Purification, characterization, crystallization and preliminary x-ray structure determination of scytalidium thermophilum bifunctional catalase and identification of its catechol oxidase activity

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2007
Sutay, Didem
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In this study, the aim was identification and classification of the enzyme having phenol oxidase activity produced by a thermophilic fungus, Scytalidium thermophilum. For this purpose, enzyme production, purification, biochemical characterization and structural analysis by X-ray crystallography studies have been performed. At the beginning of the research, this enzyme was considered as a phenol oxidase and analyzed accordingly. However, during purification, amino acid sequencing and structural studies, the enzyme was shown to be a “catalase”, with an additional catechol oxidase activity. This novel bifunctional “catalase-catechol oxidase” (CCO) was purified 10 fold with 45 % yield by anion exchange and gel filtration chromatographies. CCO was determined as a tetrameric protein having total and subunit molecular weights of 320 and 80 kDa, respectively. Isoelectric point of CCO was verified as 5.0. CCO catalase and catechol oxidase activities were characterized in terms of their kinetic behavior at different pH and temperatures. Depending on the substrate specificity and inhibitor studies of CCO, the phenol oxidase activity was determined as catechol oxidase but not tyrosinase or laccase. The best crystallization condition for CCO was determined and X-ray diffraction data was collected at the Daresbury Synchrotron Radiation Source (United Kingdom) at 2.7 Å resolution. The preliminary structure was solved by molecular replacement method using Penicilium vitale catalase structure. CCO was verified to have a tetrameric structure with two homodimers and a metal center in each polypeptide chain.
Biotechnology.
http://etd.lib.metu.edu.tr/upload/3/12608428/index.pdf
https://hdl.handle.net/11511/17260
Graduate School of Natural and Applied Sciences, Thesis

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Citation Formats
D. Sutay, “Purification, characterization, crystallization and preliminary x-ray structure determination of scytalidium thermophilum bifunctional catalase and identification of its catechol oxidase activity,” Ph.D. - Doctoral Program, Middle East Technical University, 2007.
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