高温热解过程中半纤维素结构变化及对酶解制备低聚木糖影响机理

Xylo-oligosaccharides (XOs) as prebiotics are produced by cooking coupling with enzymatic hydrolysis by xylanase mainly in industry, but the target products’ conversion is low contributing to complexity and specificity of the reaction. And the reasons are still unclear. The present project is to illuminate the effect mechanism of low conversion of xylan based on the properties of substrates and its effects on enzymatic hydrolysis: 1) make clear of corncob pentosan dissolution in high-temperature cooking process and distribution of each components DPs; 2) Multi-component of pentosan polymer in cooking liquor of corncob will be separated and analyzed. Key factors restricting the conversion of the substrate will be observed; 3) corncob hemicellulose structure and properties will be analyzed by glycosylation, glycosidic linkage analysis throughout the course of cooking. Acidophilic xylanase XynA from Penicillium janthinellum XAF01 will be applied to enzymatic hydrolysis and composition of products will be observed to further clarify 'soluble' glycan structural changes' effects; 4) The enzymatic hydrolysis of insoluble pentosan will be analyzed based on enzyme adsorption theory (such as Langmuir-type or Frendlich-type adsorption isotherm). To sum up, the purpose of this project is to reveal the mechanism of hydrolyzing by xylanase in complex system, so as to improve the preparation efficiency of XOs in industry.

高温热解耦合酶解为“超强益生元”低聚木糖当前主要的工业制备方式，但目标产物转化效率偏低，反应体系的复杂性及特殊性使得对水解规律的研究仍处于探索阶段。课题拟重点从底物性质及底物与水解酶作用规律层面考察纤维质高温热解体系中影响低聚木糖转化的机理问题：1）明确半纤维素在高温热解过程中的溶出规律及各组分聚合度的分布特点；2）分离热解液中戊聚糖高聚物并进行组分分析，筛查底物层面制约转化的关键因素；3）通过糖基、糖苷键分析推定高温热解过程中半纤维素结构及性质变化，引入微紫青霉酸性木聚糖酶XynA对酶解前后效果进行比对，进一步明确“可溶性”多聚糖结构变化对于产物转化效果的影响；4）基于酶吸附理论探究纤维质高温热解体系中“粘附态”不溶性戊聚糖的酶解规律。拟通过本研究从机理层面解释复杂水解体系中制约酶催化效率的关键作用机制，同时为低聚木糖实际生产中长期存在的主要瓶颈问题提供理论参考。

研究高温热解及分级沉淀获得到5种不同的玉米芯半纤维素组分(H1-H5)，其数均分子量分别为134400g/mol、12470g/mol、3168g/mol、1645g/mol、1218g/mol；每个组分的主要糖基为木糖，同时含有葡萄糖、阿拉伯糖、糖醛酸等木聚糖侧链糖基，不同组分所含的糖基的比例组成不同；通过比较各组分水解效果可知：分子量越大对酶解阻碍越强；当分子量相对较小时，起决定性作用的则是支链度，支链的增多会阻碍酶与底物的接触，不利于酶的水解。组分溶解性对低聚木糖转化率存在相关性，且呈正相关，线性相关系数为0.73。分枝度和多分散性指数与低聚木糖转化率存在很强的线性相关性（线性相关系数0.93），显示其为制约低聚木糖转化率的关键因素。超声耦合酶解各组分所得低聚木糖转化率各不相同，对于组分H2、H3，超声耦合酶解较单独酶解，低聚木糖转化率明显提高（56.4%-137.8%）；而以H4、H5为底物时，超声耦合酶解所得低聚木糖转化率提升不显著，且实验还发现酶解方式不同（超声耦合酶解和单独酶解），底物结构参数对低聚木糖转化率的影响程度也存在差异。研究考察了木聚糖酶对玉米芯热解残渣和玉米芯木聚糖的吸附性能，发现木聚糖酶对玉米芯热解残渣的吸附率（55.86%）远大于对玉米芯木聚糖的吸附率（21.64%）。根据吸附动力学特性，确定了木聚糖酶在玉米芯热解残渣上吸附条件为：4℃下，吸附60min。此条件下，木聚糖酶以玉米芯热解残渣为底物时的吸附等温线符合S型吸附等温线特征。且木聚糖酶酶解玉米芯木聚糖的过程中，木聚糖酶在整个水解过程中主要保持在液相中，显示酶解速率和吸附量存在正相关。