TAILIEUCHUNG - Báo cáo khoa học: Production and characterization of a secreted, C-terminally processed tyrosinase from the filamentous fungus Trichoderma reesei

A homology search of the genome database of the filamentous fungus Trichoderma reeseiidentified a new T. reeseityrosinase 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. | ễFEBS Journal Production and characterization of a secreted C-terminally processed tyrosinase from the filamentous fungus Trichoderma reesei Emilia Selinheimo1 Markku Saloheimo1 Elina Ahola2 Ann Westerholm-Parvinen1 Nisse Kalkkinen2 Johanna Buchert1 and Kristiina Kruus1 1 VTT TechnicalResearch Centre of Finland Espoo Finland 2 Protein Chemistry Research Group and Core Facility Institute of Biotechnology University of Helsinki Finland Keywords fungal secreted Trichoderma reesei tyrosinase Correspondence E. Selinheimo VTT TechnicalResearch Centre of Finland PO Box 1000 Espoo FIN-02044 VTT Finland Fax 358 20 722 7071 Tel 358 20 722 7135 E-mail Received 16 May 2006 revised 7 July 2006 accepted 27 July 2006 doi 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 cbhl 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 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 kDa than that calculated from the putative amino acid sequence 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 .

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