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Extracellular enzyme activities of laccase and cellulase and their transcriptional regulation were investigated at various growth stages in a sawdust-based substrate for Lentinula 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.Key Words cxtracellular enzyme; fruit body; Lentinula edodes; mRNA; transcriptional regulation.Lentinula edodes (Berk.) Pegler (shiitake) is the second most important cultivated mushroom in the world. Production in Japan in 1997 was estimated at 250,000 metric tons and valued 171 billion yen (US 1.6 billion). In Japan, indoor cultivation on a hardwood sawdustbased substrates is rapidly replacing traditional cultivation in bed-logs in the open air. The sawdust-based cultivation method for L. edodes has advantages such as a shorter cultivation period and better manipulation of flushing (fruit body formation) by control of environmental conditions. This method now accounts for about a half of all production and is expected to become the major cultivation method.The major nutritional components of the growth medium comprise the various lignocellulose polymers from sawdust cells and some nutritional additives. Several extracellular enzyme activities of L. edodes have been detected in the sawdust-based substrate. These include laccase, peroxidases, cellulases, hemicellulases, proteases, and various glycosidases. Biodegradation is achieved by secretion of a set of extracellular enzymes. Laccase and cellulase especially have been studied in depth since their activities revealed rapid changes during fruit body development in L. edodes (Leatham and Stahmann, 1981; Leatham, 1985;Ohga, 1992) and in Agaricus bisporus (Turner, 1974;Turner et al., 1975; * Corresponding author. E-rnail: ohgasfor(~rnbox.nc.kyushu-u.ac.jp Wood and Goodenough, 1977;Manning and Wood, 1983;Claydon et al. 1988). The growth of the mycelium and the production of fruit bodies are predominantly dependent on the efficient utilization by the extracellular enzymes of the lignocellulose polymers of the sawdustbased substrate. Increase in utilization of substrate is a target for strain manipulation, and this could be achieved by regulation of extracellular enzyme level, using strain selection methods including genetic eng...

A screening of wood-rotting basidiomycete fungi was conducted for glucose 1-oxidase (GOD) and laccase (LAC) production as well as for ligninolytic activity measured by a Rhemazol reaction. The results showed that genera rich in GOD are lignin degraders as well as effective producers of extracellular LAC. The fungi poor in GOD neither showed LAC, nor ligninolytic activity. The Björkman lignin and 3 phenolic compounds, hydroquinone and syringic and vanillic acids, were tested on the sequential activity of LAC and GOD. In the presence of LAC, quinoid intermediates formed from Björkman lignin and phenolic compounds were observed. The addition of GOD caused a diminution of the quinone level. During incubation of Björkman lignin with LAC and GOD depolymerization occurred, and in the experiments omitting GOD the quantities of low molecular products were markedly lower. Consequently, the consecutive ping-pong activity of LAC and GOD reduced the polymerization and improved the efficiency of depolymerization processes.

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