Biochemical and structural characterisation of the copper containing oxidoreductases catechol oxidase, tyrosinase, and laccase from ascomycete fungi

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dc.contributor Aalto-yliopisto fi
dc.contributor Aalto University en
dc.contributor.author Gasparetti, Chiara
dc.date.accessioned 2012-10-17T11:34:40Z
dc.date.available 2012-10-17T11:34:40Z
dc.date.issued 2012
dc.identifier.isbn 978-951-38-7935-8 (electronic)
dc.identifier.isbn 978-951-38-7934-1 (printed)
dc.identifier.issn 2242-1203 (electronic)
dc.identifier.issn 2242-119X (printed)
dc.identifier.uri https://aaltodoc.aalto.fi/handle/123456789/6003
dc.description.abstract Catechol oxidase (EC 1.10.3.1), tyrosinase (EC 1.14.18.1), and laccase (EC 1.10.3.2) are copper-containing metalloenzymes. They oxidise substituted phenols and use molecular oxygen as a terminal electron acceptor. Catechol oxidases and tyrosinases catalyse the oxidation of p-substituted o-diphenols to the corresponding o-quinones. Tyrosinases also catalyse the introduction of a hydroxyl group in the ortho position of p-substituted monophenols and the subsequent oxidation to the corresponding o-quinones. Laccases can oxidise a wide range of compounds by removing single electrons from the reducing group of the substrate and generate free radicals. The reaction products of these oxidases can react further non-enzymatically and lead to formation of polymers and cross-linking of proteins and carbohydrates, in certain conditions. The work focused on examination of the properties of catechol oxidases, tyrosinases and laccases. A novel catechol oxidase from the ascomycete fungus Aspergillus oryzae was characterised from biochemical and structural point of view. Tyrosinases from Trichoderma reesei and Agaricus bisporus were examined in terms of substrate specificity and inhibition. The oxidation capacity of laccases was elucidated by using a set of laccases with different redox potential and a set of substituted phenolic substrates with different redox potential. Finally, an evaluation of the protein cross-linking ability of catechol oxidase from A. oryzae, tyrosinases from T. reesei and A. bisporus and laccases from Trametes hirsuta, Thielavia arenaria, and Melanocarpus albomyces was performed. A novel extracellular catechol oxidase from A. oryzae (AoCO4; UniProtKB: Q2UNF9; Entrez gene ID: 5990879) was chosen for cloning and expression as representative of the newly discovered family of short tyrosinases sequences. AoCO4 gene was heterologously expressed in T. reesei. The protein produced did not show activity on L-tyrosine and 3,4-dihydroxy-L-phenylalanine (L-DOPA), which are typical substrates for tyrosinases. Consequently, the protein was classified as a catechol oxidase. AoCO4 was produced in a bioreactor and the expression resulted in high yields. The purified AoCO4 was partially processed at a Kex2/furin-type protease site and showed a molecular weight of 39.3 kDa. AoCO4 was able to oxidise a limited range of diphenolic compounds, e.g., catechol, caffeic acid, hydrocaffeic acid, and 4-tert-buthylcatechol. AoCO4 oxidised also the monophenolic compounds aminophenol and guaiacol. AoCO4 showed a pH optimum in the acidic range and was observed to be a relatively thermostable enzyme. A crystal structure of AoCO4 was solved at 2.5 Å resolution. AoCO4 was a monomer and the overall structure of AoCO4 was found to be similar to the known structures of catechol oxidases and tyrosinases. A detailed characterisation of the substrate-specificity of the extracellular tyrosinase from T. reesei (TrT) was accomplished and compared to the commercial tyrosinase from A. bisporus (AbT). TrT generally showed lower affinity than AbT on substrates that had a free amino group, such as L-tyrosine, L-DOPA, and YGG tripeptide. The reaction end product of TrT and AbT was studied via mass spectrometry and dopachrome was found to be the only reaction end product of L-DOPA oxidation catalysed by both tyrosinases. Dopachrome produced by AbT-catalysed oxidation of L-DOPA was also shown to inhibit the TrT tyrosinase by an end product inhibition mechanism. Further, when the type of inhibition of potential inhibitors for TrT was analysed with p-coumaric acid and caffeic acid as substrates, potassium cyanide and kojic acid were the strongest inhibitors of TrT. The kinetics of three laccases, with different redox potential (E°), for three p-substituted dimethoxy phenolic substrates (2,6-dimethoxyphenol, syringic acid, and methyl syringate) with different E° were determined at two pHs. The laccases studied were from M. albomyces, T. arenaria, and T. hirsuta. The enzyme from the ascomycete fungus T. arenaria was a novel laccase produced in T. reesei. T. arenaria laccase was purified, and biochemically and structurally characterised. By comparison of the three laccases it could be shown that both the difference in redox potential (ΔE°) and the pH had an effect on the kinetics. However, the effect of ΔE° was found to prevail over that of the pH for substrates with a high E°, such as methyl syringate. All oxidative enzymes studied in this work were also tested for their cross-linking ability, with α-caseins utilised as a model substrate with and without a cross-linking agent. TrT was found to be the best enzyme for cross-linking of α-caseins, whereas AbT was found to be the best enzyme for cross-linking α-caseins in the presence of catechol as a cross-linking agent. Interestingly, AoCO4 was also found to cross-link α-caseins in the presence of catechol as a cross-linking agent. en
dc.format.extent 124 + app. 55
dc.format.mimetype application/pdf
dc.language.iso en en
dc.publisher VTT Technical Research Centre of Finland en
dc.publisher VTT fi
dc.relation.ispartofseries VTT Science en
dc.relation.ispartofseries 16
dc.relation.haspart [Publication 1]: Selinheimo, E., Gasparetti, C., Mattinen, M.-L., Steffensen, C.L., Buchert, J., Kruus, K. Comparison of substrate specificity of tyrosinases from Trichoderma reesei and Agaricus bisporus. Enzyme and Microbial Technology (2009) 44:1-10. © 2008 Elsevier. By permission.
dc.relation.haspart [Publication 2]: Gasparetti, C., Faccio, G., Arvas, M., Buchert, J., Saloheimo, M., Kruus, K. Discovery of a new tyrosinase-like enzyme family lacking a C-terminally processed domain: production and characterization of an Aspergillus oryzae catechol oxidase. Applied Microbiology and Biotechnology (2010) 86:213-226.
dc.relation.haspart [Publication 3]: Kallio, J.P., Gasparetti, C., Andberg, M., Boer, H., Koivula, A., Kruus, K., Rouvinen, J., Hakulinen, N. Crystal structure of an ascomycete fungal laccase from Thielavia arenaria - common structural features of ascolaccases. FEBS Journal (2011) 278:2283-2295. © 2011 by authors.
dc.relation.haspart [Publication 4]: Gasparetti, C., Nordlund, E., Jänis, J., Buchert, J., Kruus, K. Extracellular tyrosinase from the fungus Trichoderma reesei shows product inhibition and different inhibition mechanism from the intracellular tyrosinase from Agaricus bisporus. BBA - Proteins and Proteomics (2012) 1824:598-607. © 2012 Elsevier. By permission.
dc.subject.other Biotechnology en
dc.title Biochemical and structural characterisation of the copper containing oxidoreductases catechol oxidase, tyrosinase, and laccase from ascomycete fungi en
dc.type G5 Artikkeliväitöskirja fi
dc.contributor.school Kemian tekniikan korkeakoulu fi
dc.contributor.school School of Chemical Technology en
dc.contributor.department Biotekniikan ja kemian tekniikan laitos fi
dc.contributor.department Department of Biotechnology and Chemical Technology en
dc.subject.keyword Trichoderma reesei en
dc.subject.keyword Agaricus bisporus en
dc.subject.keyword Aspergillus oryzae en
dc.subject.keyword Thielavia arenaria en
dc.subject.keyword tyrosinase en
dc.subject.keyword catechol oxidase en
dc.subject.keyword laccase en
dc.subject.keyword purification en
dc.subject.keyword characterisation en
dc.subject.keyword oxidation capacity en
dc.subject.keyword inhibition en
dc.subject.keyword three-dimensional structure en
dc.subject.keyword cross-linking en
dc.identifier.urn URN:ISBN:978-951-38-7935-8
dc.type.dcmitype text en
dc.type.ontasot Doctoral dissertation (article-based) en
dc.type.ontasot Väitöskirja (artikkeli) fi
dc.contributor.supervisor Leisola, Matti, Professor, Department of Biotechnology and Chemical Technology, Aalto University, Espoo, Finland
dc.contributor.supervisor Kruus, Kristiina, Research Professor, Bio and Process Technology, VTT Technical Research Centre of Finland, Espoo, Finland
dc.contributor.supervisor Buchert, Johanna, Research Professor, Bio and Process Technology, VTT Technical Research Centre of Finland, Espoo, Finland
dc.opn Martins, Lígia O., Professor, Instituto de Tecnologia Química e Biológica, Univesidade Nova de Lisboa, Oeiras, Portugal
dc.rev Lundell, Taina, Adjunct Professor, Department of Food and Environmental Sciences, University of Helsinki, Helsinki, Finland
dc.rev Sannia, Giovanni, Full Professor, Department of Organic Chemistry and Biochemistry, University of Naples Federico II, Napoli, Italy
dc.date.defence 2012-10-26


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