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

Coleri Cihan, Arzu; TAŞKIN KAFA, Ayşe Hümeyra; cankaya, murat; KUZUCU, Mehmet

doi:10.15671/hjbc.775093

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

Ayşe Hümeyra TAŞKIN KAFA, (Cumhuriyet Üniversitesi, Tıp Fakültesi, Tıbbi Mikrobiyoloji Anabilim Dalı, Sivas, Türkiye)

Arzu ÇÖLERİ CİHAN, (Ankara Üniversitesi, Fen Fakültesi, Biyoloji Bölümü, Ankara, Türkiye)

Mehmet KUZUCU, (Erzincan Üniversitesi, Fen-Edebiyat Fakültesi, Biyoloji Bölümü, Erzincan, Türkiye)

Murat ÇANKAYA (Erzincan Üniversitesi, Fen-Edebiyat Fakültesi, Biyoloji Bölümü, Erzincan, Türkiye)

Hacettepe Journal of Biology and Chemistry

7 0

Yıl: 2021 Cilt: 49 Sayı: 3 Sayfa Aralığı: 219 - 231 Metin Dili: İngilizce DOI: 10.15671/hjbc.775093 İndeks Tarihi: 20-11-2021

Cloning, Over-Expression, and Purification of β–Carbonic Anhydrasefrom 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 $CO_2$ 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 $Zn^{2+}$, which resulted in 5-fold increases of $CO_2$ hydration activity. Also sulfonamide showed inhibitory effect on the pure enzyme. The apparent Km and Vmax for $CO_2$ as substrate were 8.4 mM and 637 WAU/mg for DrCA respectively. The $CO_2$ hydration assay demonstrated that the specific activity of purified recombinant enzymes (DrCA) was significantly high.

Anahtar Kelime:

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

Öz:

Bu çalışmada, radyasyona ekstrem dirençli bir bakteri olan Deinococcus radiodurans’dan direnç fizyolojisinde önemli ol duğunu düşündüğümüz karbonik anhidrazın (DrCA) enziminin klonlanması, saflaştırılması ve başlangıç karakterizasyonugerçekleştirilmiştir. Ayrıca, artan dozlarda gama radyasyonun pH ile ilişkili DrCA enzim aktivitesi üzerindeki etkisi belirlen miştir. Radyasyon uygulamasından sonra DrCA aktivitesi, 800 Gy’a kadar sürekli artarak 6 kat, artmıştır, bu noktadan sonraaktivitede azalma olduğu gözlenmiştir. DrCA enzimi için maksimum $CO_2$hidrasyon aktivitesi, pH 7.0 ve 40°C’de gözlenmiştir.Homo-dimer kompleks yapısında olan DrCA enzimi hafif termostabildir. DrCA’nın $CO_2$hidrasyon aktivitesini, çeşitli metaliyonları, özellikle $Zn^{2+}$ önemli ölçüde, 5 kat artırmıştır. Ayrıca sülfonamid saf enzim üzerinde inhibe edici etki göstermiştir.DrCA’nın substrat olarak $CO_2$için belirlenen Km ve Vmax değerleri sırasıyla 8.4 mM ve 637 WAU/mg idi. $CO_2$ hidrasyon deneyisaflaştırılmış rekombinant enzimin (DrCA) spesifik aktivitesinin önemli ölçüde yüksek olduğunu göstermiştir.

Anahtar Kelime:

Belge Türü: Makale Makale Türü: Araştırma Makalesi Erişim Türü: Erişime Açık

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APA	TAŞKIN KAFA A, Coleri Cihan A, KUZUCU M, cankaya m (2021). Cloning, Over-Expression, and Purification of β–Carbonic Anhydrasefrom an Extremophilic Bacterium: Deinococcus radiodurans. , 219 - 231. 10.15671/hjbc.775093
Chicago	TAŞKIN KAFA Ayşe Hümeyra,Coleri Cihan Arzu,KUZUCU Mehmet,cankaya murat Cloning, Over-Expression, and Purification of β–Carbonic Anhydrasefrom an Extremophilic Bacterium: Deinococcus radiodurans. (2021): 219 - 231. 10.15671/hjbc.775093
MLA	TAŞKIN KAFA Ayşe Hümeyra,Coleri Cihan Arzu,KUZUCU Mehmet,cankaya murat Cloning, Over-Expression, and Purification of β–Carbonic Anhydrasefrom an Extremophilic Bacterium: Deinococcus radiodurans. , 2021, ss.219 - 231. 10.15671/hjbc.775093
AMA	TAŞKIN KAFA A,Coleri Cihan A,KUZUCU M,cankaya m Cloning, Over-Expression, and Purification of β–Carbonic Anhydrasefrom an Extremophilic Bacterium: Deinococcus radiodurans. . 2021; 219 - 231. 10.15671/hjbc.775093
Vancouver	TAŞKIN KAFA A,Coleri Cihan A,KUZUCU M,cankaya m Cloning, Over-Expression, and Purification of β–Carbonic Anhydrasefrom an Extremophilic Bacterium: Deinococcus radiodurans. . 2021; 219 - 231. 10.15671/hjbc.775093
IEEE	TAŞKIN KAFA A,Coleri Cihan A,KUZUCU M,cankaya m "Cloning, Over-Expression, and Purification of β–Carbonic Anhydrasefrom an Extremophilic Bacterium: Deinococcus radiodurans." , ss.219 - 231, 2021. 10.15671/hjbc.775093
ISNAD	TAŞKIN KAFA, Ayşe Hümeyra vd. "Cloning, Over-Expression, and Purification of β–Carbonic Anhydrasefrom an Extremophilic Bacterium: Deinococcus radiodurans". (2021), 219-231. https://doi.org/10.15671/hjbc.775093

APA	TAŞKIN KAFA A, Coleri Cihan A, KUZUCU M, cankaya m (2021). Cloning, Over-Expression, and Purification of β–Carbonic Anhydrasefrom an Extremophilic Bacterium: Deinococcus radiodurans. Hacettepe Journal of Biology and Chemistry, 49(3), 219 - 231. 10.15671/hjbc.775093
Chicago	TAŞKIN KAFA Ayşe Hümeyra,Coleri Cihan Arzu,KUZUCU Mehmet,cankaya murat Cloning, Over-Expression, and Purification of β–Carbonic Anhydrasefrom an Extremophilic Bacterium: Deinococcus radiodurans. Hacettepe Journal of Biology and Chemistry 49, no.3 (2021): 219 - 231. 10.15671/hjbc.775093
MLA	TAŞKIN KAFA Ayşe Hümeyra,Coleri Cihan Arzu,KUZUCU Mehmet,cankaya murat Cloning, Over-Expression, and Purification of β–Carbonic Anhydrasefrom an Extremophilic Bacterium: Deinococcus radiodurans. Hacettepe Journal of Biology and Chemistry, vol.49, no.3, 2021, ss.219 - 231. 10.15671/hjbc.775093
AMA	TAŞKIN KAFA A,Coleri Cihan A,KUZUCU M,cankaya m Cloning, Over-Expression, and Purification of β–Carbonic Anhydrasefrom an Extremophilic Bacterium: Deinococcus radiodurans. Hacettepe Journal of Biology and Chemistry. 2021; 49(3): 219 - 231. 10.15671/hjbc.775093
Vancouver	TAŞKIN KAFA A,Coleri Cihan A,KUZUCU M,cankaya m Cloning, Over-Expression, and Purification of β–Carbonic Anhydrasefrom an Extremophilic Bacterium: Deinococcus radiodurans. Hacettepe Journal of Biology and Chemistry. 2021; 49(3): 219 - 231. 10.15671/hjbc.775093
IEEE	TAŞKIN KAFA A,Coleri Cihan A,KUZUCU M,cankaya m "Cloning, Over-Expression, and Purification of β–Carbonic Anhydrasefrom an Extremophilic Bacterium: Deinococcus radiodurans." Hacettepe Journal of Biology and Chemistry, 49, ss.219 - 231, 2021. 10.15671/hjbc.775093
ISNAD	TAŞKIN KAFA, Ayşe Hümeyra vd. "Cloning, Over-Expression, and Purification of β–Carbonic Anhydrasefrom an Extremophilic Bacterium: Deinococcus radiodurans". Hacettepe Journal of Biology and Chemistry 49/3 (2021), 219-231. https://doi.org/10.15671/hjbc.775093