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

纤维素降解真菌系统被认为仅仅由细胞外水解酶组成。在这个概念中，纤维素通过各种纤维素酶的协同作用被水解成纤维素二糖。在此之后，纤维素二糖被认为在真菌将其作为能量来源吸收之前，通过细胞外β -葡萄糖苷酶（BGL）转化为葡萄糖。然而，白腐菌Phanerochaete chrysosporium在纤维素降解过程中产生细胞外黄血酶，即纤维素二糖脱氢酶（CDH）。在这项工作中，CDH在纤维素降解真菌系统中的重要性已经从酶和遗传学的研究中意识到。CDH将纤维素二糖氧化为纤维素内酯。因此，在CDH存在的情况下，细胞外β -葡萄糖苷酶（BGL）的催化特异性不仅应该检查纤维二糖，还应该检查纤维生物内酯。BGL既能水解纤维素二糖，也能水解纤维素内酯，但纤维素内酯的水解速率远低于纤维素二糖。此外，CDH对纤维素二糖的催化效率远高于BGL，说明在CDH存在的情况下，BGL不能将纤维素二糖作为底物。此外，纤维二糖对CDH和BGL编码基因（CDH和BGL）转录反应的影响表明，CDH在纤维二糖代谢中上调，而BGL不上调。综上所述，胞外BGL不直接参与黄孢假单胞菌降解纤维素的酶系统。此外，利用美国能源部联合基因组研究所公开的P. chrysosporium基因组信息，对真菌在纤维素降解过程中胞外培养液中分泌的蛋白质进行了蛋白质组学分析，并在2d凝胶电泳上鉴定出12种不同的酶。此外，利用基因组信息克隆了一种新的细胞外细胞色素b562的cDNA，并在酵母毕赤酵母中作为重组蛋白表达。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