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

Sutay, Didem
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.


Purification, characterization, and identification of a novel bifunctional catalase-phenol oxidase from Scytalidium thermophilum
Kocabas, Didem Sutay; Bakir, Ufuk; Phillips, Simon E. V.; McPherson, Michael J.; Ögel, Zümrüt Begüm (Springer Science and Business Media LLC, 2008-06-01)
A novel bifunctional catalase with an additional phenol oxidase activity was isolated from a thermophilic fungus, Scytalidium thermophilum. This extracellular enzyme was purified ca. 10-fold with 46% yield and was biochemically characterized. The enzyme contains heme and has a molecular weight of 320 kDa with four 80 kDa subunits and an isoelectric point of 5.0. Catalase and phenol oxidase activities were most stable at pH 7.0. The activation energies of catalase and phenol oxidase activities of the enzyme ...
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Telli, İlkin Ece; Çalık, Pınar; Department of Chemical Engineering (2004)
In this study, firstly, bioprocess characteristics for Serine Alkaline Protease (SAP) production, using recombinant Bacillus subtilis carrying pHV1431::subC, were examined. The cell concentration, substrate concentration, SAP activity and SAP synthesis rate profiles demonstrated that the system reaches to a steady state in terms of cell growth and SAP synthesis between t=15-25 h, therefore, this time interval is appropriate to employ both metabolic flux analysis and metabolic control analysis, which apply s...
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Kaya, Hande; Çalık, Pınar; Department of Chemical Engineering (2006)
In this study, the benzaldehyde lyase (BAL, EC production in E. coli BL21 (DE3) pLySs as intracellular and in Bacillus species as extracellular were investigated, and comparison of the production capacity of the enzyme in the developed recombinant microorganisms were compared. For this purpose, firstly, PCR amplified bal gene was cloned into pRSETA vector which is under the control of strong T7 promoter and expressed in E. coli BL21 (DE3) pLysS strain. With developed recombinant E. coli BL21 (DE3)...
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Angardi, Vahideh; Çalık, Pınar; Department of Chemical Engineering (2007)
In this study, the effects of oxygen transfer conditions on the synthesis of the enzyme benzaldehyde lyase as intracellular in recombinant E. coli BL21 (DE3) pLysS was investigated sistematically and a comprehensive model was developed to determine benzaldehyde lyase activity. For this purpose, the research program was carried out in mainly two parts. In the first part of study, the effects of oxygen transfer together with the mass transfer coefficient (KLa), enhancement factor E (=KLa/KLao), volumetric oxy...
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.