Purification of laccase I from Armillaria mellea

Abstract

Laccase activity accumulated during rhizomorph development in malt extract cultures of Armillaria meffeu. The activity was readily separated into two bands by PAGE under non-denaturing conditions. The less rapidly migrating activity (on PAGE) was purified by ammonium sulphate precipitation, anion-exchange chromatography and gel filtration. The purified enzyme (laccase I) showed a main polypeptide of M,. 59000. Activity of the purified enzyme was greatest with 2,6-dimethoxyphenol (DMOP) as substrate. Syringaldazine, p-phenylenediamine and other typical laccase substrates were readily oxidized. No oxidation of tyrosine was detected. The K, for DMOP was 0.178 mM and for p-phenylenediamine was 1.69 mM. The pH optimum for pphenylenediamine oxidation was 3.5. Electrophoretically purified main polypeptide of laccase I was used to raise a specific antibody. Immunoblot analysis showed that whilst the antibody bound strongly to laccase I, no binding to laccase I1 was detectable.Antibody raised against pure laccase from Agaricus bisporus reacted with laccase I from Armillaria meffeu but not with laccase 11. The isolectric pH was 4.1 for laccase I and 3.3 for laccase 11.

Biochemical characterization and molecular evidence of a laccase from the bird’s nest fungus Cyathus bulleri

“…Optimum pH for laccase production by C. bulleri was 4.5 while fungal growth was more at pH 5.5. Similar observations have also been reported for Armillaria mellea (Rehman and Thurstan, 1992) while a bimodal pH graph was observed in case of Agaricus bisporus (Wood, 1980). The major loss of laccase activity after DEAE-cellulose chromatography could be due to high adsorption of the protein to the column, which was not completely eluted under conditions used in this study.…”

“…This is in contrast to Agaricus bisporus (Wood, 1980), Armillaria mellea (Rehman and Thurstan, 1992) and basidiomycete PM1 (Coll et al, 1993) where laccase production was linear to fungal growth. Laccase production by C. bulleri was aVected by both temperature and pH.…”

Purification and characterization of extracellular laccase from Pleurotus ostreatus

“…Many laccases have been reported to be excreted extracellularly as several isozymes (F~hraeus and Reinhammar, 1967;Cheung and Marshall, 1969;Morohoshi et al, 1987;Rehman and Thurston, 1992;Coil et al, 1993). However, the laccase of P. o s t r e a t u s K16-2 appeared as a single activity peak during all chromatography steps.…”

“…Among the ligninolytic enzymes of basidiomycetes, such as laccase, lignin peroxidase, and manganese peroxidase, we have studied laccases, which are found in major white-rot fungi capable of degrading lignin. Extracellular laccases have been purified and characterized from such fungi as Trametes versicolor (Cheung and Marshall, 1969;F~ihraeus and Reinhammar, 1967;Mosbach, 1963;Rogalski et al, 1990), Ganoderma lucidum (Kumari and Sirsi, 1972), Neurospora crassa (Froehner and Eriksson, 1974), Agaricus bisporus (Wood, 1980), Aspergillus nidulans (Kurtz and Champe, 1982), Schizophyllum commune (de Vries et al, 1986), Lentinus edodes (Kofujita et al, 1991 ), Panus tigrinus (Maltseva et al, 1991), Armillaria mellea (Rehman and Thurston, 1992), basidiomycete PM 1 (CECT 2971) (Coil et al, 1993), Cryptococcus neoformans (Williamson, 1994), Trametes villosa (Yaver et al, 1996), and Pycnoporus cinnabarinus (Eggert et al, 1996). Though these laccases have similar molecular weights and similar substrate specificities, they exhibit considerably different amino acid sequences.…”

The structure and function of fungal laccases

“…It is possible that here too, laccase is responsible for making a polyphenolic glue that sticks the hyphae together. This idea must be treated with some caution, however, as in laboratory liquid cultures of Armillaria mellea, although laccase activity accumulates in the medium co-ordinately with the onset of rhizomorph formation, manipulation of medium pH can substantially alter the amount of laccase activity that accumulates without having any significant effect on the mass of rhizomorph tissue formed (Rehman & Thurston, 1992). If laccase is required for rhizomorph synthesis therefore, the amount of laccase activity produced in the medium at the optimal pH for growth (pH 5) is in very considerable excess.…”