Studies on biodegration mechanism of cellulose by white-rotting fungi.

生长在纤维素上的白色腐木真菌黄孢Phanerochaete chrysosporium产生氧化还原酶纤维素二糖脱氢酶（CDH）。本研究探讨了CDH在纤维素降解中的作用。CDH的吸附只发生在纤维素的无定形表面，而不发生在纤维素的结晶表面，说明CDH的功能并不直接参与纤维素晶体结构的分解。在纤维素上生长的黄孢霉的静态培养中，大多数CDH活性在菌丝-纤维素复合体中观察到，而不是在培养液中释放。此外，CDH在纤维素表面的定位是专门针对纤维素酶作用形成的裂缝。这一观察结果很好地证明了纤维素酶与CDH在体内的协同作用。阐述了培养条件的优化，以获得大量的CDH。在培养基中添加犊牛血清可显著提高细胞外溶液中CDH的产量。为了探究这一现象的原因，我们建立了RT-PCR检测相应mRNA水平的方法。研究了纤维素生物水解酶I （CBH I）与CDH在微晶细菌纤维素分解过程中的相互作用。结果表明，CDH氧化还原系统通过解除产物抑制来增强CBH I的活性。研究了CDH中黄素和血红素辅助因子之间电子传递的pH依赖性机制。结果表明，这种现象是由于pH值对含黄素结构域的蛋白质结构的构象变化所引起的。

The white wood-rotting fungus Phanerochaete chrysosporium grown on cellulose produces the oxidoreductase cellobiose dehydrogenase (CDH). In this study, the role of CDH in cellulose degradation was investigated.Adsorption of CDH is occurred only on the amorphous surface of cellulose, but not on the crystalline surface, suggesting that the function of CDH is not directly responsible for decomposition of the crystalline structure of cellulose.In the static cultures of P.chrysosporium grown on cellulose, most of CDH activity is observed in the mycelium-cellulose complex, but not released in the culture solution. Moreover, localization of CDH on the cellulose surface is specialized to the crack formed by the action of cellulases. This observation is a good evicdence to suggest the cooperative function between cellulases and CDH in vivo.The optimization of cultivation condition has been elaborated to obtain a large amount of CDH.The addition of calf bovine serum in the culture medium is resulted in the significant enhancement of CDH production in the extracellular solution. To investigated the reason of this phenomenon, the method using RT-PCR for measurement of the corresponding mRNA level have been established.The interaction between cellobiohydrolase I (CBH I) and CDH in the decomposition of the microcrystalline bacterial cellulose was investigated The result demonstrated that the CDH redox system enhances CBH I activity by relieving the product inhibition.The mechanism on pH dependence of electron transportation between flavin and heme cofactors in CDH was investigated. The result suggests that this phenomenon is caused by the pH depending conformational changes of the protein structure of the flavin containing domain.