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 journalArticleScientificpeer-review

29 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

Fingerprint

Aspergillus oryzae
Catechol Oxidase
Monophenol Monooxygenase
Enzymes
Fungal Genome
Furin
Dihydroxyphenylalanine
Trichoderma
Protein Folding
Circular Dichroism
Anions
Tyrosine
Cations
Chromatography
Proteins
Peptide Hydrolases
Amino Acids

Keywords

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

Cite this

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title = "Discovery of a new tyrosinase-like enzyme family lacking a C-terminally processed domain: Production and characterization of an Aspergillus oryzae catechol oxidase",
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.",
keywords = "Fungal, Catechol oxidase, Tyrosinase, Secreted, Aspergillus, Trichoderma",
author = "Chiara Gasparetti and Greta Faccio and Mikko Arvas and Johanna Buchert and Markku Saloheimo and Kristiina Kruus",
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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. / Gasparetti, Chiara (Corresponding Author); Faccio, Greta; Arvas, Mikko; Buchert, Johanna; Saloheimo, Markku; Kruus, Kristiina.

In: Applied Microbiology and Biotechnology, Vol. 86, No. 1, 2010, p. 213-226.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Discovery of a new tyrosinase-like enzyme family lacking a C-terminally processed domain

T2 - Production and characterization of an Aspergillus oryzae catechol oxidase

AU - Gasparetti, Chiara

AU - Faccio, Greta

AU - Arvas, Mikko

AU - Buchert, Johanna

AU - Saloheimo, Markku

AU - Kruus, Kristiina

PY - 2010

Y1 - 2010

N2 - 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.

AB - 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.

KW - Fungal

KW - Catechol oxidase

KW - Tyrosinase

KW - Secreted

KW - Aspergillus

KW - Trichoderma

U2 - 10.1007/s00253-009-2258-3

DO - 10.1007/s00253-009-2258-3

M3 - Article

VL - 86

SP - 213

EP - 226

JO - Applied Microbiology and Biotechnology

JF - Applied Microbiology and Biotechnology

SN - 0175-7598

IS - 1

ER -