TY - JOUR
T1 - Production and characterization of a secreted, C-terminally processed tyrosinase from the filamentous fungus Trichoderma reesei
AU - Selinheimo, Emilia
AU - Saloheimo, Markku
AU - Ahola, Elina
AU - Westerholm-Parvinen, Ann
AU - Kalkkinen, Nisse
AU - Buchert, Johanna
AU - Kruus, Kristiina
PY - 2006
Y1 - 2006
N2 - 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.
AB - 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.
KW - fungal
KW - secreted
KW - Trichoderma reesei
KW - tyrosinase
U2 - 10.1111/j.1742-4658.2006.05429.x
DO - 10.1111/j.1742-4658.2006.05429.x
M3 - Article
SN - 1742-464X
VL - 273
SP - 4322
EP - 4335
JO - FEBS Journal
JF - FEBS Journal
IS - 18
ER -