【作者】 余圆圆；

【导师】 范雪荣；

【作者基本信息】 江南大学 ， 纺织工程， 2014， 博士

【摘要】 纤维素酶在纤维素纤维织物加工中具有反应条件温和、加工过程清洁环保、处理后织物柔软性提高等优点，广泛应用于生物抛光和牛仔织物返旧整理。但是，纤维素酶处理纤维素纤维织物时，织物内部的纤维以及纤维内部被过度降解，导致织物强力损失过大。利用大分子修饰剂对纤维素酶进行改性，可以得到大体积的修饰纤维素酶。利用大体积修饰纤维素酶处理织物时，酶在织物内的渗透以及在纤维内的扩散位阻增大，水解作用更多发生在纤维和织物的表面，从而避免酶对纤维和织物造成过度损伤。本课题通过共价修饰和非共价修饰两种方式，将可逆可溶大分子修饰剂Eudragit(含Eudragit S-100和Eudragit L-100两种)与纤维素酶分子连接在一起，制备三类Eudragit修饰纤维素酶。(1) Eudragit共价修饰纤维素酶。通过碳二亚胺盐酸盐(EDC)活化Eudragit上的羧基，使其与纤维素酶上的氨基共价结合，得到大体积的Eudragit共价修饰纤维素酶。(2) Eudragit非共价修饰纤维素酶。通过修饰剂Eudragit与纤维素酶分子间的非共价作用，制备大体积的Eudragit非共价修饰纤维素酶。(3) Eudragit-纤维素酶-漆酶共修饰酶。通过EDC作用，使Eudragit、纤维素酶和漆酶共价交联，得到大体积的Eudragit-纤维素酶-漆酶共修饰酶。分别考察上述修饰酶在棉、麻织物抛光整理和牛仔织物返旧整理中的应用效果。课题详细研究了修饰酶的制备过程、修饰酶的结构和性质、修饰酶对纤维素纤维织物的作用模式以及修饰酶处理纤维素纤维织物的效果。主要研究内容和结论如下：1.研究了Eudragit修饰纤维素酶的制备。体积排阻色谱结果显示，与未修饰酶相比，共价修饰酶的最早被检测出时间和整个峰形出峰时间均提前，表明共价修饰酶的体积大于未修饰酶。红外光谱结果显示，非共价修饰酶谱图中存在明显的蛋白特征吸收峰。这些结果表明共价和非共价修饰均成功发生，酶蛋白分子和修饰剂分子连接在一起。以未修饰酶的游离氨基量和酶活为100%，制得的Eudragit S-100共价修饰纤维素酶和Eudragit L-100共价修饰纤维素酶的氨基修饰率分别为50.72%和51.98%，纤维素酶相对酶活分别为80.04%和76.88%；Eudragit S-100非共价修饰酶和Eudragit L-100非共价修饰酶的纤维素酶相对酶活分别为82.2%和78.2%；Eudragit S-100-纤维素酶-漆酶共修饰酶和Eudragit L-100-纤维素酶-漆酶共修饰酶的氨基修饰率分别为38.06%和42.24%，纤维素酶相对酶活分别为78.46%和75.62%，漆酶相对酶活分别为88.28%和86.76%。2.研究了修饰纤维素酶的蛋白二级结构，酶学性质，以及重复使用性能。圆二色谱和荧光光谱结果表明，修饰会对酶蛋白的二级结构产生影响。修饰后酶蛋白二级结构中的α-螺旋结构所占比例下降，β-折叠和β-转角结构所占比例上升，并且Eudragit L-100修饰剂对蛋白二级结构的影响大于Eudragit S-100。酶的温度和pH稳定性实验结果表明，与未修饰酶相比，Eudragit修饰酶的温度和pH稳定性发生改变。Eudragit共价修饰纤维素酶在60℃~80℃和pH6.0~9.0范围内的稳定性提高。Eudragit非共价修饰纤维素酶的最适作用温度由未修饰纤维素酶的50℃提高至60℃，最适作用pH不变。Eudragit-纤维素酶-漆酶共修饰酶的最适作用温度为50℃，最适作用pH为5.0。修饰酶的重复使用性能实验结果表明，经5次重复使用后，Eudragit共价修饰纤维素酶的酶活保留率在50%以上，Eudragit非共价修饰纤维素酶的酶活保留率在30%左右，Eudragit-纤维素酶-漆酶共修饰酶的纤维素酶酶活与漆酶酶活保留率均为40%左右，均具有一定的重复使用性能。3.研究了Eudragit S-100共价修饰纤维素酶处理对纤维结构的影响，以及EudragitS-100共价修饰纤维素酶对不同形态底物、不同纤维素纤维织物的水解作用。通过XRD、ATR-FTIR和亚甲基蓝吸附法分别对酶处理前后棉纤维的结晶度、表面区域氢键、可及性进行研究。结果表明，与Eudragit S-100共价修饰纤维素酶处理相比，未修饰纤维素酶处理对纤维无定形区、表面区域分子间氢键以及内部孔道结构的破坏更大。未修饰纤维素酶与Eudragit S-100共价修饰纤维素酶水解不同形态底物(棉织物，棉纱线，棉纤维)的实验结果表明，当底物形态分别为纤维和纱线时，未修饰酶处理棉纤维比处理棉纱线生成的还原糖量多39.90%，修饰酶处理棉纤维比处理棉纱线生成的还原糖量多71.95%；当底物形态分别为纱线和织物时，未修饰酶的水解作用不受影响，而修饰酶处理棉织物生成的还原糖量少于处理棉纱线生成的还原糖量。可以看出，织物形态对修饰酶水解作用的影响大于对未修饰酶水解作用的影响。未修饰纤维素酶与Eudragit S-100共价修饰纤维素酶水解苎麻织物、棉织物、丝光棉织物、粘胶纤维织物的实验结果显示，当织物中纤维可及性增大时，对未修饰纤维素酶水解作用影响更大，Eudragit S-100共价修饰纤维素酶对织物中纤维可及性增大的敏感性较低。4.研究了Eudragit S-100共价修饰纤维素酶在棉、苎麻织物抛光整理中的应用。研究发现，当纤维素酶酶活浓度为5.64U/mL时，未修饰和修饰纤维素酶处理棉织物的减量率分别为7.26%和5.34%、苎麻织物的减量率分别为2.79%和2.62%、棉织物中纤维铜值分别为0.04861和0.02790、苎麻织物中纤维铜值分别为0.02427和0.02286。与未修饰酶处理相比，修饰酶处理织物减量率较小，纤维铜值较低，表明未修饰酶处理对棉和苎麻织物中纤维的水解作用更强，损伤更大。当纤维素酶酶活浓度为1.88U/mL时，未修饰和修饰纤维素酶处理棉织物的强力损失分别为15.09%和5.61%、苎麻织物的强力损失分别为24.60%和10.65%。可以看出，Eudragit S-100共价修饰纤维素酶处理织物的强力损失明显低于未修饰纤维素酶处理织物的强力损失。SEM和AFM结果表明，未修饰和修饰纤维素酶处理均能去除棉和苎麻织物中纤维表面的微原纤，改善织物表面形貌，但未修饰酶处理织物中断裂纤维的数量明显多于修饰酶处理织物中断裂纤维数量。5.研究了Eudragit S-100共价修饰纤维素酶和Eudragit-纤维素酶-漆酶共修饰酶在牛仔织物返旧整理中的应用。当纤维素酶酶活浓度为5.64U/mL时，未修饰纤维素酶和Eudragit S-100共价修饰纤维素酶处理牛仔织物的CIE L值接近，均为25左右，表明褪色效果相当，织物强力损失分别达到46.21%和30.90%。在纤维素酶酶活为1.88U/mL时，未修饰纤维素酶、Eudragit S-100共价修饰纤维素酶、Eudragit S-100-纤维素酶-漆酶共修饰酶处理牛仔织物的CIE L值分别为22.91、22.68和25.76，处理牛仔织物的强力损失分别达到30.13%、13.41%和12.67%。利用Eudragit-纤维素酶-漆酶共修饰酶进行牛仔织物返旧整理，织物既达到了很好的褪色效果，又具有很高的强力保留。更多还原

【Abstract】 Cellulases are the most successfully used enzymes in textile wet processing, such asbiopolishing, and denim washing. Cellulase treatments have many advantages, such as mildreaction conditions, clean environment-friendly processing, and improved flexibility of fabric.However, the use of cellulases used in textile processes often results in a certain loss ofcellulosic fabric strength. Cellulases can easily penetrate cellulosic fibers, leading tosignificant losses of weight and tensile strength. Tensile strength loss can be minimized bycellulase modification. Enlarging the molecule of enzyme will limit the enzyme’s action morerelatively to the surface of the cellulosic fiber and fabric, and the diffusion of an enlargedenzyme molecule is significantly inhibited particularly in the interior of cellulosic fibers.In this study, cellulases were modified with Eudragit, using carbodiimide (EDC)covalently coupling and non-covalent methods. Cellulase, laccase and Eudragit wereco-modified with covalently coupling method. The modified enzymes were applied tobio-polishing of cellulosic fabrics or denim washing, respectively. Preparation of modifiedenzymes, structure and nature of modified enzymes, action mode of modified cellulase oncellulosic fabrics, effects of treatments of modified enzymes on cellulosic fabrics, had beenstudied in detail. The research contents and conclusions were as follows:1. Preparation of modified enzymes was studied.The results of gel filtration analysis revealed that the earliest detected times of covalentmodified enzymes were earlier than those of free enzymes. This result indicated that covalentmodified enzymes had a higher molecular weight than free enzymes. The typical absorptionof protein was detected in FTIR spectra of modified cellulases with non-covalent method. Theresults showed that enzymes were successfully modified with Eudragit.The modification rates of free amino of covalent-Eudragit S-100-cellulase andcovalent-Eudragit L-100-cellulase reached50.72%and51.98%, respectively. The relativecellulase activities of covalent-Eudragit S-100-cellulase and covalent-EudragitL-100-cellulase were80.04%and76.88%, respectively. The relative cellulase activities ofnon-covalent-Eudragit S-100-cellulase and non-covalent-Eudragit L-100-cellulase were82.20%and78.20%, respectively. The modification rates of free amino of EudragitS-100-cellulase-laccase and Eudragit L-100-cellulase-laccase reached38.06%and42.24%,respectively. The relative cellulase activities of Eudragit S-100-cellulase-laccase and EudragitL-100-cellulase-laccase were78.46%and75.62%, respectively. The relative laccaseactivities of Eudragit S-100-cellulase-laccase and Eudragit L-100-cellulase-laccase were88.28%and86.76%, respectively.2. The second structure of protein, temperature and pH stability, and reusability ofmodified enzymes were studied.The results of Circular dichroism and Fluorescence emission spectra indicated that thesecond structure of enzyme protein could be changed afier the modification. The proportionof helix reduced, and the proportions of strand and turns increased. Moreover, the morechange of second structure of protein caused by Eudragit L-100than Eudragit S-100. This study showed that the modification of cellulase via covalent binding onto Eudragitgained better stability, especially at pH6.0~9.0and temperature60~80℃. The cellulasemodification with non-covalent method changed its temperature profile, and the maximumactivities of the free and modified cellulases were observed at50℃and60℃, respectively.The optimum temperature and the optimum pH of Eudragit-cellulase-laccase were50℃andpH5.0, respectively.Covalent-Eudragit-cellulase retained about50%of its original activity after five cyclesof repeated uses, showing high reusability. Non-covalent-Eudragit-cellulase retained about30%of its original activity after five cycles of repeated uses. Eudragit-cellulase-laccase retainedabout40%of its original cellulase and laccase activities.3. The effects of covalent-Eudragit S-100-cellulase treatment on cotton fiber structureand the hydrolysis of different cellulosic fabrics or different forms of substrates withcovalent-Eudragit S-100-cellulase were studied.The results of XRD, ATR-FTIR and fibre accessibility indicated that the hydrolysisoccurring in the interior of the cotton fibers was limited during covalent-EudragitS-100-cellulase treatment.The results of hydrolysis of different forms of substrates (cotton fabric, cotton yarn,cotton fiber) indicated that the reducing sugars released from cotton fiber were39.90%morethan that from cotton yarn at free cellulase treatment; the reducing sugars released from cottonfiber were71.95%more than that from cotton yarn at covalent-Eudragit S-100-cellulasetreatment; the reducing sugars released from cotton yarn and fabric were close at freecellulase treatment; the reducing sugars released from cotton fabric were less than that fromcotton yarn at covalent-Eudragit S-100-cellulase treatment.The results of hydrolysis viscose fabrics, mercerizing fabrics, cotton fabrics and ramiefabrics by free cellulase or covalent-Eudragit S-100-cellulase treatments showed the moreinfluence on hydrolysis by free cellulase with the increase of fiber accessibility of the fabrics.4. The enzymatic treatments on bio-polishing of cotton and ramie fabrics using freecellulase or covalent-Eudragit S-100-cellulase were investigated.The results revealed that weight losses of cotton fabrics with free cellulase orcovalent-Eudragit S-100-cellulase treatments (cellulase activity5.64U/mL) were7.26%and5.34%, respectively; weight losses of ramie fabrics with free cellulase or covalent-EudragitS-100-cellulase treatments were2.79%and2.62%, respectively; fiber copper values ofcotton fabrics with free cellulase or covalent-Eudragit S-100-cellulase treatments were0.04861and0.02790, respectively; fiber copper values of ramie fabrics with free cellulase orcovalent-Eudragit S-100-cellulase treatments were0.02427and0.02286, respectively. Theresults showed that the cotton and ramie fibers treated with free cellulase were morehydrolyzed and more destruction than those treated with covalent-Eudragit S-100-cellulase.The results revealed that tensile strengths of cotton fabrics with free cellulase orcovalent-Eudragit S-100-cellulase treatments (cellulase activity1.88U/mL) were15.09%and5.61%, respectively; tensile strengths of ramie fabrics with free cellulase orcovalent-Eudragit S-100-cellulase treatments were24.60%and10.65%, respectively. As seen from TM-AFM and SEM images, the covalent-Eudragit S-100-cellulasetreatment could produce smooth cotton fibre surfaces without causing serious damages to thefibre surface structures.5. The enzymatic treatments of the denim fabrics using free cellulase and modifiedenzymes were investigated.At a cellulase activity of5.64U/mL, CIE L values of the denim fabrics treated with thefree and covalent Eudragit-cellulase were close to25. This result revealed that the enzymatictreatments with the free and covalent Eudragit-cellulase produced similar decolorationefficiency for the denim fabrics. The tensile strength losses of denim fabrics treated with freecellulase or covalent Eudragit-cellulase reached46.21%and30.9%, respectively.At a cellulase activity of1.88U/mL, CIE L values of the denim fabric treated with freecellulase, covalent Eudragit-cellulase and Eudragit-cellulase-laccase were22.91,22.68and25.76, respectively. The tensile strength losses of the denim fabric treated with free cellulase,covalent Eudragit-cellulase and Eudragit-cellulase-laccase were30.13%,13.41%and12.67%, respectively. The enzymatic treatments with Eudragit-cellulase-laccase produced betterdecoloration efficiency and higher tensile strength for the denim fabrics.更多还原