反胶束中漆酶催化降解愈创木酚的研究

发布时间：2018-03-22 22:21

本文选题：木质素　切入点：愈创木酚　出处：《湖南大学》2015年硕士论文　论文类型：学位论文

【摘要】：木质素是一种自然界中储量仅次于纤维素的有机高分子物质,能够以每年500亿吨左右的速度再生。造纸制浆工业每年要从植物中分离出大约1.4亿吨纤维素,同时得到5000万吨左右的木质素副产品;分散剂、吸附剂/解吸剂、石油回收助剂、沥青乳化剂等工业产品中也用到了木质素及其衍生物,迄今为止,每年有超过95%的木质素以“黑液”形式直接排入江河或浓缩后烧掉,很少得到有效利用。反胶束(reversed micelle)或逆胶束、反胶团是表面活性剂溶于非极性的有机溶剂内浓度超过临界胶束浓度(CMC)时,自发形成的稳定澄清的均相W/O纳米尺寸聚集体。反胶束中,表面活性剂的非极性疏水基团与外部非极性的有机溶剂接触,极性基团排列在内围绕成一个具有溶解极性物质能力的极性核(polar core)。极性核溶解水后,就形成了“水池”(water pool),反胶束的这种结构能够同时增溶亲水性的酶和疏水性的底物,模拟生物分子中酶反应的天然微环境,使得酶在其中表现出优于水介体中反应的活性。因此,近年来反胶束酶催化体系得到了广泛的关注和研究。本课题将亲脂性的木质素模式物愈创木酚与亲水性的漆酶溶解于反胶束溶液中,探究反胶束中漆酶对木质素的作用机制,并通过实验建立改变反应条件反胶束中漆酶的酶活变化和最佳反应条件下漆酶对愈创木酚的催化效率、建立反应的动力学模型和动力学参数,深入研究反胶束中漆酶催化愈创木酚的反应机理。本文的具体研究工作及成果包括以下内容:本课题首先构建CTAB,Tween-80,AOT以及鼠李糖脂四种反胶束体系,然后研究CTAB,Tween-80,AOT以及鼠李糖脂的最佳表面活性剂浓度、相对应的最佳含水率条件下漆酶催化愈创木酚的效率和动力学机制。并且对反胶束体系中酶催化愈创木酚降解的反应机制进行研究,测定反应产物,参照已有研究,分析漆酶催化愈创木酚的反应机制。最后引入甘油对鼠李糖脂构建的反胶束体系进行改性,构建鼠李糖脂/异辛烷/正己醇/甘油/水反胶束体系并且筛选出最佳表面活性剂浓度以及对应的最大增溶水量,测定优化反应条件下反胶束中漆酶催化愈创木酚的效率,对甘油改性前后漆酶的催化效率进行分析比较。通过对不同反胶束环境中漆酶酶活的对比分析,确定在CTAB,Tween-80,AO T以及鼠李糖脂4种表面活性剂构建的反胶束体系中,适合酶解反应的最佳表面活性剂浓度和对应的最佳含水率W0分别为:CTAB浓度20mmol/L,W0=20;Tween 80浓度50 mmo l/L,W0=25;AO T浓度30 mmo l/L,W0=10;鼠李糖脂浓度10mmo l/L,W0=30。在阳离子、阴离子和非离子几种类型的表面活性剂中,同属于阴离子表面活性剂的AOT和鼠李糖脂构建的反胶束体系的底物降解率均高于CTAB和Tween 80体系,说明阴离子表面活性剂更适宜漆酶的反应,并且同属于阴离子表面活性剂的生物表面活性剂鼠李糖脂构建的反胶束体系中漆酶的活性和稳定性均高于AOT反胶束,在最适含水率条件下制备构建的反胶束体系中,反应6h后,各反胶束体系在底物愈创木酚浓度为1mmol/L时,CTAB、Tween80、AO T和鼠李糖脂4种表面活性剂构建的反胶束体系中木质素模式物的漆酶降解效率分别为21.33%、30.85%、36.02%、65.45%。四种反胶束体系中漆酶对愈创木酚的催化反应遵循均Michaelis-Menten动力学。漆酶通过参与愈创木酚的解聚和聚合反应,将愈创木酚催化为水溶性底物4-邻甲基苯酚。通过甘油对鼠李糖脂构建的反胶束体系进行改性,可以增加反胶束体系胶团半径,有利于反胶束中漆酶分子的溶入,增强反胶束中酶构象的稳定性,有利于提高漆酶的活性和稳定性。
[Abstract]:Lignin is a kind of nature reserves after cellulose organic polymer material, to 500 tons per year. The regeneration rate of paper pulp industry each year to about 1.4 tons of cellulose were isolated from the plant, and 50 million tons of lignin by-product; dispersant, adsorption / desorption agent, oil recovery agent asphalt emulsifier and other industrial products were also used in lignin and its derivatives, so far, more than 95% of the wood known as "black liquor" form directly into rivers or concentrated burned every year, have not been effectively used. The reverse micelles (reversed micelle) or reverse micelles, reverse micelles is a surface active agent is dissolved in an organic solvent within the concentration exceeds the critical micelle concentration (CMC), homogeneous W/O nanoparticles size of the spontaneous formation of stable clarification. In reverse micelles, non-polar hydrophobic groups and surfactant Non polar organic solvent exposure outside, polar groups, arranged around the polar nucleus of a solution with polar material capability (polar core). The polar nucleus dissolved water, formed a "pool" (water pool), the structure of reverse micelles can also hydrophilic and hydrophobic solubilization enzyme the substrate, enzyme reaction simulating molecular biology in natural micro environment, the enzyme showed superior water reaction mediator in activity in them. Therefore, in recent years the reversed micellar enzymatic system has received wide attention and research. This paper will lipophilic lignin model compounds guaiacol and hydrophilic the laccase dissolved in reverse micelles, explore the laccase in reverse micelles on the lignin mechanism, and through the experiment to establish change reaction conditions in reverse micelles laccase enzyme catalytic efficiency change and the optimum reaction conditions of laccase of guaiacol, establish The kinetic model and kinetic parameters of the reaction, the catalytic reaction mechanism of guaiacol laccase in-depth study in reverse micelles of guaiacol. The specific research work and achievements of this paper include the following contents: firstly the construction of CTAB, Tween-80, AOT four and rhamnolipid reverse micellar system, then CTAB, Tween-80, AOT and the best surfactant rhamnolipid glycolipid surfactant concentration efficiency and kinetics of laccase catalyzed conditions of guaiacol guaiacol corresponding to the optimum water ratio. And study the reaction mechanism of reverse micelles enzyme catalyzed degradation of guaiacol, determination of reaction products, according to the existing research, analysis of the reaction mechanism catalyzed by guaiacol. Finally the introduction of reverse micelles the system construction of the glycerol rhamnolipid was modified to construct rhamnolipid / isooctane / hexanol / glycerol / water reverse micelles and selected the best surfactant concentration And the maximum water solubilization efficiency, catalytic determination of guaiacol under the optimal reaction conditions of laccase in reverse micelles of guaiacol, glycerol modified laccase catalytic efficiency of comparison. Through the contrastive analysis of different reverse micelles laccase activity in the environment, in CTAB, Tween-80, AO, T and rhamnolipid 4 surface active agent construction in the reverse micelles, the best surface activity suitable for enzymatic hydrolysis agent concentration and the optimum water ratio corresponding to the W0 were: CTAB concentration 20mmol/L, W0=20; Tween 80 concentration of 50 MMO l/L, W0=25 AO; T W0=10; l/L MMO 30 concentration, the concentration of rhamnolipid 10mmo l/L, W0=30. in the surface of cation. The active agent of anionic and nonionic types of substrate degradation of reverse micelles constructed belong to anionic surfactant AOT and rhamnolipid were higher than that of CTAB and Tween 80 system, that the anionic surface active Agent is more suitable for the laccase reaction, laccase reverse micelles of rhamnolipid biosurfactant and belong to anionic surfactant in the construction of the activity and stability of AOT were higher than that of reverse micelles, reverse micelles in the optimum moisture content under the condition of the preparation of construction, after reaction of 6h, the reverse micelles in the substrate guaiacol concentration of 1mmol/L, CTAB, Tween80, laccase degradation efficiency of AO T and rhamnolipid 4 surfactant constructed in the reverse micelles of lignin model compounds were 21.33%, 30.85%, 36.02%, four 65.45%. of laccase in reverse micelles catalysis of guaiacol reaction followed Michaelis-Menten kinetics. Laccase by participating in guaiacol depolymerization and polymerization, the guaiacol catalyzed water soluble o-cresol. The substrate 4- was modified by reverse micelles of glycerol on the construction of rhamnolipid can increase Reverse micelle micelle radius, in favor of laccase in reverse micelles into the molecular conformation of the enzyme, enhance the stability of reverse micelles, could enhance the laccase activity and stability.

【学位授予单位】：湖南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：O643.3;X703

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