ISSN 1330-9862 original scientific paper (FTB-1933) The Tyrosinase Produced by Lentinula boryana (Berk. & Mont.) Pegler Suffers Substrate Inhibition by L-DOPA Rodrigo Otávio de Faria 1 , Vivian Rotuno Moure 1 , Wellington Balmant 1 , Maria Angela Lopes de Almeida Amazonas 2 , Nadia Krieger 3 and David Alexander Mitchell 1* 1 Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Cx. P. 19046, Centro Politécnico, Curitiba 81531-990, Paraná, Brazil 2 Centro Nacional de Pesquisa de Florestas, Empresa Brasileira de Pesquisa Agropecuária – Embrapa Florestas, Cx. P. 319 Colombo 83411-000, Paraná, Brazil 3 Departamento de Química, Universidade Federal do Paraná, Cx. P. 19081, Centro Politécnico, Curitiba 81531-990, Paraná, Brazil Received: May 15, 2007 Revised version: June 15, 2007 Accepted: June 22, 2007 Summary We undertook a preliminary characterization of the tyrosinase produced by a strain of Lentinula boryana from Brazil, with a view to evaluate its potential for biotechnological ap- plications. The enzyme was similar to other fungal tyrosinases in many respects. When the crude extract was characterized, the tyrosinase activity was optimal at pH=6 and was not particularly thermostable, with half-lives of about 10 min and 1 min at 50 and 60 °C, re- spectively. We purified the enzyme with ammonium sulfate precipitation followed by ion exchange chromatography on a DEAE Sepharose column, obtaining a yield of 33 % and a 5.3-fold enrichment. The purified preparation gave three bands on SDS-PAGE, with molec- ular masses of 20, 27 and 47 kDa. This preparation showed substrate inhibition kinetics with L-DOPA (3,4-dihydroxy-L-phenylalanine), with a K M of 1.9 mM and a K I of 72 mM. Under the same reaction conditions, a commercial mushroom tyrosinase followed Michae- lis-Menten kinetics, with a K M of 0.51 mM. Although the present study did not identify properties that would make the tyrosinase of L. boryana more suitable in biotechnological applications than tyrosinases from other mushrooms, it has made a contribution by show- ing that the enzyme suffers substrate inhibition by L-DOPA, something that has not previ- ously been reported for mushroom tyrosinases. Key words: tyrosinase, Lentinula boryana, substrate inhibition, 3,4-dihydroxy-L-phenylalanine, L-DOPA Introduction Lentinula boryana is an edible fungus, often referred to as American shiitake, that grows in tropical and sub- tropical regions of the American continent, from the south- east of the United States of America to South America (1). During recent studies of a strain of L. boryana from Brazil, we noted the production of a dark pigment, which was characterized as a DOPA-melanin (2). Since the pro- duction of DOPA-melanin is a strong indication that the producing organism also produces the enzyme tyrosina- se, we tested the mycelium for tyrosinase activity. The test result was positive. Tyrosinase (EC 1.14.18.1; tyrosine, L-DOPA:oxygen oxidoreductase; catecholase; diphenol oxidase; polyphe- 334 R.O. DE FARIA et al.: Substrate Inhibition of L. boryana Tyrosinase, Food Technol. Biotechnol. 45 (3) 334–340 (2007) *Corresponding author; Phone: ++55 41 3361 1658; Fax: ++55 41 3266 2042; E-mail: davidmitchell@ufpr.br