| Budget Amount *help |
¥16,800,000 (Direct Cost: ¥16,800,000)
Fiscal Year 2004: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 2003: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 2002: ¥12,600,000 (Direct Cost: ¥12,600,000)
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| Research Abstract |
Fungal system of cellulose degradation has been considered to consist of merely extracellular hydrolytic enzymes. In this concept, cellulose is hydrolyzed to cellobiose by the synergistic action of various cellulases. After that, cellobiose is considered to convert in glucose by extracellular beta-glucosidases (BGL) before uptake of it in fungi as an energy source. However, the white-rot fungus Phanerochaete chrysosporium produces the extracellular flavohemoenzyme, cellobiose dehydrogenase (CDH), during cellulose degradation. In this work, importance of CDH in fungal system of cellulose degradation has been aware from both enzymatic and genetic investigations. CDH oxidizes cellobiose to cellobionolactone. In the presence of CDH, therefore, the catalytic specificity of the extracellular beta-glucosidase (BGL) should be examined not only for cellobiose, but also for cellobionolactone. BGL can hydrolyze both cellobiose and cellobionolactone, but cellobionolactone is hydrolyzed much more s
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lowly than cellobiose. In addition, the catalytic efficiency of CDH for cellobiose is extremely higher than that of BGL, indicating that BGL cannot use cellobiose as a substrate in the presence of CDH. Moreover, the effects of cellobiose on transcriptional response of CDH and BGL encoding genes (cdh and bgl) indicate that cdh is up-regulated in cellobiose metabolism, but not bgl. From all these observations, it is concluded that the extracellular BGL does not directly participate in the enzymatic system of cellulose degradation by P. chrysosporium. Moreover, using genome information of P. chrysosporium disclosed by Joint Genome Institute in US DoE, proteomic analysis of proteins secreted in the extracellular culture solution of the fungus during cellulose degradation was performed and 12 different enzymes on 2D-gel electrophoresis have been identified. In addition, using the genome information, cDNA of a novel extracellular cytochrome b562 has been cloned and expressed in the yeast Pichia pastoris as recombinant protein. Less
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