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Discovery of a new tyrosinase-like enzyme family lacking a C-terminally processed domain: production and characterization of an Aspergillus oryzae catechol oxidase

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An Erratum to this article was published on 11 February 2011

Abstract

A homology search against public fungal genome sequences was performed to discover novel secreted tyrosinases. The analyzed proteins could be divided in two groups with different lengths (350–400 and 400–600 residues), suggesting the presence of a new class of secreted enzymes lacking the C-terminal domain. Among them, a sequence from Aspergillus oryzae (408 aa, AoCO4) was selected for production and characterization. AoCO4 was expressed in Trichoderma reesei under the strong cbh1 promoter. Expression of AoCO4 in T. reesei resulted in high yields of extracellular enzyme, corresponding to 1.5 g L−1 production of the enzyme. AoCO4 was purified with a two-step purification procedure, consisting of cation and anion exchange chromatography. The N-terminal analysis of the protein revealed N-terminal processing taking place in the Kex2/furin-type protease cleavage site and removing the first 51 amino acids from the putative N-terminus. AoCO4 activity was tested on various substrates, and the highest activity was found on 4-tert-butylcatechol. Because no activity was detected on L-tyrosine and on l-dopa, AoCO4 was classified as a catechol oxidase. AoCO4 showed the highest activity within an acidic and neutral pH range, having an optimum at pH 5.6. AoCO4 showed good pH stability within a neutral and alkaline pH range and good thermostability up to 60°C. The UV–visible and circular dichroism spectroscopic analysis suggested that the folding of the protein was correct.

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Acknowledgments

The work was carried out with financial support from the Marie Curie EU-project “Enzymatic tailoring of protein interactions and functionalities in food matrix” PRO-ENZ (MEST-CT-2005-020924). Harry Boer, Päivi Mantikainen, and Evanthia Monogioudi are acknowledged for their assistance at MALDI-TOF MS and CD instruments. Also, the skilful technical assistance of Hanna Kuusinen, Riitta Isoniemi, Michael Bailey, and Gunilla Rönnholm is acknowledged.

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  1. VTT Technical Research Centre of Finland, P. O. Box 1000, Espoo, 02044 VTT, Finland

    Chiara Gasparetti, Greta Faccio, Mikko Arvas, Johanna Buchert, Markku Saloheimo & Kristiina Kruus

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  1. Chiara Gasparetti
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  2. Greta Faccio
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  3. Mikko Arvas
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Correspondence to Chiara Gasparetti.

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Chiara Gasparetti and Greta Faccio contributed equally to this work.

An erratum to this article can be found at http://dx.doi.org/10.1007/s00253-011-3110-0

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Gasparetti, C., Faccio, G., Arvas, M. et al. Discovery of a new tyrosinase-like enzyme family lacking a C-terminally processed domain: production and characterization of an Aspergillus oryzae catechol oxidase. Appl Microbiol Biotechnol 86, 213–226 (2010). https://doi.org/10.1007/s00253-009-2258-3

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