【作者】 魏衍纲；

【导师】 江华；

【作者基本信息】 南京林业大学 ， 化学工艺， 2012， 硕士

【摘要】 通过补充木聚糖酶单组分辅助纤维素酶水解稀碱预处理过的杨木，研究木聚糖酶组分对酶解过程中纤维素、半纤维素的转化规律和酶解产物结构的影响。两种不同的条件下预处理杨木，分别得到底物A（120℃，5%NaOH处理后洗涤，然后80℃，5%NaOH，2%H₂O₂处理）和底物B（80℃，5%NaOH，5%H₂O₂处理）。底物A的纤维素、半纤维素和木质素含量分别为57.8、8.5和18.5%，底物B的纤维素、半纤维素和木质素含量分别为47、9.7和24.8%。通过凝胶过滤层析分离纯化里氏木聚糖酶得到4种电泳纯组分，分子量分别为98.73×103、21.62×10³、19.67×10³和12.85×10³g/mol，它们分别为木糖苷酶、内切木聚糖酶XYNⅠ、内切木聚糖酶XYNⅡ和外切木聚糖酶。纤维素酶水解预处理过的杨木72h后，底物A、B的葡萄糖得率分别为74.88和39.2%，木糖得率分别为62.3和57.5%，分别补充添加木糖苷酶、XYNⅠ、XYNⅡ和外切木聚糖酶单组分后，底物A的葡萄糖得率分别为81.8、89.5、89.9和87.2%，提高了7.0、14.62、15.02和12.32个百分点，木糖得率分别为74.5、80.3、79.8和82.4%，提高了12.2、18.0、17.5和20.1个百分点；底物B的葡萄糖得率分别为45.3、62.7、57.8、47.7%，提高了6.1、23.5、18.6和8.5个百分点，木糖得率分别为61.7、70.1、77.1和85.5%，提高了4.2、12.6、19.6和28.0个百分点。可以看出，补充木聚糖酶单组分，可以提高两种底物的葡萄糖和木糖得率，其中XYNⅠ和XYNⅡ能显著提高葡萄糖得率，外切木聚糖酶能显著提高木糖得率；四种木聚糖酶组分对不同组成的底物的作用不同，当底物纤维素含量较低、半纤维素含量较高时，外切木聚糖酶对木糖得率的提高更加明显，说明外切木聚糖酶能更好的降解半纤维素。在纤维素酶的基础上添加XYNⅠ和XYNⅡ混合组分后，两种底物的葡萄糖得率分别为90.8和54%，提高了16和10个百分点；木糖得率分别为65和81.6%，提高了14和24.2个百分点。这说明，在底物半纤维素含量较高的情况下，XYNⅠ和XYNⅡ组分的复配对半纤维素的降解效果最好。对添加XYNⅠ和XYNⅡ的酶解液进行凝胶色谱分离，并对其中的低聚木糖组分进行红外光谱、核磁共振¹H NMR和¹³C NMR分析。可以看出，产物中4-OMe-α-D-GlcpA和（1→4）-β-D-Xylp通过C-O-C键连接，（1→4）-β-D-Xylp与木素通过糖苷键连接形成木素-碳水化合物复合体LCC，（1→4）-β-D-Xylp是通过β-糖苷键连接；由红外可知X2比X1的结构中多了一个亚甲基，由核磁共振图谱可以看出两种产物在结构上有一定区别，具体还有待进一步研究。更多还原

【Abstract】 This experiment investigated the effects of xylanase components on transformation rulesof cellulose and hemicellulose and the structure of enzyme hydrolyzate during enzymatichydrolysis process via poplar which was pretreated by xylanase single component assistedenzymatic hydrolysis of dilute alkali.Pretreatment of poplar under two different conditions, substrate A(120℃, treated by5%NaOH, then80℃, treated by5%NaOH and2%H2O2) and substrate B(80℃treated by5%NaOH and5%H2O2) were got respectively. The cellulose, hemicellulose and lignin contents ofsubstrate A and B were57.8,8.5,18.5%and47,9.7,24.8%, respectively.Four kinds of electrophoresis pure components were got when Richter xylanase wasseparated and edulcorated by gel filtration chromatography. Their molecular weights were98.73×10~3,21.62×10~3,19.67×10~3and2.85×10~3g/mol. They were xylosidase,Endo-xylanase XYNⅠand XYNⅡ, Exo-xylanase.The glucose yield of substrate A and substrate B were74.88and39.2%and the xyloseyield were62.3and57.5%after pretreated poplar hydrolysed by cellulose for72h. And afteradding xylosidase, Endo-xylanase XYNⅠand XYNⅡ, Exo-xylanase separately, the glucoseyield of substrate A were81.8,89.5,89.9and87.2%, get about7.0,14.62,15.02and12.32percentage point increase, the xylose yield were74.5,80.3,79.8and82.4%, get about12.2,18.0,17.5and20.1percentage point increase; the glucose yield of substrate B were45.3,62.7,57.8and47.7%, get about6.1,23.5,18.6and8.5percentage point increase, the xylose yieldwere61.7,70.1,77.1and85.5%, get about4.2,12.6,19.6and28.0percentage point increase.The results showed that xylanase can increase glucose yield and xylose yield of two substrates.Among them, XYNⅠand XYNⅡcan increase glucose yield obviously, Exo-xylanase canincrease xylose yield obviously. Four xylanase components have different effects on differentsubstrates. When the content of cellulose was low and the hemicellulose was higher,Exo-xylanase improved xylose yield more obviously which indicated that Exo-xylanase candigest hemicellulose better.The glucose yield of two substrates were90.8and54%, get about16and10percentagepoint increase, the xylose yield were65and81.6%, get about14and24.2percentage pointincrease by adding XYN Ⅰ and XYN Ⅱ mixed-component on the basis of the cellulose. Theresults showed that XYN Ⅰ and XYN Ⅱ mixed-component can digest hemicellulose better in thecase of the substrate with high content of hemicellulose.The enzymatic hydrolyzate was separated by gel filtration chromatography which wasadded XYN Ⅰ and XYN Ⅱ and xylo-oligosaccharides was analyzed by IR,1H and13C NMR.4-OMe-α-D-GlcpA was connected by C-O-C bond with (1→4)-β-D-Xylp in the products.(1→4)-β-D-Xylp was connected by glycosidic bond with lignin and formed Lignin-CarbohydrateComplexe(LCC).(1→4)-β-D-Xylp was connected by β-glycosidic bond;X2have one-CH2more than X1on the structure by the result of IR. The two products have little difference on thestructure by the result of1H and13C NMR and the specific differences are to be futher studied.更多还原