Functional and structural analysis of catalase-phenol oxidase from scytalidium thermophilum

Download
2010
Yüzügüllü, Yonca
Scytalidium thermophilum produces a novel phenol oxidase, which has turned out to be a bifunctional catalase-phenol oxidase (CATPO) during the course of this work, by other researchers of our group. Therefore, in the beginning of the studies, substrate specificity and inhibitor assays were conducted on the crude enzyme, followed by production, purification, cloning, expression, and mutagenesis and crystallography studies for further functional and structural analysis of CATPO. Accordingly, substrate specificity and inhibitory tests applied for crude enzyme characterisation presented the similarity of the phenol oxidase nature of CATPO essentially to catechol oxidase. Production studies were performed to investigate the effects of different factors including induction time, growth temperature, exogenous iron and hydrogen peroxide addition. In view of that, CATPO is constitutively produced in a growth associated manner. However, some phenolic compounds enhance its production. In this study, 15 phenolic compounds were tested for their ability to affect CATPO production. Of the phenolic compounds tested, catechol, resorcinol and vanillic acid caused repression of CATPO production. On the other hand, caffeic acid, myricetin and resveratrol enhanced CATPO production. As a biocatalyst, the efficiency of CATPO was examined and found to be a good candidate for getting pharmaceutically important drug intermediates. Its dual mechanism was analysed through side-directed mutagenesis. Two conserved residues (His101 and Val142) were mutated to discriminate catalase and phenol oxidase activities. Spectroscopic and mutagenesis studies exhibited the presence of heme d centre. Lastly, its structure was analysed by X-ray crystallography and found to have a tetrameric structure.

Suggestions

Analysis of phenol oxidation products by scytalidium thermophilum bifunctional catalase/phenol oxidase (catpo)
Avcı, Gülden; Karakaş, Gürkan; Ögel, Zümrüt Begüm; Department of Biotechnology (2011)
This thesis was aimed to analyze phenol oxidation by the bifunctional catalase/phenol oxidase of the thermophilic fungus Scytalidium thermophilum. Several reactive oxygen species (ROS) are continuously produced in fungi under oxidative stress. Depending on the nature of the ROS species, some are highly toxic and are rapidly detoxified by various cellular enzymatic mechanisms, including the production of catalase. S. thermophilum produces a novel bifunctional catalase-phenol oxidase (CATPO) which is capable ...
Microorganism mediated stereoselective bio-oxidation and bio-hydrogenation reactions and thiamine pyrophosphate dependent enzyme catalyzed enantioselective acyloin reactions
Sopacı, Şaziye Betül; Demir, Ayhan Sıtkı; Department of Biotechnology (2009)
In this study various microbial and enzymatic methods developed for enantioselective acyloin synthesis for preparation of some pharmaceutically important intermediates. By performing Aspergillus flavus (MAM 200120) mediated biotransformation, enantioselective bio-oxidation of meso-hydrobenzoin was achieved with a high ee value (76%). Racemic form of hydrobenzoin was also employed for the same bio-oxidation process and this bioconversion was resulted in accumulation of meso form (>90% yield) confirming the s...
Novel bioconversion reactions for the syntheses of a-hydroxy ketones
Ayhan, Peruze; Demir, Ayhan Sıtkı; Department of Biotechnology (2009)
The objective of the study presented here was to develop either enzymatic or whole cell mediated green procedures for the syntheses of a-hydroxy ketones. Production of optically active synthons is crucial for the preparation of fine chemicals. Enzymes and whole-cell biocatalysts have proven to be excellent vehicles with their chiral nature for the biotransformations. Under the light of this discussion, firstly benzaldehyde lyase [BAL, (EC 4.1.2.38)] was used in novel C-C bond formation reactions to obtain i...
Investigating the anticarcinogenic role of salix aegyptiaca l. in colorectal carcinoma
Enayat, Shabnam; Banerjee, Sreeparna; Department of Biology (2009)
In this study, extracts from bark, leaves and catkins of Salix aegyptiaca L. were investigated for their antioxidant content by 2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH) free radical quenching assay, total phenolic and total flavonoid assays. The highest antioxidant activity (19 ug/ml IC50 for inhibition of DPPH radical activity), total phenolic content (212 mg gallic acid equivalents/g of dried extract) and total flavonoid (479 mg catechin equivalents/g of dried extract) was observed in the ethanolic ex...
The effect of cysteine-43 mutation on thermostability and kinetic properties of citrate synthase from Thermoplasma acidophilum
Kocabıyık, Semra; Russel, RJM; Danson, MJ; Hough, DW (Elsevier BV, 1996-07-05)
In this study, we have substituted serine-43 by cysteine in the recombinant citrate synthase from a moderately thermophilic Archaeon Thermoplasma acidophilum, for site-specific attachment of labels and have investigated the effects of this mutation on the biochemical properties and thermal stability of the enzyme. Both wild-type and the mutant enzymes were purified to homogenity using affinity chromatography on Matrex Gel Red A. The mutant Thermoplasma citrate synthase is very similar to wild-type citrate s...
Citation Formats
Y. Yüzügüllü, “Functional and structural analysis of catalase-phenol oxidase from scytalidium thermophilum,” Ph.D. - Doctoral Program, Middle East Technical University, 2010.