1887

Abstract

SUMMARY: Two different bands with laccase activity were obtained after nondenaturing PAGE of the culture filtrate of . Immunoblot analysis revealed that antisera raised against laccase I were not reactive to laccase II. Laccase I, which exhibited faster mobility on nondenaturing polyacrylamide gel, was purified 42·9-fold with an overall yield of 10·8%. Gel filtration and SDS-PAGE revealed that laccase I is a single polypeptide with a molecular mass of approximately 64 kDa. Laccase I contained 12·5% carbohydrate by weight and 3·9 mol copper (mol protein). The absorption spectrum of laccase I showed a type 1 signal at 605 nm and EPR spectra showed that the parameters of the type 1 and type 2 Cu signals were = 2·197 and = 0·009 cm, and = 2·263 and = 0·0176 cm, respectively. The data obtained from the pH profiles suggested that two ionization groups, whose p values were 5·60–5·70 and 6·70–6·85, may play an important role in the active site of laccase I as the ligand of copper metal. The optimal pH and temperature for the activity of laccase I were 6·0–6·5 and 30–35 °C, respectively. The enzyme had affinity for various lignin-related phenolic compounds: the values for ferulic acid and syringic acid were 48 and 89 μM, respectively. EPR spectroscopic study of the action of laccase I on 3,5-dimethoxy-5-hydroxyacetophenone indicated that this enzyme catalyses single electron transfer with the formation of the phenoxy radical as an intermediate.

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1995-02-01
2021-04-17
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