Bacillus licheniformis O12'den Yeni Bir Termostabil Lakkazın Tek Adım Afinite Kromatografisi Kullanılarak Saflaştırılması, Karakterizasyonu ve Renk Giderme Potansiyeli

Yıl 2024, Cilt: 14 Sayı: 1, 333 - 344, 01.03.2024

Arzu Öztürk Kesebir , Melda Şişecioğlu , Ahmet Adıgüzel , Deryanur Kılıç , Ömer İrfan Küfrevioğlu

https://doi.org/10.21597/jist.1354190

Cited By: 1

Öz

Lakkazlar çok çeşitli substratları oksitleyebilen bakır içeren enzimlerdir. Lakkazın bu özelliği sayesinde çevre kirliliğine neden olan bazı boyarmaddelerin renk giderimi yapılabilmektedir. Bacillus licheniformis gibi bazı bakteriler doğal olarak lakkaz enzimini üretir. Çalışmada lakkaz saflaştırması için yeni bir afinite kolonu test edilmiştir. Bu amaçla uygun ortamda yetiştirilen bakterilerin ürettiği hücre dışı lakkaz sefaroz 4B-L-tirozin-ρ-aminobenzoik asit afinite kromatografisi yöntemiyle izole edildi. Saflığı SDS-PAGE yöntemiyle kontrol edildi. B. lichenisformis O12'den afinite kolonu ile izole edilen lakkazın tekstil atık suyundaki bazı boyarmaddelerin renk giderici etkisi araştırıldı. Bu prosedürde herhangi bir mediyatör kullanılmadı. Sonuç olarak lakkaz %38,3 verimle 4,82 kat saflaştırıldı. Saflaştırılan enzimin moleküler ağırlığı SDS-PAGE yöntemiyle ~70 kDa olarak belirlendi. Enzim, pH 4.0'da ve 92°C sıcaklıkta optimum aktivite gösterdi. Enzimin, 60°C ve 92°C'de 12 saatlik inkübasyondan sonra bile %100 aktivitesini koruduğu görüldü. Kinetik parametreler ABTS, 2,6-DMP ve guaiacol gibi lakkaz substratları ile belirlendi. Ortama sadece izole edilen lakkaz eklenerek, herhangi bir redoks aracısı kullanılmadan %35 Reaktif siyah, %31 Asit siyahı 1, %28 Metilen mavisi ve %15 Asit kırmızısı 27 boyar maddelerinde renk giderimi elde edildi. B. licheniformis O12 lakkaz enziminin bu özellikleri, onu çeşitli biyoteknolojik ve endüstriyel uygulamalarda kullanım için potansiyel bir aday enzim haline getirmektedir.

Anahtar Kelimeler

Bacillus licheniformis, Lakkaz, Afinite Kromatografisi, Karakterizasyon, Renk giderimi, Saflaştırma

Proje Numarası

218Z032

Purification and Characterization of a New Thermostable Laccase from Bacillus licheniformis O12 Using One-Step Affinity Chromatography and Its Potential for Decolorization

Öz

Laccases are copper-containing enzymes that can oxidize a wide variety of substrates. Thanks to this feature of laccase, some dyes that cause environmental pollution can be decolorized. Some bacteria, such as Bacillus licheniformis, naturally produce the enzyme laccase. A new affinity column was tested in this study. For this purpose, the extracellular laccase sepharose 4B-L-tyrosine-ρ-aminobenzoic acid produced by bacteria grown in suitable media was isolated by affinity chromatography method. Its purity was checked by SDS-PAGE method. The decolorization effect of some dyestable in textile wastewater of laccase isolated from B. lichenisformis O12 by affinity column was investigated. No mediator was used in this procedure. .As a result, laccase was purified 4.82-fold purification with a yield of 38.3% respectively, The molecular weight of the purified enzyme was determined as ~70 kDa by the SDS-PAGE method. The enzyme showed optimum activity at pH 4.0 and temperature 92°C. The enzyme was found to retain 100% activity even after 12 hours of incubation at 60°C and 92°C. The kinetic parameters were determined with laccase substrates such as ABTS, 2,6-DMP, and guaiacol. The purified laccase was decolorized with varied efficiencies such as 35% of Reactive black, 31% of Acid black 1, 28% of Methylene blue, and 15% of Acid red 27 without the use of any redox mediators. These properties of B. licheniformis O12 laccase enzyme make it a potential candidate enzyme for use in various biotechnological and industrial applications.

Anahtar Kelimeler

Bacillus licheniformis, Laccase, Affinity chromatography, Characterization, Decolorization, Purification

Destekleyen Kurum

TÜBİTAK

Proje Numarası

218Z032

Teşekkür

This project This study was funded by TUBITAK project number 218Z032. Thank you for the institutional contributions of TUBITAK..

Kaynakça

• Afreen, S. Shamsiz, T.N. Baig, M.A. Ahmadı, N. Fatıma, S. Qureshi, M.I. (2017). A novel multicopper oxidase (laccase) from cyanobacteria: Purification, characterization with potential in the decolorization of anthraquinonic dye, PLoS One, 12, e0175144.
• Asgher, M. Bhatti, H.N. Ashraf, M. Legge, R.L. (2008). Recent developments in biodegradation of industrial pollutants by white rot fungi and their enzyme system, Biodegradation, 19 771–783.
• Baldrian, P. (2006). Fungal laccases – occurrence and properties, FEMS Microbiol. Rev., 30 215-242.
• Baltaci, M.O. Genc, B. Arslan, S. Adiguzel, G. Adiguzel, A. (2017). Isolation and Characterization of Thermophilic Bacteria from Geothermal Areas in Turkey and Preliminary Research on Biotechnologically Important Enzyme Production, Geomicrobiol., J. 34 53-62.
• Bilal, M. Asgher, M. Parra-Saldivar, R. Hu, H. Wang, W. Zhang, X. Iqbal, H.M.N. (2017). Immobilized ligninolytic enzymes: an innovative and environmental responsive technology to tackle dye-based industrial pollutants–a review, Sci. Total Environ., 576 646–659.
• Bozoglu, C. Adıguzel, A. Nadaroglu, H. Yanmis, D. Gulluce, M. (2013). Purification and Characterization of Laccase from newly isolated Thermophilic Brevibacillus sp. (Z1) and its applications in removal of Textile Dyes, Res. J. Biotechnol., Vol. 8 (9).
• 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, 248.
• Chefetz, B. Chen, Y. Hadar, Y. (1998). Purification and Characterization of Laccase from Chaetomium thermophilium and Its Role in Humification, Environ. Microbiol., 64, 3175–3179.
• Diamantidis, G. Effosse, A. Potier, P. Bally, R. (2000). Purification andcharacterization of the first bacterial laccase in the rhizosphericbacterium Azospirillum lipoferum, Soil Biol. Biochem., 32 919-927.
• Dube, E. Shareck, F. Hurtubise, Y. Beauregard, M. Daneault, C. (2008). Decolourization of recalcitrant dyes with a laccase from Streptomyces coelicolor under alkaline conditions, Ind J. Microbiol.Biotechnol., 35 1123–1129.
• Dwivedi, U.N. Singh, P. Pandey, V.P. Kumar, A.J. (2010). Structure-Function Relationship among Bacterial, Fungal and Plant Laccases, Mol. Catal. B: Enzym. 68, 117–128.
• Fang, Z. Li, T. Wang, Q. Zhang, X. Peng, H. Fang, W. Hong, Y. Ge, H. Xiao, Y. (2011). A bacterial laccase from marine microbial metagenome exhibiting chloride tolerance and dye decolorization ability, Appl Microbiol Biotechnol., 89 1103–1110.
• Forootanfar, H. Faramarzi, M.A. (2015). Insights into laccase producing organisms, fermentation states, purification strategies, and biotechnological applications, Biotechnol. Prog. 31 1443–1463.
• Giardina, P. Faraco, V. Pezzella, C. Piscitelli, A. Vanhulle, S. Sannia, G. (2010). Laccases: a never-ending story, Cell. Mol. Life Sci. 67 369–385.
• Guan, Z.B. Zhang, N. Song, C.M. Zhou, W. Zhou, L.X. Zhao, H. Xu, C.W. (2014). Molecular Cloning, Characterization, and Dye-Decolorizing Ability of a Temperatureand pH-Stable Laccase from Bacillus subtilis X1, Appl. Biochem. Biotechnol., 172, 1147–1157.
• Heinfling, A. Martinez, A.T. Martinez, M.J. Bergbauer, M. Szewzyk, U. (1998). Purification and characterization of peroxidases from the dye-decolorizing fungus Bjerkandera adusta, FEMS Microbiol. Lett, 165.
• Idris, A.S. Pandey, A. Rao, S.S. Sukumaran, R.K. (2017). Cellulase production through solid-state tray fermentation, and its use for bioethanol from sorghum stover, Bioresour. Technol., 242, 265–271.
• Jaiswal, N. Pandey, V.P. Dwivedi, U.N. (2015). Purification of a thermostable alkaline laccase from papaya (Carica papaya) using affinity chromatography, Process Biochem., 72 326-332.
• Jean, S.J. Lim, S.J. (2017). Purification and Characterization of the Laccase Involved in Dye Decolorization by the White-Rot Fungus Marasmius scorodonius, J. Microbiol. Biotechnol., 27 1120–1127.
• Jeon, S.J. Park, J.H. (2020). Refolding, characterization, and dye decolorization ability of a highly thermostable laccase from Geobacillus sp. JS12, Protein Expr. Purif, 173, 105646.
• Kesebir, A.O. Kilic, D. Sisecioglu, M, Adıguzel, A. Kufrevioglu, O.I. (2021). Recombinant laccase production from Bacillus licheniformis O12: Characterization and its application for dye decolorization, Biologia, 76 3429–3438.
• Kim, H.W. Lee, S.Y. Park, H. Jeon, S.J. (2015). Expression, refolding, and characterization of a small laccase from Thermus thermophilus HJ6, Protein Expr. Purif., 114 37–43.
• Koschorreck, K. Richter, S.M. Ene, A.B. Roduner, E. Schmid, R.D. Urlacher, V.B. (2008). Cloning and characterization of a new laccase from Bacillus licheniformis catalyzing dimerization of phenolic acids, Appl. Microbiol. Biotechnol. 79, 217–224.
• Kumar, V.V. Sathyaselvabala, V. Premkumar, M.P. Vidyadevi, T.https://www.sciencedirect.com/science/article/pii/S1381117711002402 - ! Sivanesan, Si.https://www.sciencedirect.com/science/article/pii/S1381117711002402 - ! (2012). Biochemical characterization of three phase partitioned laccase and its application in decolorization and degradation of synthetic dyes, J. Mol. Catal., B Enzym., 74 63– 72.
• Laemmli, U.K. (1970). Cleavage of structural proteins during in assembly of the heat of bacteriophage T4, Nature, 227 680–685.
• Lorenzo, M. Moldes, D. Sanromán, M.A. (2006). Effect of heavy metals on the production of several laccase isoenzymes by Trametes versicolor and on their ability to decolourise dyes, Chemosphere, 63, 912–917.
• Mehandia, S. Sharma, S.C. Arya, S.K. (2020). Isolation and characterization of an alkali and thermostable laccase from a novel Alcaligenes faecalis and its application in decolorization of synthetic dyes, Biotechnol. Rep., 25 e00413.
• Miyazaki, K. (2005). A hyperthermophilic laccase from Thermus thermophilus HB27, Extremophiles, 9, 415–425.
• Sadhasivam, S. Savitha, S. Swaminathan, K. Feng-Huei, L. (2008). Production, purification and characterization of mid-redox potential laccase from a newly isolated Trichoderma harzianum WL1,Process Biochem 2008, 43, 736–742.
• Moon-Jeong, H. Hyoung-Tae, C. Hong-Gyu, S. (2005). Purification and Characterization of Laccase from the White Rot Fungus Trametes versicolor, J Microbiol, 43 555–560.
• Murugesan, K. Kim, Y.M. Jeon, J.R. Chang, Y.S. (2009). Effect of metal ions on reactive dye decolorization by laccase from Ganoderma lucidum, Journal of Hazardous Materials, 168 523-529.
• Rezaei S. Shahverdi AR. Faramarzi M.A. (2017). Isolation, one-step affinity purification, and characterization of a polyextremotolerant laccase from the halophilic bacterium Aquisalibacillus elongatus and its application in the delignification of sugar beet pulp, Bioresour. Technol., 230 67–75.
• Rudakiya, D.M. Patel, D.H. Gupte, A. (2020). Exploiting the potential of metal and solvent tolerant laccase from Tricholoma giganteum AGDR1 for the removal of pesticides, Int. J. Biol. Macromol., 144, 586–595.
• Sadeghian-Abadi, S. Rezaei, S. Yousefi-Mokri, M. Faramarzi, M.A. (2019). Enhanced production, one-step affinity purification, and characterization of laccase from solid-state culture of Lentinus tigrinus and delignification of pistachio shell by free and immobilized enzyme, J. Environ. Manage., 244 235–246.
• Saito, K. Ikeda, R. Endo, K. Tsujino, Y. Takagi, M. Tamiya, E.J. (2012). Isolation of a novel alkaline-induced laccase from Flammulina velutipes and its application for hair coloring, Biosci. Bioeng., 113 575–579.
• Santhanam, N. Vivanco, J.M. Decker, S.R. Reardon, K.F. (2011). Expression of industrially relevant laccases: prokaryotic style, Trends Biotechnol. 29 480–489.
• Singh, G. Bhalla, A. Kaur, P. Capalash, N. Sharma, P. (2011). Laccase from prokaryotes: a new source for an old enzyme, Rev. Environ. Sci. Biotechnol. 10 309–326.
• Shujing, S. Yonghui, Z. Youxiong, Q. Bixian, L, Kaihui, H. Liping, X. (2013). Purification and characterization of fungal laccase from Mycena purpureofusca, Chiang Mai J Sci, 40, 151–160.
• Sunil, S.M. Renuka, P.S. Pruthvi, K. Swetha, M. Malini, S. Veema, S.M. (2011). Isolation, Purification, and Characterization of Fungal Laccase from Pleurotus sp. SAGE-Hindawi Enzyme,, Research Article ID 248735.
• Trubitsina, L.I. Tishchenko, S.V. Gabdulkhakov, A.G. Lisov, A.V. Zakharova, M.V. Leontievsky, A.A. (2015). Structural and functional characterization of two-domain laccase from Streptomyces viridochromogenes, Biochimie, 112 151e159.
• Vantamiru, A.B. Kaliwal, B.B. (2016). Purification and characterization of laccase from Marasmius species BBKAV79 and effective decolorization of selected textile dyes, 3 Biotech, 6 189.
• Xiao, Y. Tu, X. Wang, J. Zhang, M. Cheng, Q. Zeng, W. Shi, Y. (2003). Purification, molecular characterization and reactivity with aromatic compounds of a laccase from basidiomycete Trametes sp. strain AH28-2, Appl Microbiol Biotechnol, 60, 700–707.
• Xiao, X. Xu, C.C. Wu, Y.M. Cai, P.J. Li, W.W. Du, D.L. Yu, H.Q. (2012). Biodecolorization of Naphthol Green B dye by Shewanella oneidensis MR-1 under anaerobic conditions, Bioresour. Technol., 110 86–90.
• Yang, J. Lin, Q. Ng, T.B. Ye, X. Lin, J. (2014). Purification and Characteriza tion of a Novel Laccase from Cerrena sp. HYB07 with Dye Decolorizing Ability, PLoS One, 9 e110834.
• Yoshida. H. (1883). Chemistry of Lacquer (Urushi) Part 1, J. Chem. Soc. 43 472–486.