【作者】 吴淑芳；

【导师】 李忠正； 丁少军；

【作者基本信息】 南京林业大学 ， 林产化学加工工程， 2005， 博士

【摘要】 以往对纤维素酶结构及功能研究的出发点主要致力于提高纤维素酶对生物质的降解能力,这需要多组分纤维素酶的协同作用。近年来,纤维素酶在植物纤维改性中的应用受到关注。大量研究结果表明,内切型纤维素酶在植物纤维酶法改性中起着关键作用。由于天然纤维素酶来源广泛,组分复杂,其分离纯化相当困难,使单一组分酶的应用受到一定的限制。内切型纤维素酶EGI是用基因工程方法得到的重组酶,其活性高,纯度高,最适pH为7.5,加之其制备方便,在植物纤维改性中有很好的应用前景。EGI最终的高效应用尚需作进一步的基础研究。 本研究即从构建无纤维素吸附区的内切纤维素酶EGI-CD着手,通过对EGI和EGI-CD酶对不同性质的底物吸附及水解性能的比较,讨论了EGI不溶性纤维素在底物上的吸附性能及其对水解的影响,以及纤维素吸附区(CBD)在EGI对植物纤维水解及改性中的作用。 研究主要得到了以下结果: 1.利用基因工程方法成功构建了无纤维素吸附区的内切纤维素酶EGI-CD,为研究纤维素吸附区(CBD)对EGI酶的吸附性能和底物特异性的影响提供了条件。 2.纤维素酶在不溶性纤维素底物上的吸附因底物结构的不同而异。吸附平衡时,EGI或EGI-CD对磷酸润胀纤维素、滤纸和微晶纤维素的吸附率分别为64.2%、43.5%、33.6%或38.8%、14.5%和0,因此CBD的去除影响酶在不溶性纤维素底物上的吸附,并且底物结晶度高则影响大。这一结果为EGI酶在基因水平上的改造指出了方向。 3.EGI对于不溶性纤维素底物的水解能力主要取决于酶在底物上的吸附程度。CBD的吸附不仅提供了酶与底物接触的机会,CBD本身也会使纤维发生变化,尽管这种变化不会导致底物水解,但对催化域的水解具有促进作用。 4.一定量的内切纤维素酶EGI在较短的时间内即可水解底物产生还原糖,但随着时间的延长,其产生的还原糖量趋于稳定,反映了EGI酶仅作用于纤维表面,具有植物纤维改性所需要的酶的特性。 5.EGI酶可明显提高混合办公废纸的油墨脱出率。脱墨前采用NaOH预处理可使油墨脱出率达到90%以上。靛蓝牛仔织物经EGI处理后后织物表面雪花点增多、立体感增强、色光好,并且对强度没有明显的损伤。更多还原

【Abstract】 Most of post work on cellulase structures and its functions was focused on how to enhance its biomass lignocellulosic decomposability, which needed many groups of cellulase to synergic action. Recently it was paid more attention to the application of cellulase on modification of plant fibre. Many results showed that endoglucanase(EG) played key role on modification of plant fibre. Because of diversity of nature cellulase sources, complexity of multi-components and difficult of separation , application of single cellulase component was much restricted. EGI one kind of endoglucanase was obtained by gene engineering as recombinant enzyme. Due to its high activity, high purity, optimal pH value 7.5 and convenient production, EGI had brilliant future on modification of plant cellulose. But further study was still needed for EGI final effective application.In this paper major work was done on the construction of expression vector containing of endoglucanase EGI-CD and it’s heterogeneous expression in Pichia pastoris. With the comparison of adsorption and hydrolysis of different substrates by EGI and EGI-CD, the influcence of CBD on the adsorption and on its hydrosis of enzyme on substrates were discussed in order to improve its more economically application of EGI on the industrial condition with good modification efficiency. Major results as following:1. EGI-CD was successfully obtained by gene engineering, and used as material for the study of the influence of the CBD on adsorption and specificities of EGI on different substrates.2. Adsorption of cellulase on insoluble cellulose was depended on the structures of substrates.The influcence of CBD on adsorption of EGI on cellulose was positively relation to crystallization of cellulose. The percentages adsorption of EGI or EGI-CD on swollen cellulose、 filter paper and crystalline cellulose were 64.2%, 43.5%, 33.6% or 38.8%, 14.5%, 0, respectively. The results indicated deletion of CBD decreased adsorption rate of EGI on insoluble cellulose substrate. Such results provided direction for EGI evolution by gene engineering.3. Hydrosis of insoluble cellulose by EGI mainly lied on the adsorption of enzyme on substrates. The adsorption of EGI-CD without CBD only provided the contact opportunity of enzyme and substrates. While the adsorption of EGI with CBD not only provided the contact opportunity of enzyme and substrates, but also caused the cellulos structure alteration. Although such alteration could not result in the hydrosis of substrates directly, it promoted the hydrosis of catalytic domain of enzyme.4. EGI could release reducing sugar from cellulose within short time. But with the timeincrease, the quantity of reducing sugar trended stabilization. These results showed EGI just attacked on cellulose surface, indicated EGI had the characteristic for modification of plant cellulose.5. EGI could obviously improve the deinking efficiency of mixed office waste (MOW). The pretreatment of NaOH before deinking could make deinking efficiency more than 90%. EGI treatment improved the surface properties of denim fabric with more snowflake, more third dimension and better color, but no effect on the strength of denim fabric.更多还原