Proteolytic activities in two wood-decaying basidiomycete fungi, Serpula lacrymans and Coriolus versicolor

Abstract

Proteolytic enzyme activities of the wood-decaying basidiomycetes Serpula lacrymans and Coriolus versicolor, have been characterized using azocasein as substrate and by electrophoretic analysis with gelatin-containing polyacrylamide gels (gelatin-SDS-PAGE). In S. /acryinans# intracellular and extracellular azocaseinase activity was optimal at pH 5.6 and was inhibited by pepstatin A. Gelatin-SDS-PAGE revealed two highly active proteinases, 51 and 54 (apparent M, 65000 and 30000, respectively) and two less active enzymes, S2 and 53 (apparent M, 47000 and 43000, respectively). S1, the predominant intracellular proteinase, was present at all ages of the mycelium (tested up t o 3 months). It is active over a broad pH range, with highest activity around neutral pH. As 51 was partially inhibited by 1,lO-phenanthroline, the enzyme was considered t o be a metalloproteinase although EDTA and phosphoramidon had no effect. A proteinase apparently identical t o S 1 was also detected in the medium of older cultures. 54 is a pepstatinsensitive aspartic proteinase; its activity was highly pH-dependent and it was inactive in gelatin gels at pH 50 and above. S2 and 53 were identified as intracellular metalloproteinases, present in relatively young and growing cultures. They were distinct from S1 as they were inhibited by EDTA and phosphoramidon. During starvation-induced autolysis of S. lacrymans, proteinase 51 was the only enzyme present throughout (and the intracellular azocaseinase activity increased), which suggested a likely role of 51 in intrahyphal protein mobilization. 54 is more likely to play a part in extracellular digestion of protein. The azocaseinase activities of cultures of C. versicolor were optimal at pH 7-0 (intracellular) and pH 5 6 (extracellular). Mycelial extracts gave one major band of proteinase activity in gelatin gels, C1 (apparent M, 62-64000). Since the activity was sensitive to inhibitors of both serine and metalloproteinases, there may have been overlapping bands due t o enzymes of both types. Extracellular samples gave a more complex pattern, (five bands, C2-C6, M, 50000-100000). C2 and C4 are PMSF-sensitive proteinases, C5 and C6 are probably metalloproteinases, while C3, which was most active at pH 4.0, was unaffected by any of the inhibitors tested, including pepstatin A. No aspartic proteinase equivalent t o 5. lacrymans 54 appeared t o be produced by C. versicolor. From the information gained about the intracellular or extracellular location of these enzymes, and the conditions under which they are active, an in vivo role may be tentatively ascribed to some of them.

The role of wood decay fungi in the carbon and nitrogen dynamics of the forest floor

“…Starvation-induced proteinases have been investigated with a view to developing a suitable reporter for identifying sites in the mycelium where autolysis and regression are induced. A serine proteinase activity was identified in Serpula lacrymans with characteristics consistent with a role in mobilizing intracellular protein stores from cytoplasm, as well as a lysosomal-type activity optimal at low pH (Watkinson et al, 2001;Wadekar et al, 1995). Genes for serine proteinases of several cord-forming wood decay fungi have been cloned (Eastwood & Tlalka, unpublished; Eastwood & Higgins, unpublished) and show homology to other fungal serine proteinases with putative roles in internal protein mobilization (Kingsnorth et al, 2001).…”

Characteristics of intracellular peptidase and proteinase activities from the mycelium of a cord-forming wood decay fungus, Serpula lacrymans

“…Addition of chelators did not inhibit activity, which could be interpreted to mean that the metal has a stabilising rather than a catalytic role. Investigation of S. lacrymans intracellular proteases by SDS-PAGE and azocaseinase assay showed relatively low serine protease activity, although a serine protease sensitive to PMSF was found in the wood decay fungus Coriolus versicolor (Wadekar, North & Watkinson 1995). A serine proteinase characterised from Agaricus bisporus fruiting bodies (Burton et al 1993) was implicated in protein hydrolysis for mobilisation of amino acids from stipe to cap during maturation of harvested sporophores (Burton et al 1997), a similar function to that postulated for the S. lacrymans serine proteinase described here.…”

“…Ultimately, purification of enzymes could lead to development of a cellular reporter for an autolysis-inducing signal. Wadekar, North & Watkinson (1995) characterised four distinct proteinase activities of S. lacrymans by using SDS-PAGE to characterise bands in terms of inhibitor sensitivity and pH optima, and hydrolysis of azocasein to quantify net proteolytic activity in mycelial extracts. The purpose of using a range of synthetic substrates in this investigation was to differentiate the different protease activities present in mycelium, and to quantify and further characterise them, as a basis for investigating their physiological roles.…”

Nutritional characteristics and physiological studies on the growth and development of Phlebiopsis gigantea

“…These fungi have been alienated into brown, white, and soft-rot fungi, which are able to grow abundantly on wood which is a rich source of carbon, but deficient in nitrogen source. The growth of these fungi needs perceptive control of their nitrogen economy which is essential for proteinase activity in wood, and for the intracellular restructuring of nitrogen from mycelial protein (Wadekar et al, 1995) Phlebiopsis gigantea (syn. P. gigantea, P. gigantea) belongs to the phylum Basidiomycota is a white rot fungi.…”