利用生物质资源生产生物燃料和大宗化学品，是解决当前能源和环境危机最重要的方式之一。作为世界上唯一可大规模再生的资源，生物质资源，尤其是木质纤维素原料并未被完全的开发利用。其中，纤维素酶的成本过高成为限制木质纤维素综合利用，尤其是限制木质纤维素转化乙醇技术工业化的关键瓶颈。如何提高木质纤维素的降解效率，减少纤维素酶用量成为了关键问题。作为一种重要的纤维素酶生产菌株，草酸青霉分泌的纤维素酶有酶系较全，稳定性较高等优点，是世界范围内该领域研究最为广泛的菌株之一。本实验室在上世纪筛选获得了能够稳定生产纤维素酶的草酸青霉菌株，通过几十年的诱变及改造，其生产分泌的纤维素酶能够有效地降解木质纤维素，但是距离工业化生产仍有不小的距离。如何提高草酸青霉纤维素酶对木质纤维素的降解效率，尤其是高固含量下的降解效率，减少纤维素酶用量，从而降低生产成本，成为纤维素乙醇工业化的关键。基于以上背景，本论文以草酸青霉纤维素酶为主要研究对象，分别在工艺因素、纤维素酶系组成、纤维素酶辅助因子和单酶组分性质等方面，对草酸青霉纤维素酶降解能力的不足进行了分析，并提出对应的解决策略，以期提高其对木质纤维素原料的降解效率。本论文的主要研究内容和结果如下： 1.传质限制在高固含量条件下对纤维素转化率的影响及解决策略 在高固含量下进行木质纤维素酶水解有非常大的优势。但是，随着反应体系中固含量的提高，纤维素转化率会逐渐降低。其中，随着固含量提高而出现的传质限制可能是引起这一效应的重要因素。使用脱木素木糖渣做底物，当固含量上升到20％左右时，传质限制取代葡萄糖抑制，成为对酶水解最主要的抑制因素。木质纤维素的液化能够有效的促进其糖化效率的提高。高固含量下的传质效率影响因素包括机械因素和原料性质。机械因素包括反应器装料量和搅拌速度。当固含量超过15％时，传质效率与装料量和搅拌速度紧密相关。酶水解所采用的原料性质对传质效率影响很大。同等固含量下，不同预处理加工后的玉米秸秆在酶水解时的粘度差异很大，进而导致水解效率的差异。为了解决传质限制的问题，本研究分别尝试采用了预混法、补料法和新型卧式反应器来改善传质效果，发现酶水解的固含量在15％以上时，其纤维素转化率均得到有效提高。其中，卧式反应器的提升效率最为明显。 2.原料特异性木质纤维素酶酶系组成优化及机理研究 纤维素酶是一种复合酶系，包括内切葡聚糖酶(EG)，外切葡聚糖酶(纤维二糖水解酶，CBH)和β-葡萄糖苷酶(BG)等。纤维素的酶水解就是通过几种酶协同作用，共同将其降解为可溶性的葡萄糖的过程。不同的原料对纤维素酶系组成的偏好有所不同。选择草酸青霉纤维素酶中含量较高的三种酶，EGII、CBHI和BGI，采用混料设计的方式分析了针对不同预处理纤维素原料在不同固含量下的最佳酶配比。结果发现，预处理方式对高效降解所需最佳酶配比有非常大的影响。酸处理和亚硫酸铵处理分别需要更多的CBHI和EGII。随着固含量的提高，最佳酶配比也发生显著改变。上述差异主要与预处理后原料的性质，如残留的木素含量和纤维素的结晶形态有关。利用最佳配比的酶系进行纤维素原料的降解，纤维素转化率能得到显著的提高。基于以上理论分析，发现经不同预处理后的纤维素原料需要不同的产酶菌株改造策略，才能达到最佳的降解效率。 3.化合物和辅助协同蛋白对木质纤维素降解的影响研究 在高固含量的酶水解中，需要比低固含量更高的酶加量才能达到预期的转化效率。为了降低酶解过程中纤维素酶加量，我们将四种化合物和三种辅助蛋白分别添加进酶解体系中，分析了其对纤维素转化的影响及酶用量降低的效果。结果发现，预处理后原料性质和固含量对几种添加物的效果有非常大的影响。对于酸处理后脱去了木聚糖，但残留木素较多的木糖渣来说，PEG 6000的效果最为显著，而且对纤维素转化率的促进效果几乎不受固含量的影响。在20％固含量的木糖渣的酶水解中，添加PEG 6000可以降低大约一半左右的酶用量。裂解性多糖单加氧酶对亚硫酸铵处理原料的作用效果最为显著，在20％固含量酶水解时也可以降低约一半的酶用量。通过几种添加物对纯纤维素和不同复杂木质纤维素原料酶水解的作用，发现预处理方式对其促进效果影响显著。 4.草酸青霉纤维素酶降解结晶纤维素限制因素的解析及改进策略 工业纤维素酶大部分是由里氏木霉生产的。草酸青霉纤维素酶在木糖渣的降解中，产糖速率和最终纤维素转化率都明显低于里氏木霉纤维素酶。但是，在对亚硫酸铵预处理小麦秸秆的酶解中，两者的效率基本一致。对于主要含有结晶I型纤维素的微晶纤维素来说，两种纤维素酶的降解效率也明显不同，其中有56％的纤维素难以被草酸青霉纤维素酶降解。研究显示，两种纤维素酶降解效率的差异，取决于两酶系中CBHI降解结晶纤维素效率的差异。通过功能域置换发现，里氏木霉CBHI中的非催化结构区域(linker+CBM)决定了其降解结晶纤维素的高效性。两者非催化结构区域之间的差异也部分解释了里氏木霉纤维素酶能够相对高效地降解天然结晶纤维素原料的现象，揭示了非催化结构区域在决定纤维素酶降解能力中起到重要的作用。加入来源于里氏木霉的CBHI，能够有效提升草酸青霉纤维素酶系降解结晶纤维素的能力。 关键词：草酸青霉；纤维素酶；传质限制；配比优化；添加物；纤维素结合结构域。

The use of biomass to produce biofuels and bulk chemicals is one of the most important ways to solve the current energy and environmental crisis. As the only renewable resource in large-scale, biomass resources, especially lignocellulosic materials, have not been fully exploited. Among them, the high cost of cellulase becomes the key bottleneck to restrict the comprehensive utilization of lignocellulose, especially the industrial conversion of lignocellulose to ethanol. How to improve the degradation efficiency of lignocellulose and reduce the dosage of cellulase has become the key problem. As an important cellulase-producing strain, cellulase from Penicillium oxalicum has many advantages, such as complete enzyme system and high stability. It is one of the most widely studied strains in this research field in the world. Cellulase production from Penicillium oxalicum has been studied since 1980s, through mutagenesis and genetic engineering. Lignocelluloses could be degraded with cellulase efficiently, but not fulfilled the demand of industrialized production. How to improve the biodegradation efficiency of cellulase, especially at high solid content, and how to reduce cellulase dosage and process cost, are critical to the industrialization of cellulose ethanol. Based on the background above, cellulase from P.oxalicum was taken as the main research object. In this study, we analyzed respectively process optimization, composition of cellulase complex, cellulose non-degrading enzymes and degradation ability of enzyme components. The solving strategies were developed to improve the degradation efficiency of lignocellulosic feedstocks. The main research contents and results are as follows: 1.Identifying and overcoming the effect of mass transfer limitation on decreased yield in enzymatic hydrolysis of lignocellulose at high solid concentrations There are lots of advantages with enzymatic hydrolysis at high solids concentrations such as higher product concentration and economic competitiveness. However, cellulose conversion decreased significantly with increasing solid concentrations during enzymatic hydrolysis of insoluble lignocellulosic materials. Mass transfer limitation due to high viscosities at high solid loadings instead of glucose inhibition was shown to be the potential determining factor in decreased yield at the solid concentration of delignified corncob residue up to 20% dry matter (DM) content. Two mass transfer efficiency-related factors, mixing speed and flask filling, were shown to correlate closely with cellulose conversions at solid loadings higher than 15% DM. The role of substrate characteristics in mass transfer performance was also significant, which was revealed by the saccharification of two com stover substrates with different pretreatment methods at the same solid loading. Several approaches including premix, fed-batch operation, and use of horizontal rotating reactor were shown to be valid in facilitating cellulose conversion via improving mass transfer efficiency at solid concentrations higher than 15% DM. The horizontal rotating reactor was proved to be more efficient. 2.Efficient enzymatic hydrolysis of cellulosic material with different pretreatment at high solids concentrations via optimized enzyme components Cellulase complex include endoglucanase (EG), cellobiohydrolase (CBH) and β-glucosidase (BG). To improve the enzymatic hydrolytic efficiency of different cellulosic materials, especially at high solids concentrations, the mixture design was used to evaluate the optimal mixture of EGII, CBHI and BGI according to the maximum cellulose conversions. The final optimal mixture was distinctly different between substrates from different pretreatments, which was aggravated during hydrolysis at high solids concentration. CBHI and EGII was preponderant during hydrolysis of cellulosic materials with acid and sulfite pretreatment respectively, especially at high solids concentrations. This was resulted from the nonproductive adsorption by lignin in acid-pretreated material and change of crystalline structure in sulfite pretreated material. With the optimal mixture combination, the cellulose conversions could be significantly improved, especially at high solids concentrations. Replacement test showed that overexpression of the most critical components could effectively improve the hydrolytic efficiency and alleviate solids effect at high solids concentrations. 3.Addition of chemicals and auxiliary proteins at high solid loading to improve enzymatic efficiency on substrates with different pretreatments High dosage of enzyme is required to achieve effective lignocellulose hydrolysis, especially at high solids loadings, which is a significant barrier to large-scale bioconversion of lignocellulose to fuels and chemicals. Here, we screened four chemical additives and three accessory proteins for their effects on the enzymatic hydrolysis of various lignocellulosic materials by cellulase preparation from P. oxalicum. The effects were found to be highly dependent on the composition and solids loadings of substrates. For xylan-extracted lignin-rich corncob residue, the enhancing effect of PEG 6000 was most pronounced and negligibly affected by solids content. More than half of enzyme demand could be reduced with PEG 6000 addition during the hydrolysis at 20% DM. Lytic polysaccharide monooxygenase enhanced the hydrolysis of ammonium sulfite wheat straw pulp, and its addition reduced about half of protein demand at the solids loading of 20% DM. Supplementation of the additives in the hydrolysis of pure cellulose and complex lignocellulosic materials revealed that their effects are tightly linked to pretreatment strategies. 4.Analysis and overcoming of limiting factors for the degradation of crystalline cellulose by cellulase from P.oxalicum The low efficiency of cellulases is a major bottleneck for industrial bioconversion of lignocellulosic materials. Commercial cellulase preparations are mainly produced by the fungus Trichoderma reesei. The cellulase mixturesecreted by T. reesei showed both higher rate and greater final yield in the hydrolysis of corncob residue than that of P. oxalicum, while the difference was negligible on ammonium sulfite wheat straw pulp. The two cellulase preparations also haddifferent performances on the hydrolysis of Avicel which consists of crystalline cellulose I, with about 56% of Avicel hard to degrade by P. oxalicum cellulases. CBHI was identified as a determinant of this disparity of hydrolysis efficiency between cellulase mixtures. The addition of T. reesei CBHI, but not those from P. oxalicum and Aspergillus niger, efficiently continued the hydrolysis of partially hydrolyzed Avicel that was less reactive to P. oxalicum cellulases. Further domain exchange experiment attributed the superior hydrolyzing and binding abilities ofT. reesei CBHI on Avicel to its interdomain linker and cellulose-binding domain CBM1. The results in part explained the superior performance of T. reesei cellulases on the degradation of native crystalline cellulose, and highlighted the important role of cellulose-binding region in determining the degree of hydrolysis by cellulase mixtures. Keywords: Penicillium oxalicum; Cellulase; Mass transfer limitation; Cellulase composition optimization; Additives; Cellulose binding domain.