Discovery of a new tyrosinase-like enzyme family lacking a C-terminally processed domain: production and characterization of an Aspergillus oryzae catechol oxidase.
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 degrees C. The UV-visible and circular dichroism spectroscopic analysis suggested that the folding of the protein was correct.
VTT Technical Research Centre of Finland, P. O. Box 1000, Espoo, 02044 VTT, Finland. chiara.gasparetti@vtt.fi
Chiara
C
Gasparetti
Greta
G
Faccio
Mikko
M
Arvas
Johanna
J
Buchert
Markku
M
Saloheimo
Kristiina
K
Kruus
D000595:Amino Acid Sequence; D001236:Aspergillus oryzae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001709:Biotechnology; D020134:Catalytic Domain; D004156:Catechol Oxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature; D014242:Trichoderma / Q000201:enzymology / Q000235:genetics
D005656:Fungal Proteins; D004156:Catechol Oxidase; D014442:Monophenol Monooxygenase