Catalase from a local Bacillus licheniformis species isolated from pamukkale hot springs: Cloning, expression, purification and characterization

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Tezin Türü: Yüksek Lisans

Tezin Yürütüldüğü Kurum: Pamukkale Üniversitesi, Fen-Edebiyat Fakültesi, Biyoloji Bölümü, Türkiye

Tezin Onay Tarihi: 2011

Tezin Dili: Türkçe

Öğrenci: Mehmet Özkarslı

Danışman: Alaattin Şen

Özet:

In this study; Catalase enzyme was cloned, expressed, purified and characterized from a local Bacillus licheniformis species isolated from Pamukkale hot springs. Firstly; we made PCR with suitable primers from isolated Bacillus lichenformis and followed by cloning this product into pCR8/GW/TOPO cloning vector and transformed into TOP10 competent E. coli cells. Catalase gene was confirmed with sequence analysis and then transfered into pDest expression vector with the ease of LR Clonase enzyme and transformed into BL21-AI cells. Catalase gene has 1,458 nucleotides and 485 amino acids. Subsequently, catalase was purified by applying DEAE and hydroxyapatide colon chromatographies. Purified catalase has four subunits with apparent monomer molecular weight of 60 kDa and exhibited considerable activity over a broad pH and temperature ranges from pH 5.0 to pH 11.0 and from 20oC to 50oC, respectively. Catalase enzyme has Km of 21,5 mM and Vmax of 8,333 mmol/min/mg protein values. The absorption spectrum of catalase exhibited a Soret band at 406 nm, which is typical of a heme-containing catalase. Enzyme was not reduced with sodium dithionite and potasium cyanide. Enzyme protect its stability at high detergent concentrations. As a result, it is suggested that Bacillus licheniformis catalase has a potential to be used as an industrial enzyme.