高底物浓度木质纤维原料三段酶水解技术及其机制的研究

The enzymatic hydrolysis has been identified as a major technical bottleneck in the entire lignocellulose-to-ethanol bioconversion process. In general, it is likely that increasing substrate loading in the hydrolysis will lead to increased sugar concentration and higher final ethanol content after fermentation. This approach will bring economic savings to the bioconversion process, such as reducing capital and operational cost for hydrolysis and fermentation, and minimizing energy consumption during distillation/evaporation and other downstream processes. However, the hydrolysis time and the coordinated action of cellulases always were overlooked during the hydrolysis of high solid concentration, and the enzymatic hydrolysis has not still get the theoretical foundations. Residue from plant extraction, be rich in Yunnan province, was as feedstock, and dilute acid pretreatment and three-stage hydrolysis that the substrate concentration was raised to 30% by fed-batch, were carried out in our research. The product of enzyme-substrate compound, the structure of residues produced enzymatic hydrolysis, the end-product inhibition were the main metewand during the three-stage hydrolysis. To further exploit this technology, the mechanism of three-stage enzymatic hydrolysis at high substrate concentration was showed by the kinetic equation, deduced by isothermal adsorption equation and the kinetics equation of hydrolytic reaction. On the basis of this, the integrated technology of dilute acid pretreatment and three-stage hydrolysis was built. If our work was financially supported, the result will bring the biomass conversion research a step closer to industrial implementation.

纤维素酶水解是木质纤维原料制备燃料乙醇的技术瓶颈。高底物浓度纤维酶水解可提高体系中糖浓度和乙醇得率，进而降低纤维素乙醇的生产成本。但过程中水解时间与纤维酶各组分间的协同水解关系没能引起足够重视，无法为建立高效纤维素酶水解技术提供全面的理论支持。本项目以云南省丰富的植物提取剩余物为研究对象，通过分批补料方式使底物浓度提高至30%，采用稀硫酸预处理和纤维素酶三段水解技术，以酶与底物复合物的形成、酶解残渣的结构特性和产物反馈抑制为切入点，并根据等温吸附方程和表面水解动力学方程构建高底物浓度下三段酶水解的动力学方程，揭示高底物浓度下纤维酶三段水解的机制。在此基础上，建立稀酸预处理和高底物浓度三段酶水解的集成技术。研究成果可以建立酶水解性能高的预处理工艺和低成本、高效率纤维素酶水解的技术方法和工艺，为工业放大提供理论依据和指导原则。

高底物浓度纤维酶水解可提高体系中糖浓度和乙醇得率，进而降低纤维素乙醇的生产成本。本项目以木质纤维素为研究对象，采用蒸汽爆破、稀硫酸预处理和稀碱-芬顿试剂预处理的木质纤维素作为酶水解的底物，通过分批补料的方式使水解底物浓度从15％提高至30％。以酶与底物复合物的形成、酶解残渣的结构特性、产物反馈抑制和木质素对纤维素酶水解效率的影响为切入点，并根据等温吸附方程和表面水解动力学方程构建高底物浓度下分段酶水解的动力学方程，揭示高底物浓度下纤维酶分段水解的机制。在此基础上，建立稀硫酸预处理及蒸汽爆破预处理和高底物浓度分段酶水解的集成技术。目前正式发表研究论文5篇, 其中SCI 期刊1篇 （JCR 1区，影响因子5.25），中文核心期刊3篇，发明专利1项；另有中文核心2篇已录用（未正式发表），学术会议论文3篇。主要研究任务完成情况如下：.1.以蒸汽爆破玉米秸秆为原料，在30%底物浓度下进行纤维素酶一段水解，当酶用量为15 FPU/g纤维素和30 FPU/g纤维素时，72 h纤维素酶水解得率分别为44.7％和59.9％；.2.以底物浓度为30％蒸汽爆破玉米秸秆时，在（9＋9＋12 h）三段酶水解过程中，第一段、第二段和第三段的水解得率分别为29.81％、22.58％和18.02％，30 h水解得率为70.41％，比一段水解72 h的水解得率51.64％提高了36％，并且水解时间缩短了42 h；.3.在高底物浓度的（9＋9＋12 h）三段水解过程中，随着反应过程中产物的去除，酶反应速率呈迅速增加趋势；.4.在高底物浓度的纤维素酶分段水解过程中，每一段水解结束后，酶解残渣中的酶蛋白含量较高，滤纸酶活也较高。由于每一段水解时间较短，残渣吸附的纤维素酶具有较高的再反应性，可继续进行下一段的水解。.5.（9＋9＋12 h）三段水解过程中各段底物结晶度呈现先逐渐增加再缓慢降低的趋势。.6. 在纤维素酶水解木质纤维原料的过程中，随着纤维素和半纤维素的降解，使得木质素上的纤维素酶吸附位点暴露，在第二段和第三段水解过程中仍然有新鲜纤维素酶不可逆吸附到木质素上。