堆肥菌株A.fumigatus Z5多糖单加氧酶功能及其木质纤维素分解增效机制研究

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.

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。

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。本研究从A.fumigatus Z5中克隆出多糖单加氧酶基因pmo-1，pmo-5，pmo-8，并利用毕赤酵母表达载体pPICZαA上的醇氧化酶AOX1的启动子和信号肽，使其在毕赤酵母X33中成功表达。结果表明，PMO1，PMO5，PMO8在酵母中均成功表达，其中PMO5蛋白的表达量最高，表达量为1.5 mg/mL，酶活为1.5U，蛋白分子量约为25kDa；利用亲和层析、离子色谱以及分子筛对PMO5蛋白进行了纯化，并获得了色谱纯的PMO5蛋白，达到蛋白结晶的要求；利用悬滴法对纯化的PMO5蛋白进行蛋白结晶条件的摸索，并获得了最佳的蛋白结晶条件（0.2 M Magnesium chloride hexahydrate，25% w/v Polyethylene glycol 3,350，0.1 M BIS-TRIS pH 6.0），在该结晶条件下，可以获得较好的PMO-5蛋白的晶体，达到X-ray衍射的要求；利用同步辐射光源对获得的蛋白晶体进行衍射分析，获得2 Å以下的衍射数据，PMO5蛋白中的大部分的原子都能够得到正确。异源表达的多糖单加氧酶PMO-5可以与二价铜离子结合的非常好，并形成蛋白金属离子复合物，是一种铜离子依赖型的多糖单加氧酶。多糖单加氧酶作用与溶胀纤维素的主要产物有C1 位氧化的纤维五糖（845）和纤维六糖（1007），非氧化型纤维四糖（667），非氧化型纤维五糖（829），非氧化型纤维六糖（991），非氧化型纤维七糖（1153）以及C1位置双氧化的纤维七糖（1229）；多糖单加氧酶的活性位点分析结果表明，多糖单加氧酶PMO5的活性位点为H86，Y175，而突变子H86-A，Y175-A的ITC分析结果表明，突变任何一个关键位点，PMO5都失去了与铜离子的结合能力，进一步验证了PMO5的活性中心为H86，Y175。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。