“漆酶-木聚糖酶”双功能作用预水解液促半纤维素转化糠醛机理研究

The pre-hydrolysis liquor of a dissolving pulp production process is rich in hemicelluloses, which is the main resource of producing furfural. In industrial production, the low yield of furfural is due to the occurrence of side reactions during the components of lignin, hemicellulose and furfural. .In this study, "laccase - xylanase" bifunctional enzymes were used on pre-hydrolysis liquor from the aspects of reaction mechanism and the change of base material content. Laccase acted on lignin, removing lignin drag factor from the system, and providing more exposed sites for the xylanase reaction on the hemicellulose. The techniques of gel permeation chromatography, gas chromatography combined with infrared spectroscopy and nuclear magnetic resonance (NMR) were used to explore the depolymerization, decarboxylic reaction, changes of functional groups of hemicelluloses from xylanase reaction, as well as lignin polymerization, precipitation and the changes of molecular structure and content caused by laccase oxidation at the molecular level. .To reveal the reaction mechanism and cooperative regulation rule of the enzymes on the organic matter (Lignin, hemicellulose polysaccharide/ monosaccharide, furfural, etc.) of the pre-hydrolysis liquor in the process of furfural conversion, and to improve the yield of furfural finally,to achieve a two-step furfural green conversion method, to open up a new technical route which using enzyme engineering reacts on multi-component complex system,to remove the drag factors and improve the penta-sugar content for furfural preparation at the same time, and provide a useful theoretical guidance and reference for the traditional furfural industry and pre-hydrolysis liquor of a dissolving pulp production of papermaking industry.

溶解浆预水解液富含半纤维素，是生产糠醛的主要原料。工业生产中，糠醛收率低的原因是体系中木素、半纤维素和糠醛等组分之间副反应的发生。.本研究从反应机理以及基质内容物改变等方面着手，采用 “漆酶-木聚糖酶”双功能作用预水解液。漆酶作用于木素，脱除体系中的木素拖累因子，为木聚糖酶反应作用于半纤维素提供了更多的展开位点。采用凝胶渗透色谱、气质联用、红外光谱和核磁共振等多种技术，从分子水平上深入探索木聚糖酶酶解半纤维素解聚规律、脱羧反应、官能团变化规律，以及漆酶氧化木素聚合沉积及其木素分子结构和含量变化规律，揭示酶解预水解液转化糠醛过程有机物（木素、半纤维素多糖/单糖、糠醛等）反应机制和联动调控规律，最终提高糠醛得率，开辟一条利用新型酶工程作用于多组分复杂体系的技术路线，去除拖累因子和提高制备糠醛的五碳糖含量同步进行，将为传统糠醛行业及造纸行业溶解浆厂预水解液的应用开发提供有益的理论指导与应用参考。

溶解浆预水解液（PHL）富含糖等生物质资源，可生产糠醛。然而，木素的存在使得PHL高值化利用过程中副反应增加。因此，本研究提出了一种新的工艺，即漆酶诱导木素聚合以提高木素的去除率。漆酶处理最佳工艺条件为时间3h、漆酶用量800 uL/30mL、pH 4、温度40 ℃。当漆酶用量增加，木质素去除率提高；pH在4时，木质素的去除率达到56.95%。回归方程分析影响预水解液中木素脱除率的因素顺序为：pH＞漆酶用量＞处理温度＞处理时间。凝胶渗透色谱结果表明，漆酶处理的PHL木质素的分子量比原木质素增加了160%左右。傅里叶红外光谱分析了漆酶木聚糖酶处理前后木素官能团的变化，结果表明，漆酶处理的PHL木质素的特征官能团振动比原木质素增强且代表性更明显。核磁共振进一步分析了酶处理前后木素各官能团的结构及其结构单元间的连接方式，图谱证实了PHL中只含有紫丁香基结构单元（Ｇ型）和愈创木基结构单元（Ｓ型）并且Ｓ型的信号强度都要高于愈创木基单元Ｇ型。木聚糖酶处理预水解液，当反应时间4h，pH值5.5，用量200 uL/30mL，温度55 ℃时，产生的单糖木糖含量最高。然后利用高压反应釜在硫酸催化作用下探究采气速度、硫酸浓度和温度对脱木素后的预水解液糠醛转化的影响。通过单因素法确定了预水解液转化糠醛的最佳工艺条件为温度180 ℃、H2SO4浓度 0.25%、采气速度 15 mL/min、木糖浓度 17.8 g/L，这时糠醛收率达到最大值71.89%。通过 GC/MS 分析，粗糠醛溶液中糠醛的相对含量为 93.32%，主要副产物为 5-甲基糠醛。