Purification and characterization of a new member of the laccase family from the white-rot basidiomycete Coriolus hirsutus

Laccase produced by Coriolus hirsutus was purified to electrophoretic homog eneity by acetone precipitation, DEAE Sepharose CL-6B, Sephacryl S-200 HR, Hitrap SP, and Mono S chromatography. The purification was 14.5-fold with a n overall yield of 32.3%. The enzyme is a monomeric glycoprotein with 11% c arbohydrate content, an isoelectric point of 7.4, and a molecular mass of 7 3 kDa. The N-terminal amino acid sequence showed low homology to those of t he laccases of other white-rot basidiomycetes. Spectroscopic analyses revea led a typical laccase active site in the C, hirsutus enzyme, as all three C u centers were identified. The absorption spectrum showed a type 1 signal a t around 600 nm and a type 3 signal near 330 nm. Type 3 Cu showed fluoresce nce emission near 418 nm and an excitation maximum at 332 nm. The EPR spect rum yielded parameters for the type 1 and type 2 Cu of g(II) = 2.191 and A( II) = 0.0097 cm(-1), and g(II) = 2.222 and A(II) = 0.0198 cm(-1), respectiv ely. The highest rate of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) oxidation for the enzyme was reached at 45 degreesC, and the pH optima of the enzyme varied and was substrate dependent in the range of 2.5 to 4.0. T he enzyme oxidized a variety of the usual laccase substrates, including lig nin-related phenols and had highest affinity toward guaiacol. Under standar d assay conditions, the apparent K-m value of the enzyme toward guaiacol wa s 10.9 muM. The enzyme catalyzed single electron transfer via the phenoxy r adical as an intermediate and was completely inhibited by L-cysteine and so dium azide but not by EDTA. (C) 2000 Academic Press.