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

降解纤维素的真菌系统被认为只由胞外水解酶组成。在这个概念中，纤维素在各种纤维素酶的协同作用下被水解成纤维二糖。在此之后，纤维二糖被认为是被细胞外β-葡萄糖苷酶(BGL)转化为葡萄糖，然后才被真菌作为能源吸收。然而，白腐菌黄孢原毛平革菌在降解纤维素的过程中会产生胞外黄素酶，即纤维二糖脱氢酶(CDH)。在这项工作中，CDH在降解纤维素的真菌系统中的重要性已经从酶学和遗传学研究中意识到。CDH将纤维二糖氧化为纤维二内酯。因此，在CDH存在的情况下，胞外β-葡萄糖苷酶(BGL)的催化专一性不仅要检测纤维二糖，还要检测纤维二内酯。…对纤维二糖和纤维二内酯均有较强的水解力，但对纤维二糖的水解力更强比纤维二元糖更卑微。此外，CDH对纤维二糖的催化效率远远高于BGL，说明在CDH存在下，BGL不能以纤维二糖为底物。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。这些结果表明，胞外BGL并不直接参与黄孢原毛霉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布的黄孢假单胞菌基因组信息，对该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-GELE上鉴定了12种不同的酶。此外，利用基因组信息，克隆了一种新的胞外细胞色素b562基因，并在毕赤酵母中以重组蛋白的形式表达。较少

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