Purification and Some Properties of Tyrosinase from Aspergillus flavipes 56003

G. S. Gukasyan

Armenian Institute of Applied Chemistry (ARIAC), ul. Bagratunyats 70, Yerevan, 375029 Armenia; fax: (+374) 390-7155; E-mail: ARIAC@servis.arminco.com

Received June 9, 1998; Revision received October 1, 1998

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A method for isolation and purification of tyrosinase from the fungus Aspergillus flavipes 56003 was developed. The method includes extraction with water, concentration on DEAE-cellulose, gel-filtration on Acrylex P-150, and ion-exchange chromatography on DEAE-Toyopearl 650M. The tyrosinase was purified to apparent homogeneity according polyacrylamide gel electrophoresis and ultracentrifugation. The tyrosinase is a 130-kD protein with pI 4.6. It contains two copper atoms. The K_m and V_max for tyrosine hydroxylation are 0.3 mM and 1300 µmoles/min per mg at pH 6.8, and for dehydrogenation of 3,4-dihydroxyphenylalanine (DOPA) they are 5 mM and 16000 µmoles/min per mg, respectively. Hydroxylation of monophenols has a characteristic lag period. The rate of tyrosine and DOPA oxidation is maximal at pH 6.0-6.8. The half-life of the enzyme at 50°C is 40 min. The hydroxylase activity of the tyrosinase is more stable at neutral pH, whereas the dehydrogenase activity is more stable at acidic pH (4.0). The absorption spectrum of the enzyme has a maximum at 290 mn and a shoulder in the 320-400-nm region.

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KEY WORDS: tyrosinase, Aspergillus

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