果胶酶促降解中的超声波作用途径及机理研究

发布时间：2018-10-12 08:53

【摘要】：果胶是一种存在于高等植物细胞壁中的酸性杂多糖,来源广泛,廉价易得。近年来,越来越多的研究者开始关注果胶的构效关系,并通过化学方法、酶法和物理方法等手段对果胶进行降解以获得具有生物活性的改性果胶。但是,化学方法污染严重,难以控制;物理方法降解效率较低,且多停留在实验阶段;酶法虽具有高效、专一性强等优点,但由于酶的造价昂贵导致生产成本提高。近年来,研究者开始将超声波应用于酶促反应中促进聚合物的降解,该方法具有高效率、高选择性和绿色环保等优点。但是,关于超声波在果胶酶促反应中的应用至今鲜有报道。本研究围绕超声波在果胶酶促降解反应中的应用展开,借助化学分析方法、动力学和热力学模型、蛋白质及多糖结构分析手段,系统研究了超声波对参与酶促反应的果胶酶的改性作用、果胶底物的预处理作用及其对酶促反应过程的影响。主要研究内容如下:(1)游离果胶酶的超声辐射改性效果及机理探究通过研究不同超声波条件下果胶酶活性的变化规律发现,低强度、短时间超声波作用可提高果胶酶的活性,而过高的超声波强度或过长的处理时间都会导致果胶酶的失活。实验得到果胶酶的最适改性条件为:超声波强度4.5 WmL-1、作用时间15 min;经超声改性的果胶酶催化的水解反应速率提高,反应的活化能(Ea)、焓变(ΔH)、熵变(ΔS)、自由能变(ΔG)均降低,说明超声作用提高了果胶酶的催化活性,使酶促反应更易进行;超声波处理后,果胶酶催化效率提高、与底物之间的亲和力增强;超声波处理不改变果胶酶的最适温度,但可以提高其在20-60 ℃温度范围内的反应稳定性;果胶酶在40-60 ℃温度范围内的失活动力学可通过两相模型进行拟合,超声波处理可提高果胶酶的热稳定性;超声波处理后,果胶酶蛋白荧光强度下降,二级结构中β-折叠含量上升、无规卷曲含量下降,说明酶蛋白的空间结构变得更加整齐有序、其活性中心可能受到一定影响,超声改性作用在24h内具有不可逆性。通过研究超声波在果胶酶改性中的自由基效应和机械效应发现:超声波强度越强、作用时间越长、操作温度越低,反应体系中羟自由基含量越高,超声波自由基效应越强;浓度为4 mM的硫脲可有效屏蔽超声波空化产生的自由基效应,因此可添加到处理体系中用作自由基清除剂;果胶酶溶液的浓度不会影响最佳超声波改性条件,但酶浓度越低,果胶酶在超声波场中的改性效果越显著;在果胶酶的超声辐射改性过程中,超声波的自由基效应和机械效应都起到了一定作用,其中在酶的活化阶段,超声波的机械效应起主导作用;在酶的失活阶段,超声波的自由基效应起主导作用。(2)超声波辅助果胶酶固定化及固定化酶超声辐射改性的研究固定化果胶酶的制备通过包埋交联法进行,固定化率在海藻酸钠浓度为2%、氯化钙溶液浓度为0.15 M的条件下达到最大值;当超声波作用于果胶酶的固定化过程中时,果胶酶的固定化率在强度为9.0 W mL-1、作用时间为20 min的超声波条件下取得最大值;当超声波作用于固定化酶改性时,果胶酶的活性在强度为4.5WmL-1、作用时间为10min的超声波条件下取得最大值。固定化载体的结构在超声波作用下变得膨胀松散、凝胶基质孔径增大,使果胶酶更容易作用于果胶底物;超声波作用可提高固定化果胶酶的催化效率及其与底物间的亲和力;固定化和超声波的作用均不改变果胶酶的最适温度和最适pH,但固定化果胶酶对高温和高pH的耐受性增强,而超声波作用可增强果胶酶在每个温度和pH下的反应活性;固定化显著增强了果胶酶的热稳定性和反应稳定性,同时使其获得了重复利用性;超声波的作用略微降低了固定化果胶酶的稳定性,但消极影响较小。(3)超声波预处理果胶底物对酶促反应及其产物的影响研究选择强度为18.0 WmL-1、处理时间为30 min的超声波条件作为果胶的预处理条件;经超声波预处理后果胶的酶促降解效率及其与果胶酶之间的亲和力显著增强;超声波预处理可显著降低果胶底物的分子量、分散性系数和甲酯化度,从而为果胶酶解反应提供更适宜的底物;果胶经预处理后,其酶解产物基本结构不变,主链降解更完全,半乳糖含量提高,抗癌活性明显增强。(4)超声波和果胶酶在果胶降解中的协同效应及机理研究强度为4.5 W mL-1、处理时间为10 min的超声波条件对果胶的酶促降解促进效果最强;超声波作用于酶促反应可提高果胶酶的催化效率及其与底物之间的亲和力,并对果胶酶的结构进行了有利改性;果胶分子量的超声降解、酶促降解和超声波-果胶酶联用降解过程均符合二级动力学模型,表明三个反应中果胶的降解是随机进行的;通过定义协同性系数的概念,分别从还原糖产量增加和果胶分子量降解的角度探索了超声波和果胶酶的共同作用效果,发现二者在果胶降解过程中产生协同效应,且温度越低、协同效应越强;超声波和果胶酶的联用可显著降低果胶的甲酯化度,但可保持其乙酰化度;酶解反应和声酶解反应均通过切断果胶的同型半乳糖醛酸聚糖(HG)区域引起降解,并可较好地保留其鼠李半乳糖醛酸聚糖Ⅰ(RG-Ⅰ)区域;超声波和果胶酶联用可完全降解果胶纳米结构中的聚合体和多分支结构,降解产物以短的线性单片段和单分支结构的形式存在。本研究的结果为改性果胶的制备提供了新的思路,并为超声波在酶促反应各个环节中的应用提供了理论基础,对于扩大超声波在食品工业中的应用范围具有重要意义。
[Abstract]:Pectin is an acidic heteropolysaccharide present in the cell wall of higher plants, which is widely available and cheap. In recent years, more and more researchers have begun to focus on the structural effect of pectin and degrade pectin by chemical methods, enzymatic methods and physical methods to obtain modified pectin with biological activity. However, the chemical method has serious pollution and is difficult to control; the degradation efficiency of the physical method is lower, and the multiple stays in the experimental stage; the enzymatic method has the advantages of high efficiency, strong specificity and the like, but the production cost is improved due to the high cost of the enzyme. In recent years, researchers have begun to apply ultrasound to enzymatic reactions to promote the degradation of polymers, which has the advantages of high efficiency, high selectivity and environmental protection. However, the application of ultrasonic wave in the enzymatic reaction of pectinase has been seldom reported. The application of ultrasonic wave in the enzymatic degradation reaction of pectinase was studied. By means of chemical analysis, dynamics and thermodynamic model, protein and polysaccharide structure analysis, the modification effect of ultrasonic wave on the enzymatic reaction of pectinase was studied. The pretreatment of pectin substrate and its effect on enzymatic reaction process. The main research contents are as follows: (1) The effect and mechanism of ultrasonic radiation modification of free pectinase can improve the activity of pectinase by studying the change of pectinase activity under different ultrasonic conditions. too high ultrasonic intensity or too long processing time can lead to deactivation of the pectinase. The optimum modification conditions of pectinase were as follows: ultrasonic intensity 4.5 WmL-1, action time 15 min, hydrolysis reaction rate increased by ultrasonic-modified pectinase, activation energy of reaction (pH value), temperature change (SO3H), entropy change (SOG), and free energy change (WYG) all decreased. The method has the advantages that the catalytic activity of the pectinase is improved by the ultrasonic action, the enzymatic reaction is more easily carried out, the catalytic efficiency of the pectinase is improved after the ultrasonic treatment, the affinity between the pectinase and the substrate is enhanced, the optimum temperature of the pectinase is not changed by the ultrasonic treatment, but the reaction stability of the pectinase in the temperature range of 20-60 DEG C can be improved; the loss activity mechanics of the pectinase in the temperature range of 40-60 DEG C can be fitted through two-phase models; the ultrasonic treatment can improve the thermal stability of the pectinase; and after the ultrasonic treatment, the fluorescence intensity of the pectinase protein decreases, In the secondary structure, the fold increase and random curl content decrease, indicating that the spatial structure of the enzyme protein becomes more and more orderly, and the active center of the enzyme protein may be affected, and the ultrasonic modification effect can be inexcitable in 24h. By studying the free radical effect and mechanical effect of ultrasonic wave in pectase modification, the stronger the ultrasonic intensity, the longer the action time, the lower the operating temperature, the higher the hydroxyl radical content in the reaction system, the stronger the ultrasonic free radical effect. The free radical effect generated by the ultrasonic cavitation can be effectively shielded by the solution with the concentration of 4 mM, so that the free radical scavenger can be added into the treatment system, the concentration of the pectase solution does not influence the optimal ultrasonic modification condition, but the lower the enzyme concentration, The more significant the modification effect of pectinase in the ultrasonic field, the free radical effect and the mechanical effect of the ultrasonic wave play a certain role in the process of ultrasonic radiation modification of the pectase, wherein the mechanical effect of the ultrasonic wave plays a leading role in the activation stage of the enzyme, The free radical effect of ultrasound plays a leading role. (2) The preparation of immobilized pectinase was carried out by embedding cross-linking method, the concentration of immobilized pectinase was 2%, and the concentration of calcium chloride solution was 0. 15M. When the ultrasonic wave acts on the immobilized enzyme, the immobilized rate of the pectinase is 9.0 W mL-1, the action time is 20 min, and the maximum value is obtained; when the ultrasonic wave acts on the immobilized enzyme, the activity of the pectinase is 4. 5WmL-1, The maximum value was obtained under ultrasonic conditions with an action time of 10min. the structure of the immobilized carrier becomes loose under the action of ultrasonic waves, the pore diameter of the gel matrix is increased, the pectase can be more easily applied to the pectin substrate, and the ultrasonic action can improve the catalytic efficiency of the immobilized pectinase and the affinity between the immobilized pectinase and the substrate; The effect of immobilization and ultrasonic did not change the optimum temperature and the optimum pH of pectinase, but the tolerance of immobilized pectinase to high temperature and high pH was enhanced, and the ultrasonic action could enhance the reaction activity of pectinase at each temperature and pH. The immobilization of pectinase significantly enhanced the thermal stability and the reaction stability of the pectinase, and the repeated use of the pectinase was also achieved; the effect of the ultrasonic wave slightly reduced the stability of the immobilized pectinase but had a small negative effect. (3) the influence of the ultrasonic pretreatment pectin substrate on the enzymatic reaction and the product thereof is 18. 0WmL-1, the processing time is 30 minutes, and the ultrasonic condition is used as the pretreatment condition of the pectin; the enzymatic degradation efficiency of pectin after ultrasonic pretreatment and the affinity between the pectin and the pectinase are greatly enhanced; the ultrasonic pretreatment can obviously reduce the molecular weight, the dispersity coefficient and the methyl esterification degree of the pectin substrate, thereby providing a more suitable substrate for the enzymolysis reaction of the pectinase; After the pectin is pretreated, the basic structure of the enzymolysis product is unchanged, the degradation of the main chain is more complete, the galactose content is improved, and the anti-cancer activity is obviously enhanced. (4) The synergistic effect and mechanism of ultrasonic and pectase in pectin degradation were 4. 5 W mL-1, the treatment time was 10 min, and the enzymatic degradation of pectin was the strongest. The ultrasonic action on the enzymatic reaction can improve the catalytic efficiency of the pectinase and the affinity between the pectinase and the substrate, and the structure of the pectinase is advantageously modified, the ultrasonic degradation of the pectin molecular weight, the enzymatic degradation and the degradation process of the ultrasonic-pectinase meet the secondary kinetic model, It is shown that the degradation of pectin in three reactions is random; by defining the concept of synergistic coefficient, the co-action effect of ultrasonic and pectinase is explored from the viewpoint of increasing the yield of reducing sugar and the degradation of pectin molecular weight, and the synergistic effect is found in the process of pectin degradation. and the lower the temperature, the stronger the synergistic effect, the combination of the ultrasonic wave and the pectinase can remarkably reduce the degree of methyl esterification of the pectin, and the combination of the ultrasonic wave and the pectinase can completely degrade the polymer and the multi-branch structure in the pectin nano structure, and the degradation products exist in the form of short linear single-piece sections and single-branch structures. The results of this study provide a new idea for the preparation of modified pectin, and provide a theoretical basis for the application of ultrasonic wave in each link of enzymatic reaction. It is of great significance to expand the application scope of ultrasonic wave in food industry.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TS201.25

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