果膠酶促降解中的超聲波作用途徑及機(jī)理研究
發(fā)布時(shí)間：2018-10-12 08:53
【摘要】：果膠是一種存在于高等植物細(xì)胞壁中的酸性雜多糖,來(lái)源廣泛,廉價(jià)易得。近年來(lái),越來(lái)越多的研究者開(kāi)始關(guān)注果膠的構(gòu)效關(guān)系,并通過(guò)化學(xué)方法、酶法和物理方法等手段對(duì)果膠進(jìn)行降解以獲得具有生物活性的改性果膠。但是,化學(xué)方法污染嚴(yán)重,難以控制;物理方法降解效率較低,且多停留在實(shí)驗(yàn)階段;酶法雖具有高效、專一性強(qiáng)等優(yōu)點(diǎn),但由于酶的造價(jià)昂貴導(dǎo)致生產(chǎn)成本提高。近年來(lái),研究者開(kāi)始將超聲波應(yīng)用于酶促反應(yīng)中促進(jìn)聚合物的降解,該方法具有高效率、高選擇性和綠色環(huán)保等優(yōu)點(diǎn)。但是,關(guān)于超聲波在果膠酶促反應(yīng)中的應(yīng)用至今鮮有報(bào)道。本研究圍繞超聲波在果膠酶促降解反應(yīng)中的應(yīng)用展開(kāi),借助化學(xué)分析方法、動(dòng)力學(xué)和熱力學(xué)模型、蛋白質(zhì)及多糖結(jié)構(gòu)分析手段,系統(tǒng)研究了超聲波對(duì)參與酶促反應(yīng)的果膠酶的改性作用、果膠底物的預(yù)處理作用及其對(duì)酶促反應(yīng)過(guò)程的影響。主要研究?jī)?nèi)容如下:(1)游離果膠酶的超聲輻射改性效果及機(jī)理探究通過(guò)研究不同超聲波條件下果膠酶活性的變化規(guī)律發(fā)現(xiàn),低強(qiáng)度、短時(shí)間超聲波作用可提高果膠酶的活性,而過(guò)高的超聲波強(qiáng)度或過(guò)長(zhǎng)的處理時(shí)間都會(huì)導(dǎo)致果膠酶的失活。實(shí)驗(yàn)得到果膠酶的最適改性條件為:超聲波強(qiáng)度4.5 WmL-1、作用時(shí)間15 min;經(jīng)超聲改性的果膠酶催化的水解反應(yīng)速率提高,反應(yīng)的活化能(Ea)、焓變(ΔH)、熵變(ΔS)、自由能變(ΔG)均降低,說(shuō)明超聲作用提高了果膠酶的催化活性,使酶促反應(yīng)更易進(jìn)行;超聲波處理后,果膠酶催化效率提高、與底物之間的親和力增強(qiáng);超聲波處理不改變果膠酶的最適溫度,但可以提高其在20-60 ℃溫度范圍內(nèi)的反應(yīng)穩(wěn)定性;果膠酶在40-60 ℃溫度范圍內(nèi)的失活動(dòng)力學(xué)可通過(guò)兩相模型進(jìn)行擬合,超聲波處理可提高果膠酶的熱穩(wěn)定性;超聲波處理后,果膠酶蛋白熒光強(qiáng)度下降,二級(jí)結(jié)構(gòu)中β-折疊含量上升、無(wú)規(guī)卷曲含量下降,說(shuō)明酶蛋白的空間結(jié)構(gòu)變得更加整齊有序、其活性中心可能受到一定影響,超聲改性作用在24h內(nèi)具有不可逆性。通過(guò)研究超聲波在果膠酶改性中的自由基效應(yīng)和機(jī)械效應(yīng)發(fā)現(xiàn):超聲波強(qiáng)度越強(qiáng)、作用時(shí)間越長(zhǎng)、操作溫度越低,反應(yīng)體系中羥自由基含量越高,超聲波自由基效應(yīng)越強(qiáng);濃度為4 mM的硫脲可有效屏蔽超聲波空化產(chǎn)生的自由基效應(yīng),因此可添加到處理體系中用作自由基清除劑;果膠酶溶液的濃度不會(huì)影響最佳超聲波改性條件,但酶濃度越低,果膠酶在超聲波場(chǎng)中的改性效果越顯著;在果膠酶的超聲輻射改性過(guò)程中,超聲波的自由基效應(yīng)和機(jī)械效應(yīng)都起到了一定作用,其中在酶的活化階段,超聲波的機(jī)械效應(yīng)起主導(dǎo)作用;在酶的失活階段,超聲波的自由基效應(yīng)起主導(dǎo)作用。(2)超聲波輔助果膠酶固定化及固定化酶超聲輻射改性的研究固定化果膠酶的制備通過(guò)包埋交聯(lián)法進(jìn)行,固定化率在海藻酸鈉濃度為2%、氯化鈣溶液濃度為0.15 M的條件下達(dá)到最大值;當(dāng)超聲波作用于果膠酶的固定化過(guò)程中時(shí),果膠酶的固定化率在強(qiáng)度為9.0 W mL-1、作用時(shí)間為20 min的超聲波條件下取得最大值;當(dāng)超聲波作用于固定化酶改性時(shí),果膠酶的活性在強(qiáng)度為4.5WmL-1、作用時(shí)間為10min的超聲波條件下取得最大值。固定化載體的結(jié)構(gòu)在超聲波作用下變得膨脹松散、凝膠基質(zhì)孔徑增大,使果膠酶更容易作用于果膠底物;超聲波作用可提高固定化果膠酶的催化效率及其與底物間的親和力;固定化和超聲波的作用均不改變果膠酶的最適溫度和最適pH,但固定化果膠酶對(duì)高溫和高pH的耐受性增強(qiáng),而超聲波作用可增強(qiáng)果膠酶在每個(gè)溫度和pH下的反應(yīng)活性;固定化顯著增強(qiáng)了果膠酶的熱穩(wěn)定性和反應(yīng)穩(wěn)定性,同時(shí)使其獲得了重復(fù)利用性;超聲波的作用略微降低了固定化果膠酶的穩(wěn)定性,但消極影響較小。(3)超聲波預(yù)處理果膠底物對(duì)酶促反應(yīng)及其產(chǎn)物的影響研究選擇強(qiáng)度為18.0 WmL-1、處理時(shí)間為30 min的超聲波條件作為果膠的預(yù)處理?xiàng)l件;經(jīng)超聲波預(yù)處理后果膠的酶促降解效率及其與果膠酶之間的親和力顯著增強(qiáng);超聲波預(yù)處理可顯著降低果膠底物的分子量、分散性系數(shù)和甲酯化度,從而為果膠酶解反應(yīng)提供更適宜的底物;果膠經(jīng)預(yù)處理后,其酶解產(chǎn)物基本結(jié)構(gòu)不變,主鏈降解更完全,半乳糖含量提高,抗癌活性明顯增強(qiáng)。(4)超聲波和果膠酶在果膠降解中的協(xié)同效應(yīng)及機(jī)理研究強(qiáng)度為4.5 W mL-1、處理時(shí)間為10 min的超聲波條件對(duì)果膠的酶促降解促進(jìn)效果最強(qiáng);超聲波作用于酶促反應(yīng)可提高果膠酶的催化效率及其與底物之間的親和力,并對(duì)果膠酶的結(jié)構(gòu)進(jìn)行了有利改性;果膠分子量的超聲降解、酶促降解和超聲波-果膠酶聯(lián)用降解過(guò)程均符合二級(jí)動(dòng)力學(xué)模型,表明三個(gè)反應(yīng)中果膠的降解是隨機(jī)進(jìn)行的;通過(guò)定義協(xié)同性系數(shù)的概念,分別從還原糖產(chǎn)量增加和果膠分子量降解的角度探索了超聲波和果膠酶的共同作用效果,發(fā)現(xiàn)二者在果膠降解過(guò)程中產(chǎn)生協(xié)同效應(yīng),且溫度越低、協(xié)同效應(yīng)越強(qiáng);超聲波和果膠酶的聯(lián)用可顯著降低果膠的甲酯化度,但可保持其乙�；�;酶解反應(yīng)和聲酶解反應(yīng)均通過(guò)切斷果膠的同型半乳糖醛酸聚糖(HG)區(qū)域引起降解,并可較好地保留其鼠李半乳糖醛酸聚糖Ⅰ(RG-Ⅰ)區(qū)域;超聲波和果膠酶聯(lián)用可完全降解果膠納米結(jié)構(gòu)中的聚合體和多分支結(jié)構(gòu),降解產(chǎn)物以短的線性單片段和單分支結(jié)構(gòu)的形式存在。本研究的結(jié)果為改性果膠的制備提供了新的思路,并為超聲波在酶促反應(yīng)各個(gè)環(huán)節(jié)中的應(yīng)用提供了理論基礎(chǔ),對(duì)于擴(kuò)大超聲波在食品工業(yè)中的應(yīng)用范圍具有重要意義。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：TS201.25
本文編號(hào)：2265526
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：TS201.25
【相似文獻(xiàn)】
相關(guān)期刊論文 前10條
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