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

Chiara Gasparetti (Corresponding Author), Greta Faccio, Mikko Arvas, Johanna Buchert, Markku Saloheimo, Kristiina Kruus

    Research output: Contribution to journalArticlepeer-review

    31 Citations (Scopus)

    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.
    Original languageEnglish
    Pages (from-to)213-226
    Number of pages14
    JournalApplied Microbiology and Biotechnology
    Volume86
    Issue number1
    DOIs
    Publication statusPublished - 2010
    MoE publication typeA1 Journal article-refereed

    Keywords

    • Fungal
    • Catechol oxidase
    • Tyrosinase
    • Secreted
    • Aspergillus
    • Trichoderma

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