Production and characterization of a secreted, C-terminally processed tyrosinase from the filamentous fungus Trichoderma reesei

Emilia Selinheimo (Corresponding Author), Markku Saloheimo, Elina Ahola, Ann Westerholm-Parvinen, Nisse Kalkkinen, Johanna Buchert, Kristiina Kruus

Research output: Contribution to journalArticleScientificpeer-review

85 Citations (Scopus)

Abstract

A homology search of the genome database of the filamentous fungus Trichoderma reesei identified a new T. reesei tyrosinase gene tyr2, encoding a protein with a putative signal sequence. The gene was overexpressed in the native host under the strong cbh1 promoter, and the tyrosinase enzyme was secreted into the culture supernatant. This is the first report on a secreted fungal tyrosinase. Expression of TYR2 in T. reesei resulted in good yields, corresponding to approximately 0.3 and 1 g·L−1 tyrosinase in shake flask cultures and laboratory‐scale batch fermentation, respectively. T. reesei TYR2 was purified with a three‐step purification procedure, consisting of desalting by gel filtration, cation exchange chromatography and size exclusion chromatography. The purified TYR2 protein had a significantly lower molecular mass (43.2 kDa) than that calculated from the putative amino acid sequence (61.151 kDa). According to N‐terminal and C‐terminal structural analyses by fragmentation, chromatography, MS and peptide sequencing, the mature protein is processed from the C‐terminus by a cleavage of a peptide fragment of about 20 kDa. The T. reesei TYR2 polypeptide chain was found to be glycosylated at its only potential N‐glycosylation site, with a glycan consisting of two N‐acetylglucosamines and five mannoses. Also, low amounts of shorter glycan forms were detected at this site. T. reesei TYR2 showed the highest activity and stability within a neutral and alkaline pH range, having an optimum at pH 9. T. reesei tyrosinase retained its activity well at 30°C, whereas at higher temperatures the enzyme started to lose its activity relatively quickly. T. reesei TYR2 was active on both l‐tyrosine and l‐dopa, and it showed broad substrate specificity.
Original languageEnglish
Pages (from-to)4322-4335
Number of pages14
JournalFEBS Journal
Volume273
Issue number18
DOIs
Publication statusPublished - 2006
MoE publication typeA1 Journal article-refereed

Keywords

  • fungal
  • secreted
  • Trichoderma reesei
  • tyrosinase

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