PURIFICATION AND CHARACTERIZATION OF LACCASE FROM LENTINUS-EDODES

An extracellular laccase produced in wheat-bran culture by Lentinus edodes (Berk.) Sing. was purified successively by diethylaminoethyl (DEAE)-Sepharose ion-exchange chromatography, ion exchange high-performance liquid chromatography (HPLC) and gel-filtration HPLC to be electrophoretically pure. The molecular weight of the enzyme was 65000 with a pI of about 3.0. The enzyme oxidized guaiacol, o- and p-phenylendiamines, syringaldazine, ABTS ((2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonate) )) and catechol in the absence of added H2O2, whereas substrates for tyrosinase and polyphenol oxidase were not changed. Spectral analysis indicated that the enzyme contains copper ions. The phenolic beta-O-4 lignin substructure model compound, syringylglycerol-beta-guaiacyl ether (I), was degraded by the enzyme. By gas chromatography-mass spectrometry of the degradation products, 2,6-dimethoxy-p-benzoquinone (II), 2,6-dimethoxy-p-hydroquinone (III), and 1-(3,5-dimethoxy-4-hydroxyphenyl)-2- (2-methoxyphonoxy) -3-hydroxypropanone (IV) were identified, and 2-(2-methoxyphenoxy)-3-hydroxypropanal (VI) was suggested to be a counter part of III. The structure of the degradation products indicates that the enzyme catalyzes alkyl-aryl splitting of Substrate I.