木质纤维高固酶解体系的糖化机制及其过程的分析与调控

Nowadays, renewable energy production, such as bioethanol, from lignocellulose has been attracted extensive attention due to energy and environment crisis. However, high solid enzymatic hydrolysis of pretreated lignocellulose with low efficiency is the major obstacle for the commercialization of bio-ethanol production. Therefore, to overcome this key technical bottleneck, the inhibition of enzymatic hydrolysis and pretreatment degradation products to cellulase main components (glucan endo, exo and cellobiase) were firstly studied in this project, which will be highly helpful to understand the mechanism of cellulase inhibition. Furthermore, the effects of cellulose consistency, the ratios of non-cellulose materials and water in solid substrate on cellulase non-effective adsorption were then examined. Based on these experimental researches, lumped cellulase adsorption kinetics was developed to explore the cellulase non-effective adsorption mechanism. Meanwhile, effects of above factors on cellulase penetration speed into cellulose cell walls were detected by fluorescent protein labeling method, making possible to identify the relationship between the properties of high solid enzymatic hydrolysis system and its hydrolysis efficiency. Finally, by systematic research on the relationships between rheological properties of high solid enzymatic hydrolysis system, cellulase invalid adsorption and product inhibition, comprehensive mechanism of lignocellulosic enzymatic hydrolysis at high solid consistency could be fully revealed. Thereafter, under the guidance of this mechanism, systematically analyzing and effectively controlling the process of high solid saccharification would be realized through some innovations, including the design of circulating bio-reactor coupling flash and ultrafiltration membrane, which can provide theoretical guidance and technical support to the commercialization of bio-ethanol production from woody biomass as soon as possible.

能源紧缺和环境问题致使木质纤维燃料乙醇的研究成为当今国际关注的热点。然而，高浓酶解效率低下是生物乙醇走向商业化的重要技术瓶颈。因此，本项目首先研究预处理降解产物和酶解产物对纤维素酶主要组分（葡聚糖内切、外切和纤维二糖酶）的抑制行为，揭示产物对纤维素酶的抑制机制；其次，考察纤维素、非纤维类物质和水在底物中的比例对纤维素酶主要组分在底物上产生无效吸附的影响，建立集总吸附动力学，探明无效吸附对酶水解效率的影响机制；同时，利用荧光蛋白标记考察上述因素对纤维素酶主要组分向纤维细胞入侵速率的影响，明确高固酶解体系特性与酶解效率之间的关系；最终，系统研究高固酶解体系流变特性、无效吸附和产物抑制等因素的相互关系，阐明木质纤维高固酶解体系的糖化机制。基于阐明的机制，利用耦合闪蒸和超滤膜的循环式生物反应器等手段，实现对木质纤维高固体系糖化过程的有效调控与强化，为生物乙醇尽快实现商业化提供理论指导和技术支持。

为降低化石能源消耗对环境造成的影响，生物质能源（如燃料乙醇）的开发及商业化已成为可再生能源产业中重要的研究领域。然而，为了降低燃料乙醇蒸发浓缩的能耗，提高固体纤维高浓酶解效率是解决上述问题的重要手段之一。鉴于此，采用酸性亚硫酸盐（简称SPORL）和热水（LHW）等化学法对不同原料（竹材、桉木等）进行预处理后，本项目对预处理固体基质和预处理液进行表征、对高浓酶解体系流变特性以及酶对固体基质吸附行为进行分析后发现，影响固体基质低高浓酶解的主要因素主要来自于三方面：（1）固体基质表面沉积的木质素以及固体基质酶解产物浓度；（2）预处理液中碳水化合物降解所产生的抑制物，主要包括糠醛（F）和羟甲基糠醛（HMF）；（3）在常规的酶水解时间范围内，由底物浓度提高而引起纤维素酶和水解产物的传质效率的降低足以制约其酶水解效率的提高，而影响传质效率的残余物主要是预处理过程中大量残留的非纤维素组分（半纤维素和木质素）。鉴于此，一方面，项目开发了无酸催化的醇-水（HBAW和EWS）两步预处理；另一方面，筛选出天然、廉价和高效的木质素屏蔽剂——脱脂豆粉中的大豆蛋白。无酸催化的醇-水（HBAW和EWS）两步预处理不仅可最大限度地对木质生物质原料进行组分清洁分离，预处理后固体基质中纤维素含量较高；同时，该预处理因无酸催化，所产生的碳水化合物降解产物浓度也较低，对发酵微生物的抑制作用较弱。结合上述HBAW和EWS两步预处理，结合超声处理，使固体基质内外表面的木质素尽可能暴露出来，然后采用筛选的大豆蛋白对木质素进行屏蔽；进一步基于同步及半同步糖化和发酵等手段消除酶解产物对纤维素酶的抑制，最终达到完全提高木质纤维高固酶解效率的目的。经上述处理，辅助添加少了半纤维素酶，当固体基质底物浓度为20%时，固体基质中纤维素的酶解转化率达到80%以上，葡萄糖总体回收率在75%以上，达到项目预期研究的目的。

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