Investigation on function and gene expression of multiple cellulolytic enzymes from filamentous fungi.

真菌纤维素降解系统一直被认为仅由胞外水解酶组成。在这个概念中，纤维素通过各种纤维素酶的协同作用水解成纤维二糖。之后，纤维二糖被认为在真菌中作为能量来源摄取之前通过细胞外β-葡糖苷酶（BGL）转化为葡萄糖。然而，白腐真菌黄孢原毛平革菌在纤维素降解过程中产生胞外黄血红素酶，纤维二糖脱氢酶（CDH）。在这项工作中，CDH的重要性，真菌系统的纤维素降解已意识到从酶和遗传学的调查。CDH将纤维二糖氧化为纤维生物内酯。因此，在CDH的存在下，细胞外β-葡糖苷酶（BGL）的催化特异性不仅应检查纤维二糖，而且还应检查纤维二糖内酯。BGL能水解纤维二糖和纤维二糖内酯，但纤维二糖内酯水解得更多。 ...更多信息 比纤维二糖低此外，CDH对纤维二糖的催化效率远高于BGL，表明BGL在CDH存在下不能利用纤维二糖作为底物。此外，纤维二糖对CDH和BGL编码基因（cdh和bgl）的转录响应的影响表明，cdh在纤维二糖代谢中上调，而bgl不上调。从所有这些观察，可以得出结论，胞外BGL不直接参与的酶系统的纤维素降解的P. chrysosporium。此外，利用美国能源部联合基因组研究所公布的黄孢原毛平革菌基因组信息，对该菌胞外培养液中纤维素降解过程中分泌的蛋白质进行了蛋白质组学分析，并在二维凝胶电泳上鉴定出12种不同的酶。此外，利用基因组信息，一个新的胞外细胞色素b562的cDNA已被克隆，并在酵母中表达的重组蛋白。少

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 … More 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