木质纤维素分解是堆肥过程中最重要的生化反应，该过程主要通过微生物分泌的木质纤维酶的水解完成，木质纤维素酶活力大小将直接影响堆肥效率。多糖单加氧酶（PMOs）作为糖苷水解酶家族（GH61）成员之一，在木质纤维素分解过程中发挥了重要作用。本研究拟采用荧光定量PCR方法研究菌株A.fumigatus Z5中GH61家族基因及碳源代谢调控因子creA、aceI、xynL的转录差异；通过构建酵母表达载体，异源表达菌株Z5中pmos基因，经亲和层析纯化获得重组PMOs蛋白并研究其酶学特性；以pPK2和pDHt/SK 质粒为骨架构建二元载体pDHt/pmos::hph，定点敲除pmos基因并验证其功能；基于电子顺磁共振、等温滴定量热技术，研究PMOs识别与绑定纤维素过程。本研究将有助于阐明PMOs的功能，揭示催化木质纤维素分解及与木质纤维素酶协同作用提高催化效率的机制，为提高堆肥效率提供理论依据。

Degradation of lignocelluloses is the major biochemical reaction during the composting process, which is catalyzed by various lignocellulases secreted by microorganisms, and there is a positive correlation between the lignocellulase activity and composting efficiency. Polysaccharide monooxygenase (PMOs) being defined as a glycoside hydrolase family member (GH61) play an important role during lignocellulose degradation process. In order to reveal the regulation relationship between GH61 genes and carbon metabolic regulation factors, fluorescence quantitative PCR method will be used to quantify the transcriptional level of different GH61 genes and the carbon metabolic regulation factors creA, aceI, xynl. The highest expression GH61 gene of A.fumigatus Z5 with the PMOs function module will be expressed in Pichia pastoris X33 under methanol induction condition. The recombinant PMOs will be purified by use nickel column affinity chromatography, and the basic enzymatic characteristics of the recombinant PMOs will be determined after being identified by LC-MS/MS. Gene disruption by homologous recombination was performed with the plasmid pDHt/pmos::hph, and this plasmid was constructed by inserting a 3.2-kb SacI/ApaI fragment from pPK2 containing the hph gene flanked by pmos sequence, into the SacI/ApaI-restricted plasmid pDHt/SK. Analysis of the extracellular proteins secreted by wide type and the mutant will be carried out by two-dimensional difference gel electrophoresis (2D-DIGE). Prediction of PMOs possible coordination structure and oxidation potential will be carried out by electron paramagnetic resonance technology (EPR). The isothermal titration calorimetry technology (ITC) will be applied to reveal the cellulose recognition and binding process by PMOs. The surface plasmon resonance technology (SPR) will be used to study the interactive process between PMOs and cellobiose dehydrogenase (CDH), which will help to shed light on the mechanism driving the lignocellulose decomposition. The lignocellulose degradation capacity comparation of the wild type and mutant will be carried out under pure culture conditions by use rice straw as sole carbon source, and sampling will occur at 14 and 28 days after inoculation. The crystallinity index of various samples will detected by X-ray analysis, and the changes in surface morphology of various samples taken at different time points will be carried out by use of atomic force microscope. This study will help to elucidate the function of PMOs in detail, reveal the mechanism of improved catalytic efficiency by the synergistic effect of lignocellulose degradation with cellulose, and in turn provide a theoretical basis for composting efficiency enhancement.