Ekstremofilik bir bakteri olan Deinococcus radiodurans’dan β–Karbonik Anhidraz’ın klonlanması, Aşırı İfadesi ve Saflaştırılması

Yıl 2021, , 219 - 231, 17.05.2021

Ayşe Hümeyra Taşkın Kafa , Arzu Cihan , Mehmet Kuzucu , Murat Çankaya

https://doi.org/10.15671/hjbc.775093

Öz

Bu çalışmada, radyasyona ekstrem dirençli bir bakteri olan Deinococcus radiodurans’dan direnç fizyolojisinde önemli olduğunu düşündüğümüz karbonik anhidrazın (DrCA) enziminin klonlanması, saflaştırılması ve başlangıç karakterizasyonu gerçekleştirilmiştir. Ayrıca, artan dozlarda gama radyasyonun pH ile ilişkili DrCA enzim aktivitesi üzerindeki etkisi belirlenmiştir. Radyasyon uygulamasından sonra DrCA aktivitesi, 800 Gy’a kadar sürekli artarak 6 kat, artmıştır, bu noktadan sonra aktivitede azalma olduğu gözlenmiştir. DrCA enzimi için maksimum CO2 hidrasyon aktivitesi, pH 7.0 ve 40°C’de gözlenmiştir. Homo-dimer kompleks yapısında olan DrCA enzimi hafif termostabildir. DrCA’nın CO2 hidrasyon aktivitesini, çeşitli metal iyonları, özellikle Zn2+ önemli ölçüde, 5 kat artırmıştır. Ayrıca sülfonamid saf enzim üzerinde inhibe edici etki göstermiştir. DrCA’nın substrat olarak CO2 için belirlenen Km ve Vmax değerleri sırasıyla 8.4 mM ve 637 WAU/mg idi. CO2 hidrasyon deneyi saflaştırılmış rekombinant enzimin (DrCA) spesifik aktivitesinin önemli ölçüde yüksek olduğunu göstermiştir.

Anahtar Kelimeler

Deinococcus radiodurans, karbonik anhidraz, iyonlaştırıcı radyasyon

Cloning, Over-Expression, and Purification of β–Carbonic Anhydrase from an Extremophilic Bacterium: Deinococcus radiodurans

Öz

In this study, the cloning, purification and initial characterization of carbonic anhydrase (DrCA) enzyme which we consider to be important in the resistance physiology from extremely radioresistant bacteria Deinococcus radiodurans is performed.
In addition, the effect of increased gamma irradiation doses on pH-related DrCA enzyme activity was determined. DrCA activity after radiation treatment showed that the activity continuously increased by 6 fold, up to the first 800 Gy, which a decrease in activity was observed thereafter. The maximum CO2 hydration activity for DrCA enzyme was observed at pH 7.0 and 40°C. DrCA enzyme, homo-dimer complex, is slightly thermostable. The activity of DrCA was significantly enhanced by several metal ions, especially Zn2+, which resulted in 5-fold increases of CO2 hydration activity. Also sulfonamide showed inhibitory effect on the pure enzyme. The apparent Km and Vmax for CO2 as substrate were 8.4 mM and 637 WAU/mg for DrCA respectively. The CO2 hydration assay demonstrated that the specific activity of purified recombinant enzymes (DrCA) was significantly high.

Anahtar Kelimeler

Deinococcus radiodurans, carbonic anhydrase, ionizing radiation

Kaynakça

• 1. A.W. Anderson, H.C. Nordon, R.F. Cain, G. Parrish, D.E Duggan et al., Studies on a radioresistant micrococcus: I. Isolation, morphology, cultural characteristics and resistance to gamma radiation, Food Technol., 10 (1956) 575-578.
• 2. D.E Duggan, A.W. Anderson, P.R. Elliker, R.F. Cain, Ultraviolet exposure studies on a gamma radiation resistant micrococcus isolated from food a,b,c, J. Food Sci., 24 (1959) 376-382.
• 3. V. Mattimore, J.R. Battista, Radioresistance of Deinococcus radiodurans:functions necessary to survive ionizing radiation are also necessary to survive prolonged desiccation, J. Bacteriol., 178 (1996) 633-637.
• 4. J.R. Battista, Against all odds: the survival strategies of Deinococcus radiodurans, Annu. Rev. Microbiol., 51 (1997) 203-224.
• 5. M.M. Cox, J.R. Battista. Deinococcus radiodurans-the consummate survivor, Nat. Rev. Microbiol., 3 (2005) 882-892.
• 6. O. Alvizo, L.J. Nguyen, C.K. Savile, J.A. Bresson, S.L. Lakhapatri, et al., Directed evolution of an ultrastable carbonic anhydrase for highly efficient carbon capture from flue gas, Proc. Natl. Acad. Sci. U. S. A., 111 (2014) 16436-16441. 7. C. Capasso, C.T. Supuran, An overview of the alpha-, beta- and gamma-carbonic anhydrases from Bacteria: can bacterial carbonic anhydrases shed new light on evolution of bacteria?, J. Enzyme Inhib. Med. Chem. 30 (2015) 325-332.
• 8. S.K. Smith, J.G. Ferry, Prokaryotic carbonic anhydrases, FEMS Microbiol. Rev., 24 (2000) 335-366.
• 9. A. Di Fiore, V.A. lterio, S.M. Monti, G. De Simone, K. D'Ambrosio, Thermostable Carbonic Anhydrases in Biotechnological Applications, Int. J. Mol. Sci. 16 (2015) 15456‐15480.
• 10. C. Capasso, C.T. Supuran, An overview of the alpha-, beta- and gamma-carbonic anhydrases from Bacteria: can bacterial carbonic anhydrases shed new light on evolution of bacteria?, J. Enzyme Inhib. Med. Chem., 30 (2015) 325-332. 11. A. Aspatwar, S. Haapanen, S. Parkkila, An Update on the Metabolic Roles of Carbonic Anhydrases in the Model Alga Chlamydomonas reinhardtii, Metabolites, 13 (2018). 12. I.R. Booth, Regulation of cytoplasmic pH in bacteria, Microbiol. Rev., 49 (1985) 359.
• 13. C. Ferradini, J.P. Jay-Gerin, The effect of pH on water radiolysis: a still open question-a minireview, Res. Chem., 26 (2000) 549-565. 14. W.D. Swiatla, Computation of the effect of pH on spur chemistry in water radiolysis at elevated temperatures, Nukleonika, 53 (2008) 31-37.
• 15. A.J. Esbaugh, B.L. Tufts, The structure and function of carbonic anhydrase isozymes in the respiratory system of vertebrates, Respir. Physiol. Neurobiol., 154(2006) 185–198.
• 16. Aspatwar, A., Haapanen, S., and Parkkila, S., An Update on the Metabolic Roles of Carbonic Anhydrases in the Model Alga Chlamydomonas reinhardtii, Metabolites, 13 (2018).
• 17. C. Ward, J. Meehan, M. Gray, I.H. Kunkler, S.P. Langdon, D.J. Argyle, Carbonic Anhydrase IX [CAIX], Cancer, and Radiation Responsiveness, Metabolites, 10 (2018).
• 18. L. Dubois, S. Peeters, N.G. Lieuwes, N. Geusens, A. Thiry, S. Wigfield, F. Carta, et al., Specific inhibition of carbonic anhydrase IX activity enhances the in vivo therapeutic effect of tumor irradiation, Radiother. Oncol. 99 (2011) 424–431.
• 19. R.B. Kapust, J. Tozser, T.D. Copeland, D.S. Waugh, The P1′ specificity of tobacco etch virus protease, Biochem Biophys Res Commun., 294 (2002) 949–955.
• 20. E. Gasteiger, C. Hoogland, A. Gattiker, M.R. Wilkins, R.D. Appel, A. Bairoch, Protein Identification and Analysis Tools on the ExPASy Server. In The Proteomics Protocols Handbook; Humana Press: New York, NY, (2005) 571–607.
• 21. S. Panda, G. Chandra, Physicochemical characterization and functional analysis of some snake venom toxin proteins and related non-toxin proteins of other chordates, Bioinformation, 8 (2012) 891–896.
• 22. R. Mohan, S. Venugopal, Computational structural and functional analysis of hypothetical proteins of Staphylococcus aureus, Bioinformation, 8 (2012) 722-728.
• 23. K. Pramanik, P.K. Ghosh, S. Ray, A. Sarkar, S. Mitra, T.K. Maiti, An In Silico Structural, Functional and Phylogenetic Analysis with Three Dimensional Protein Modeling of Alkaline Phosphatase Enzyme of Pseudomonas aeruginosa, J. Genet. Eng. Biotechnol., 15 (2017) 527–537.
• 24. K. Debashree, M. Saurov, T. Bhaben, In-Silico Comparative Structural Modeling of Carbonic Anhydrase of the Marine Diatom Thalassiosira pseudonana Dates, J. Res. Bioinform., 1 (2012) 9–15.
• 25. K. Pramanik, P.K. Ghosh, S. Ray, A. Sarkar, S. Mitra, T.K. Maiti. An In Silico Structural, Functional and Phylogenetic Analysis with Three Dimensional Protein Modeling of Alkaline Phosphatase Enzyme of Pseudomonas aeruginosa, J. Genet. Eng. Biotechnol., 15 (2017) 527–537.
• 26. K. Pramanik, T. Soren, S. Mitra, T.K. Maiti. In Silico Structural and Functional Analysis of Mesorhizobium ACC Deaminase. Comput. Biol. Chem., 68(2017) 12–21.
• 27. P. Jaya, V.K. Nathan, P. Ammini, Characterization of marine bacterial carbonic anhydrase and their CO2 sequestration abilities based on a soil microcosm, Prep. Biochem. Biotechnol., 49 (2019) 891-899.
• 28. A.B. Murray, M. Aggarwal, M. Pinard, D. Vullo, M. Patrauchan, C.T. Supuran, R McKenna, Structural Mapping of Anion Inhibitors to β-Carbonic Anhydrase psCA3 from Pseudomonas aeruginosa, ChemMedChem., 13 (2018) 2024–2029. 29. S.R. Lotlikar, S. Hnatusko, N.E. Dickenson, S.P. Choudhari, W.L. Picking, M.A. Patrauchan, Three functional β-carbonic anhydrases in Pseudomonas aeruginosa PAO1: role in survival in ambient air, Microbiology, (2013) 1748-1759.
• 30. A. Eminoğlu, D. Vullo, A. Âşık, D.N. Çolak, C.T. Supuran, S. Çanakçı, A.O. Beldüz, Cloning, expression and biochemical characterization of a β-carbonic anhydrase from the soil bacterium Enterobacter sp. B13, J. Enzyme Inhib. Med. Chem., 31 (2016) 1111-1118.
• 31. P. Jaya, V.K. Nathan, P. Ammini. Characterization of marine bacterial carbonic anhydrase and their CO2 sequestration abilities based on a soil microcosm, Prep. Biochem. Biotechnol., 49 (2019) 891-899.
• 32. F. Chen, W. Jin, H. Gao, Z. Guo, H. Lin, J. Li, K. Hu, X. Guan, et al., Cloning, Expression and Characterization of Two Beta Carbonic Anhydrases from a Newly Isolated CO2 Fixer, Serratia marcescens Wy064, Indian J. Microbiol., 59 (2019) 64-72. 33. C.T. Supuran, Bacterial carbonic anhydrases as drug targets: towards novel antibiotics?, Front Pharmacol., 2 (2011) 1–6.
• 34. R.A. Ynalvez, Y. Xiao, A.S. Warda, K. Cunnusamy, J.V. Moroney. Identification and characterization of two closely related β-carbonic anhydrases from Chlamydomonas reinhardtii, Physiol. Plant., 133 (2008) 15–26.
• 35. R. Ramanan, K. Kannan, N. Vinayagamoorthy, K. Ramkumar, S. Sivanesan, T. Chakrabarti, Purification and characterization of a novel plant-type carbonic anhydrase from Bacillus subtilis. Biotechnol. Bioproc. E., 14 (2009) 32–37.
• 36. S.R. Lotlikar, S. Hnatusko, N.E. Dickenson, S.P. Choudhari, W.L. Picking, M.A. Patrauchan, Three functional β-carbonic anhydrases in Pseudomonas aeruginosa PAO1: role in survival in ambient air, Microbiology., 159 (2013) 1748-1759.
• 37. F. Chen, W. Jin, H. Gao, Z. Guo, H. Lin, J. Li, K. Hu, X. Guan, V.C. Kalia, J.K. Lee, L. Zhang, Y. Li, Cloning, Expression and Characterization of Two β Carbonic Anhydrases from a Newly Isolated CO2 Fixer, Serratia marcescens Wy064. Indian J. Microbiol., 59 (2019) 64-72.