Christopher F. Thurston

Accumulation of serine proteinase in senescent sporophores of the cultivated mushroom, Agaricus bisporus

Serine proteinase activity was assayed during sporophore maturation and postharvest senescence in Agaricus bisporus. Serine proteinase accumulated largely in stipe tissue early during postharvest storage and in the later stages of maturation of unpicked sporophores. Western blot staining correlated with enzyme activity indicating de novo synthesis. The enzyme could not be detected by Western blotting in freshly harvested sporophores at an early maturation stage. Small increases in activity were detected in the cap tissue during the final stages of senescence, postharvest and in unpicked sporophores. The distribution of the serine proteinase in senescent sporophore tissues (high in stipe, low in cap) was confirmed by histochemical and immunologically stained tissue prints.

Transcriptional regulation of laccase and cellulase genes in the mycelium of Agaricus bisporus during fruit body development on a solid substrate

The cultivated mushroom Agaricus bisporus was grown and fruited in wheat straw compost. RNA was extracted from samples of mycelium growing in compost during colonization and fruit body development. Laccase gene (combined lcc1 and lcc2) expression and cel3 gene (cellobiohydrolase) expression were determined by Northern blot analysis and by competitive RT-PCR. The level of laccase transcripts was highest in colonized compost (prior to the onset of fruiting), declined during the period of fruit body enlargement and increased again after harvesting and during the second flush of fruit body production. No transcripts of the cel3 gene were detectable in colonized or pre-fruiting cultures, but the level became detectable and rose to a maximum at the veil-break stage of fruiting, declined after harvesting and rose again during the second flush. Gene expression for laccase and cellobiohydrolase correlated with the known changes of laccase and cellulase enzyme activities during the fruiting life cycle.

Purification of laccase I from Armillaria mellea

Laccase activity accumulated during rhizomorph development in malt extract cultures of Armillaria mellea. 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 r 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 m for DMOP was 0·178 mM and for p-phenylenediamine was 1·69 mM. The pH optimum for p-phenylenediamine 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 II was detectable. Antibody raised against pure laccase from Agaricus bisporus reacted with laccase I from Armillaria mellea but not with laccase II. The isolectric pH was 4.1 for laccase I and 3·3 for laccase II.

Purification and characterization of a serine proteinase from senescent sporophores of the commercial mushroom Agaricus bisporus

A proteinase has been purified from the stipes of senescent sporophores of the mushroom Agaricus bisporus. The proteinase was inhibited by PMSF. It has a broad pH optimum, 6.5-11.5, and a narrow substrate specificity, requiring both a hydrophobic amino acid in the P1 position and a minimum peptide chain length. The apparent molecular mass of the proteinase was 27 kDa when determined by SDS-PAGE and 14.1 kDa when measured by gel filtration. The isoelectric point of the proteinase was 9.0. Polyclonal antibodies have been raised to the proteinase. The proteinase from A. bisporus has similar properties to, and 60% N-terminal sequence identity with, proteinase K from the fungus Tritirachium album.

Enzyme production by Mycena galopus mycelium in artificial media and in Picea sitchensis F1 horizon needle litter

Mycena galopus is among the most important leaf litter decomposers in UK coniferous and angiosperm woodlands, having the potential to utilise all the major constituents of plant litter. Even so, the enzyme or combination of enzymes produced by M. galopus responsible for lignin depolymerisation was previously unknown. A range of media from liquid and semi-solid cultures to more natural substrata was tested to determine whether laccase was produced by an isolate of M. galopus, M9053. Malt extract liquid medium (MEL) with 2,5-xylidine favoured laccase production as compared with the same medium containing the inducers veratryl alcohol, veratryl aldehyde, veratric acid, homoveratric acid, vanillic acid or p-anisic acid. A semi-solid medium of cereal bran in phosphate buffer and a solid medium of Picea sitchensis F1 horizon needle litter were also not as effective as MEL with 2,5-xylidine as an inducer. Compared with six other isolates of the same species grown in MEL without inducers, M9053 exhibited rates of laccase activity fairly typical for M. galopus. An isolate from a dark coloured basidiome of M. galopus, but not var. nigra, exhibited the greatest activity while var. candida showed relatively low laccase activity. Marasmius androsaceus exhibited peak laccase production several days later than M. galopus. In addition, a manganese-dependent peroxidase that was responsible for 15% (in MEL culture fluid) and 39% (in needle litter extract III) of ligninolytic activity was produced by M9053. A further peroxidase was found to be the major ligninolytic constituent in MEL extracts (53%), and had a similar contribution to total activity (29%) as laccase (32%) in needle litter fraction III. Mycena galopus produced water- and buffer-extractable mannases and xylanases when grown on needle litter.

Regulation of transcription of the cell gene in Agaricus bisporus

Northern analysis showed that accumulation of Agaricus bisporus cell mRNA was regulated by two independent mechanisms: (i) induction by cellulose; and (ii) repression by glucose and other sugars, Isolated A. bisporus nuclei were transcriptionally active. Nuclei Isolated from cellulose‐grown mycelium synthesized six times more cell mRNA than nuclei from glucose‐grown mycelium. The start point of transcription (tsp) was identified by primer extension and S1 nuclease analysis. Putative glucose‐, and cAMP‐responsive elements as well as regions with homology to promoter regions of other fungal cellulase genes were detected both upstream and downstream from the tsp of the cel1 gene.

Extracellular proteinases from the mycelium of the cultivated mushroom Agaricus bisporus

The edible mushroom, Agaricus bisporus, when cultivated commercially derives its nitrogen from the proteins present in compost, much of which is firmly bound to the lignin-humic complex. The proteinases involved in mobilization of nitrogen were studied in both the filtrates of liquid culture and mushroom compost. Proteinase activities were detected in liquid culture filtrates with a range of substrate specificities. One activity with chymotrypsin-like specificity was found by Western blotting to be the 27 kDa serine proteinase previously purified from senescent sporophores. Serine proteinase activity and level in the filtrates rapidly increased during mycelial growth on liquid medium containing humic fraction (extracted from mushroom compost) as sole nitrogen source. Activity and level remained low in filtrates of cultures grown on glutamate or casein. Non-denaturing gel analysis revealed the serine proteinase to be the most prominent proteolytic activity in compost filtrates. Regulation of the activity in compost was indicated by a doubling during the early stages of sporophore growth.

Transcriptional regulation of laccase and cellulase in relation to fruit body formation in the mycelium of Lentinula edodes on a sawdust-based substrate.

Extracellular enzyme activities of laccase and cellulase and their transcriptional regulation were investigated at various growth stages in a sawdust-based substrate forLentinula edodes. Changes of laccase and cellulase activities revealed a clear relationship with fruit body development stages. Laccase and cellulase activities were regulated at the level of gene transcription. The level of laccase mRNA was maximal at the fully colonized stage and declined during fruit body development. Cellulase mRNA began to accumulate at the pin (miniature fruit bodies) formation stage. Cellulase mRNA transcripts were maximally expressed at the veil-break stage of fruit body development. This tendency was clearer in the fruiting cultures with the wide-range-weather strains than in non-fruiting cold-weather strains. Transcription of laccase and cellulase genes was also affected by the water conditions of the sawdust-based substrate. Primordia initiation occurred when the water potential of the medium was high for rapid mRNA transcription by the mycelium.

Correlation of exons with functional domains and folding regions in a cellulase from Agaricus bisporus

Abstract  The cellulase gene cel3 has been isolated from Agaricus bisporus and sequenced. The 5′-end of the cel3 transcript was determined by primer extension and S1 nuclease protection. Putative regulatory elements have been identified in the cel3 promoter and 3′-untranslated regions. The cel3 coding region is interrupted by six short introns, two of which separate the coding regions for the three modules in the CEL3 protein: cellulose-binding domain, linker region, and catalytic domain. Three of the remaining four introns are positioned in regions coding for loops between structural moieties. Intron positions are conserved between cel3 and other related cellulases.

The molecular genetics of cultivated mushrooms

The types, economic significance and methods of production of the principal cultivated mushrooms are described in outline. These organisms are all less than ideal for conventional genetic analysis and breeding, so molecular methods afford a particular opportunity to advance our understanding of their biology and potentially give the prospect of improvement by gene manipulation. The sequences described are limited to those found in GenBank by August 1999. The gene sequences isolated from the white button mushroom Agaricus bisporus, the shiitake Lentinula edodes, the oyster mushrooms Pleurotus spp., the paddy straw mushroom Volvariella volvacea and the enotake Flammulina velutipes are described. The largest group are genes from A. bisporus, which includes 29 for intracellular proteins and 12 for secreted proteins. In comparison, only a total of 26 sequences can be reported for the other cultivated species. A. bisporus is also the only cultivated species for which molecular karyotyping is already supported by reliable markers for all 13 of its chromosomes.