeting signal. Frontiers in Plant Science, 8, 1–11.
35. [35] Teng, X.-L., Chen, N., Xiao, X.-G. 2016. Identification of a catalase-phenol oxidase in betalain biosynthesis in red amaranth (Amaranthus cruentus). Frontiers in Plant Science, 6, 1228.
36. [36] Park, H.-S., Jun, S.-C., Han, K.-H., Hong, S.-B., Yu, J.-H. 2017. Chapter Three - Diversity, Application, and Synthetic Biology of Industrially Important Aspergillus Fungi. Advances in Applied Microbiology, 100, 161–202.
37. [37] Asan, A. 2004. Aspergillus, Penicillium and related species reported from Turkey. Mycotaxon, 89, 155–157.
38. [38] Wang, H., Tokusige, Y., Shinoyama, H., Fujii, T., Urakami, T. 1998. Purification and characterization of a thermostable catalase from culture broth of Thermoascus auratiacus. Journal of Fermentation and Bioengineering 85, 169–173.
39. [39] Koclar Avci, G., Coruh, N., Bolukbasi, U., Ogel, Z. B. 2013. Oxidation of phenolic compounds by the bifunctional catalase–phenol oxidase (CATPO) from Scytalidium thermophilum. Applied Microbiology and Biotechnology, 97, 661–672.
40. [40] Bender, K. S. Buckley, D. H., Madigan, M. T., Martinko, J. M., Stahl, D. A. 2017. Brock Mikroorganizmaların Biyolojisi. Çeviri Editörü: Çökmüş, C. Ondördüncü baskıdan çeviri, Palme Yayınevi, Ankara, ISBN: 9786053555964.
41. [41] Lushchak, V. I. 2011. Adaptive response to oxidative stress: Bacteria, fungi, plants and animals. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology. 153, 175–190.
42. [42] Yüzügüllü, Y., Ögel, Z. B., Bakır Bölükbaşı, U., Çoruh, N., Karakaş, G. 2011. Production of a novel bifunctional catalase-phenol oxidase of Scytalidium thermophilum in the presence of phenolic compounds. Turkish Journal of Biology, 35, 697–704.
43. [43] Mulvey, M. R., Switala, J., Borys, A., Loewen, P. C. 1990. Regulation of transcription of katE and katF in Escherichia coli. Journal of Bacteriology, 172, 6713–6720.
44. [44] Noventa-Jordão, M. A., Couto, R. M., Goldman, M. H. S., Aguirre, J., Iyer, S., Caplan, A., Terenzi, H. F., Goldman, G. H. 1999. Catalase activity is necessary for heat-shock recovery in Aspergillus nidulans germlings. Microbiology, 145, 3229–3234.
45. [45] Kawasaki, L., Wysong, D., Diamond, R., Aguirre, J. 1997. Two divergent catalase genes are differentially regulated during Aspergillus nidulans development and oxidative stress. Journal of Bacteriology, 179, 3284–3292.
46. [46] Yuzugullu Karakus, Y., Acemi, A., Işık, S., Duman, Y. 2018. Purification of peroxidase from Amsonia orientalis by three-phase partitioning and its biochemical characterization. Separation Science and Technology, 53, 756–766.
47. [47] Singh, R. K., Gourinath, S., Sharma, S., Roy, I., Gupta, M. N., Betzel, C., Srinivasan, A., Singh, T.P. 2001. Enhancement of enzyme activity through three-phase partitioning: crystal structure of a modified serine proteinase at 1.5 Å resolution. Protein Engineering, 14, 307–313.
48. [48] Wati, R. K., Theppakorn, T., Benjakul, S. Rawdkuen, S. 2009. Three-phase partitioning of trypsin inhibitor from legume seeds. Process Biochemisrty, 44, 1307–1314.
49. [49] Chelikani, P., Fita I., Loewen, P. C. 2004. Diversity of structures and properties among catalases. Cellular and Molecular Life Sciences, 61, 192–208.
50. [50] Diaz, A., Rangel, P., Oca, Y. M., Lledias, F., Hansberg, W. 2001. Molecular and kinetic study of catalase-1, a durable large catalase of Neurospora crassa. Free Radical Biology and Medicine, 31, 1323–1333.
51. [51] Calandrelli, V., Gambacorta, R. I., Carratore, V., Lama, L. 2008. A novel thermo-alkali stable catalase–peroxidase from Oceanobacillus oncorhynchi subsp. incaldaniensis: purification and characterization. World Journal of Microbiology and Biotechnology, 24, 2269–2275.
52. [52] Shima, S., Netrusov, A., Sordel, M., Wicke, M., Hartmann, G.C., Thauer, R. K. 1999. Purification, characterization and primary structure of a monofunctional catalase from Methanosarcina barkeri. Archives of Microbiology, 171, 317–323.
53. [53] Tauber, H. 1953. Oxidation of pyrogallol to purpurogallin by crystalline catalase. Journal of Biological Chemistry, 205, 395–400.