【作者】 杜敏；

【导师】 李新平；

【作者基本信息】 陕西科技大学 ， 制浆造纸工程， 2013， 博士

【摘要】 植物纤维原料是地球上储量最丰富的可再生自然资源，它的充分、有效利用对节约能源、保护环境都具有非常重要的意义。利用纤维素酶和半纤维素酶水解纤维原料，一方面，可使纤维表面和内部结构得到一定程度的活化，使纤维性能得到改善；另一方面，可直接获得低聚糖和单糖，进一步发酵可以生产乙醇、甲醇等生物质燃料，因而酶水解纤维原料在养殖、食品、酿酒、纺织、洗涤、造纸、能源等工业中都具有广泛的应用价值。但是，由于酶的种类繁多，酶的组成、结构、催化机理等都存在较大差异，再加上原料本身结构的复杂性，使得纤维原料在酶水解过程中，其结构和性能的变化比较复杂，从而影响纤维原料酶改性技术的应用。本课题主要是以漂白针叶木纤维为原料，通过分析比较复合纤维素酶、内切纤维素酶和木聚糖酶在不同的水解程度下对纤维形态、结构、性能的影响，研究经不同的酶水解后纤维结构和性能变化的一般规律及其机理，旨在加强对酶水解过程中纤维原料结构和性能变化的控制，为植物纤维原料酶改性技术的工业化应用提供理论指导，进而推动酶水解纤维原料的全面有效应用。研究了酶水解对纤维得率的影响，结果显示：经复合纤维素酶Celluclast1.5L水解后，随着酶用量的增加或酶水解时间的延长，纤维得率急速下降，当酶用量为10.0FPU/g，水解时间为48h时，纤维得率仅为55.34%，说明复合纤维素酶对纤维素具有很强的水解作用，可以使无定形区和结晶区纤维素都发生水解。内切纤维素酶Novozym476对纤维的水解能力远低于Celluclast1.5L，当Novozym476用量为50.0CMCU/g时，处理2h后仅有5%左右的纤维素发生水解。经木聚糖酶Pulpzyme HC水解后，纤维得率基本上没什么变化，说明木聚糖酶对漂白针叶木纤维的水解作用非常有限。研究了酶水解对纤维形态的影响。结果显示：酶水解前，纤维表面带有许多细小纤丝，纤维素酶Celluclast1.5L和Novozym476都会优先作用于这些细小纤丝，使其发生水解，使得纤维表面变得光滑，比表面积减少；随着纤维素酶Celluclast1.5L用量的增加，纤维表面出现起皮、表层剥落现象，进一步增加用量，纤维出现明显的缺口和断裂，纤维比表面积增加，纤维平均长度急剧下降，细小纤维含量明显增加，纤维卷曲率和扭结指数下降；而经纤维素酶Novozym476水解后，纤维平均长度变化不大，但纤维卷曲率和扭结指数有所增加。木聚糖酶Pulpzyme HC水解处理后纤维形态没有明显变化。研究了酶水解对漂白针叶木纤维纤维素分子量和聚集态结构的影响，结果显示：漂白针叶木纤维经纤维素酶Celluclast1.5L或Novozym476水解后，随着酶用量的增加，纤维素聚合度逐渐降低，在Celluclast1.5L用量为10.0FPU/g时，聚合度降低到694，较对照样下降了40.38%；在Novozym476用量为50.0CMCU/g时，聚合度下降到711，较对照样下降了38.92%。说明在这两种纤维素酶的作用下，纤维素大分子链都会发生较多断裂，使组成纤维素大分子链的葡萄糖基数量减少，纤维素分子量减小，由此可见，在纤维素的酶水解过程中，导致纤维素聚合度和分子量下降的主要是内切葡聚糖酶的作用。漂白针叶木纤维经木聚糖酶Pulpzyme HC水解后，纤维素聚合度和分子量基本保持不变。从X-射线衍射分析和红外光谱分析结果显示，纤维素酶水解不会引起纤维素大分子结构变化，水解过程中也没有新的官能团产生，纤维素晶型未发生改变，仍属于典型的纤维素I晶型，但在两种纤维素酶的作用下，纤维素结晶度出现了不同的变化。随着纤维素酶Celluclast1.5L用量的增加，纤维素结晶度呈现先增加后降低再增加再降低的周期性变化，结晶区纤维素和无定形区纤维素同时受到酶的作用发生水解。在内切纤维素酶Novozym476的作用下，纤维素结晶度一开始略有降低，但随着酶用量的增加，纤维素结晶度逐渐增加后又降低，整体呈现增长的变化趋势。经木聚糖酶Pulpzyme HC水解后，随着酶用量的增加，纤维素结晶度逐渐增加。研究了酶水解对漂白针叶木纤维性能的影响，结果显示：随着纤维素酶Celluclast1.5L用量的增加，水解后纤维悬浮液滤水性先增加后降低，纤维保水值先降低后增加，当酶用量为20.0FPU/g时，保水值增加到204.19%，较对照样增加了47.73%；随着酶用量的增加，纤维表面的Zeta电位绝对值先降低后增加，表面自由能逐渐降低，纤维亲水性降低，亲油性增加；酶水解后纤维热稳定性有所下降。随着纤维素酶Novozym476用量的增加，水解后纤维悬浮液滤水性缓慢增加，纤维保水值则略有降低；纤维表面Zeta电位绝对值逐渐降低，表面自由能增加，纤维亲水性增加，亲油性降低。酶水解后纤维热稳定性降低。木聚糖酶Pulpzyme HC水解处理对纤维悬浮液滤水性影响不大，但随着其用量的增加，纤维保水值略有降低，纤维表面Zeta电位绝对值逐渐降低，表面自由能增加，纤维亲水性增加，亲油性降低；另外水解后纤维热稳定性略高于对照样。研究了酶水解对漂白针叶木纤维打浆和成纸性能的影响，结果显示：漂白针叶木纤维经纤维素酶Celluclast1.5L或Novozym476预处理后，在高打浆转数下打浆，随着酶用量的增加，浆料游离度逐渐降低，说明复合纤维素酶和内切纤维素酶预处理都能增加打浆过程中纤维切断、润胀、细纤维化等作用效果，起到一定的降低打浆能耗的作用；在较低的打浆转数下，纤维素酶预处理对降低打浆能耗贡献不大。纤维素酶酶促打浆和机械打浆对纤维具有不同的作用效果：在同样的游离度下，经酶促打浆的纤维较薄、纤维发生较多切断，浆料中纤维碎片含量较多。在未经机械打浆的情况下，随着纤维素酶Cellulast1.5L用量的增加，成纸松厚度、抗张指数、柔软度、透气度等指标均呈现先增加后降低的变化趋势，其中抗张指数在酶用量为0.1FPU/g时达到最大值21.77N.m/g，较对照样增加了26.36%，而当酶用量为10.0FPU/g时，抗张指数下降到10.97N.m/g，较对照样下降了39.89%；成纸内结合强度则随着酶用量的增加而逐渐增加。在纤维素酶Celluclast1.5L和Novozym476酶促打浆过程中，如果酶用量较低，在低转数下打浆，成纸抗张指数、内结合强度和松厚度都增加；在酶用量较高时，由于长纤维表面或内部的分子链发生断裂，纤维表面会产生缺口或松动，在机械力的作用下造成纤维变薄、变短、浆料中碎片含量增多，纤维自身强度下降，因此成纸抗张指数下降，且随着酶用量的增加，下降幅度逐渐增加。纤维素酶酶促打浆对提高成纸透气度是不利的。木聚糖酶Pulpzyme HC水解对降低纤维打浆能耗基本没什么作用，但木聚糖酶Pulpzyme HC酶促打浆有利于提高成纸抗张指数和内结合强度。对比研究了酶深度水解和盐酸水解对纤维结构、性能的不同影响，结果显示：漂白针叶木纤维经盐酸水解后，纤维素聚合度可以下降到极限聚合度200左右，而且纤维得率在90%以上，而经纤维素酶Celluclast1.5L深度水解后，纤维素聚合度保持在700左右，而纤维得率则急剧下降。说明虽然稀酸水解和纤维素酶水解都是使纤维素大分子上的β-1,4-糖苷键发生断裂，但具体发生断裂的位置有很大的区别。酶水解纤维素纤维和酸水解纤维素纤维具有相似的纤维形态，纤维平均长度都下降到只有0.1~0.2mm，但是两者经粉碎后纤维素颗粒在微观形态上存在较大差别：酸水解纤维素为椭圆形的颗粒，粒径较小，完全失去了纤维细胞壁原有的结构，符合微晶纤维素的颗粒特性；酶水解纤维素颗粒粒径较大，具有较完全的纤维细胞壁结构，不符合微晶纤维素颗粒特性。酶水解纤维素和酸水解纤维素具有相似的结晶结构，结晶度都较水解前增加，两者结晶度大小区别不大。酸水解纤维素较酶水解纤维素具有稍高一点的耐热性能。更多还原

【Abstract】 Fiber raw material is the most abundant renewable naturalresources on the earth， the effective use of it has a very importantsignificance to energy conservation and the protection of theenvironment. Using of cellulase or hemicellulase to hydrolyze the fibermaterials, on one hand, it could activate the fiber surface and internalstructure to a certain degree, and improve the fiber performance; on theother hand, it could obtain oligosaccharides and monosaccharidesdirectly, which can produce biomass fuels such as ethanol, methanol byfurther fermentation. So the enzymatic hydrolysis of fiber materials hasa wide value of application in farming, food, wine, textiles, washing,papermaking, energy and other industries. However, due to the widevariety of enzymes, the large difference of its composition, structure,catalytic mechanism, and coupled with the complex structure of thefiber material itself, in the process of enzymatic hydrolysis, thestructure and properties of fiber changed complexly, it would affect theapplication of fiber enzymatic modification technology. This taskmainly used bleached softwood fiber as the raw material, by analyzingand comparing the influence of the composite cellulase, endo-cellulaseand xylanase to the morphology, structure and performance of the fiberin a different hydrolysis degree, studied the general rule and mechanismof the changes of fiber structure and properties with enzymatichydrolysis. It aimed at strengthening the control of the change of thefiber structure and properties in the enzymatic hydrolysis, providingtheoretical guidance for the industrial application of fiber enzymatichydrolysis, and promoting the application of fiber enzymatic hydrolysisin full range effectively. The effect of enzymatic hydrolysis to fiber yield was investigated.The results showed that by the hydrolysis of complex cellulase namedas Celluclast1.5L, with the increasing of the enzyme dosage or thehydrolysis time, the fiber yield declined rapidly. Hydrolyzed in thedosage of10.0FPU/g for48h, the fiber yield was just55.34%. Itindicates that the complex cellulase has a strong hydrolysis on cellulose,and it can hydrolyze the cellulose both in amorphous region and incrystallization region. Endo-cellulase named as Novozym476had a farweaker hydrolysis on the fiber than Celluclast1.5L. Hydrolyzing for2hours with Novozym476in the dosage of50.0CMCU/g, only about5%cellulose was hydrolyzed. There was not essentially change in the fiberyield when hydrolyzed with xylanase named as Pulpzyme HC, itindicates that the function of xylanase on bleached softwood fiber isvery limited.The effect of enzymatic hydrolysis to fiber morphology wasinvestigated. The results showed that there are many tiny fibrils on fibersurface without enzymatic hydrolysis. both of Celluclast1.5L andNovozym476took role on these tiny fibrils firstly and made them behydrolyzed, thus the fiber surface became smooth, and the fiber specificsurface area reduced. With the increasing enzyme dosage of theCelluclast1.5L, there was peeling and stripping appeared on the fibersurface. While the enzyme dosage increased furthermore, the fiberappeared significant gaps and breaking. Thus the fiber specific surfacearea increased, average length of fiber declined sharply, the content offine increased significantly, and curl rate and kink index of fiberdecreased. The average length of fiber hydrolyzed with Novozym476was not changed significantly, but curl rate and kink index of fiberincreased. Pulpzyme HC had no significant effect on fiber morphology.The effect of enzymatic hydrolysis on molecular weight andaggregation structure of bleached softwood fiber were investigated. Theresults showed that hydrolyzed by Celluclast1.5L or Novozym476,with the enzyme dosage increasing, the degree of polymerization (DP)of the cellulose decreased gradually. When the dosage of Celluclast 1.5L was10.0FPU/g, the DP of cellulose declined to694, dropped by40.38%compared with the control sample; when the dosage ofNovozym476was50.0CMCU/g, the DP of cellulose declined to711,dropped by38.92%compared with the control sample. It illustrates thatin the function of complex cellulase or endo-cellulase, themacromolecular chain of cellulose would break in a high degree, andthe molecular weight of cellulose declined. It shows that in the processof enzymatic hydrolysis, it is endo-glucanase that mainly leads the DPand molecular weight of cellulose to decrease. Bleached softwood fiberhydrolyzed by Pulpzyme HC, the DP and the molecular weight ofcellulose was essentially unchanged.Shown from the results of X-ray diffraction (XRD) and FourierTransform infrared spectroscopy (FTIR), that the enzymatic hydrolysiswould not cause the change of macromolecule structure, and there hadno new functional groups generated during the hydrolysis process. Thecellulose crystal form was not changed, and it is still the cellulose I.However, the crystallinity of cellulose went through different variationswith the function of two cellulases. After hydrolyzing by Celluclast1.5L, the crystallinity presents a periodic variation with the increase ofthe enzyme dosage. It indicates that the celluloses in crystallizationregion and amorphous region are hydrolyzed at the same time in thefunction of Celluclast1.5L. With the function of endo-cellulaseNovozym476, the crystallinity decreased initially, but it would increaseand then decreased with the enzyme dosage increasing, but it showed agrowth trend as a whole. After hydrolyzing by xylanase Pulpzyme HC,the crystallinity gradually increased with the increase of the enzymedosage.The effect of enzymatic hydrolysis on the properties of bleachedsoftwood fiber was investigated. The results showed that with theincreasing dosage of Celluclast1.5L, the filtration performance of fibersuspension increased initially and then decreased, while the waterretention value (WRV) of fiber decreased first and then increased. In theenzyme dosage of20.0FPU/g, the WRV of fiber increased to204.19%, increased by47.73%compared with the control sample. With theenzyme dosage increasing, absolute Zeta potention of fiber surface firstdecreased and then increased, fiber surface free energy decreasedgradually, and the hydrophilicity of fiber decreased while thelipophilicity increased. After enzymatic hydrolysis with Celluclast1.5L,the thermal stability of fiber declined.With the increasing dosage of cellulase Novozym476, thefiltration performance of fiber suspension gradually increased while theWRV of fiber decreased. Absolute Zeta potention of fiber surfacedecreased and the surface free energy of fiber increased gradually whilethe enzyme dosage increased, and the hydrophilicity of fiber increased.After enzymatic hydrolysis with Novozym476, the thermal stability offiber declined.There had no significant effect to the filtration performance offiber hydrolyzed with xylanase Pulpzyme HC. While the enzyme dosageincreased, the WRV of fiber decreased slightly, the absolute Zetapotention of fiber surface decreased gradually, the surface free energyof fiber increased gradually and the hydrophilicity of fiber increased. Inaddition, the thermal stability of hydrolysis fiber was slightly higherthan the control sample.The effect of enzymatic hydrolysis to fiber refining performanceand paper sheet properties was investigated. The results showed thatpretreated by cellulase of Celluclast or Novozym476and then refinedby higher PFI revolutions, the pulp freeness decreased with theincreasing of the enzyme dosage. it indicates that the pretreatment withcomposite cellulase or endo-cellulase in refining process wouldincrease the effect of the fiber cut、swelling、fibrillation and so on, andit would play a role in reduce the energy consumption of refining. Butin the lower PFI revolutions, cellulase pretreatment contribute little toreduce the energy consumption of refining. Cellulase enzymaticrefining had different effect on fiber morphology compared withmechanical refining. In the same pulp freeness, enzymatic refining would make the fiber thinner、more incision and there would have muchmore fragments in the pulp.In the case of without mechanical refining, the paper properties ofthickness, tensile index, softness and air permeability all increased atfirst and then decreased with the increasing of the enzyme dosage ofCellulast1.5L. In the enzyme dosage of0.1FPU/g, the tensile index ofpaper reached the maximum of21.77N·m/g and increased by26.36%compared with the control sample. While in the enzyme dosage of10.0FPU/g, the tensile index of paper declined to10.97N.m/g anddecreased by39.89%compared with the control sample. The internalbond strength of paper increased gradually with the increasing of theenzyme dosage.In the process of enzymatic refining used Celluclast1.5L orNovozym476, while in the case of less enzyme dosage, the tensileindex, internal bond strength and bulk of paper all increased with lowerPFI revolutions; In the case of higher enzyme dosage, for the reasonthat the surface or internal molecular chain of cellulose was broken, thesurface of the fiber would have a gap or loose, so the fibers would bethinner, shorten with the mechanical force, which led to the decreasingof the fiber strength, so the tensile index of paper decreased. The higherof the enzyme dosage, much more declined of the tensile index.Enzymatic refining with cellulase was not conducive to improve paper’sair permeability.Hydrolyzing by xylanase Pulpzyme HC had no beneficial to reducethe energy consumption of refining, but the enzymatic refining withPulpzyme HC would help to improve the tensile index and internalbonding strength of paper.The different effect on structure and performance of fiber withdepth enzymatic hydrolysis and acid hydrolysis was investigated. Theresults showed that hydrolyzed by hydrochloric acid, the DP of bleachedsoftwood fiber reduced to limit DP of about200rapidly, and fiber yieldwere more than90%. Hydrolyzed by the cellulase Celluclast1.5Ldeeply, the DP of cellulose maintained at about700, while the fiber yield sharply declined. It shows that although both of the hydrolysiswith dilute acid hydrolysis and cellulase would break the beta-1,4-glycosidic bond on the cellulose macromolecules, but there had quitedifference in the location of the breaking occurs. Depth enzymatichydrolyzed fiber and acid hydrolyzed fiber have a similar morphology,the average length of fibers dropped to only0.1to0.2mm. however,after crushing, there had a big difference between particles in the formof microscopic: the acid hydrolyzed cellulose were oval particles withsmall size, and it lost the original structure of the fiber cell wallcompletely, so it met with the particle characteristics ofmicrocrystalline cellulose. The enzymatic hydrolyzed cellulose had abigger size, and it had a quilt complete cell wall structure of fiber anddid not meet with the characteristics of microcrystalline cellulose.Depth enzymatic hydrolyzed cellulose and acid hydrolyzed cellulosehad similar crystal structure; the crystallinity of them was similar andincreased compared with the control sample. Acid hydrolyzed cellulosehas a slightly higher thermal stability than the enzymatic hydrolyzedcellulose.更多还原