果膠酶促降解中的超聲波作用途徑及機理研究
發(fā)布時間：2018-10-12 08:53
【摘要】：果膠是一種存在于高等植物細胞壁中的酸性雜多糖,來源廣泛,廉價易得。近年來,越來越多的研究者開始關注果膠的構效關系,并通過化學方法、酶法和物理方法等手段對果膠進行降解以獲得具有生物活性的改性果膠。但是,化學方法污染嚴重,難以控制;物理方法降解效率較低,且多停留在實驗階段;酶法雖具有高效、專一性強等優(yōu)點,但由于酶的造價昂貴導致生產成本提高。近年來,研究者開始將超聲波應用于酶促反應中促進聚合物的降解,該方法具有高效率、高選擇性和綠色環(huán)保等優(yōu)點。但是,關于超聲波在果膠酶促反應中的應用至今鮮有報道。本研究圍繞超聲波在果膠酶促降解反應中的應用展開,借助化學分析方法、動力學和熱力學模型、蛋白質及多糖結構分析手段,系統(tǒng)研究了超聲波對參與酶促反應的果膠酶的改性作用、果膠底物的預處理作用及其對酶促反應過程的影響。主要研究內容如下:(1)游離果膠酶的超聲輻射改性效果及機理探究通過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),低強度、短時間超聲波作用可提高果膠酶的活性,而過高的超聲波強度或過長的處理時間都會導致果膠酶的失活。實驗得到果膠酶的最適改性條件為:超聲波強度4.5 WmL-1、作用時間15 min;經超聲改性的果膠酶催化的水解反應速率提高,反應的活化能(Ea)、焓變(ΔH)、熵變(ΔS)、自由能變(ΔG)均降低,說明超聲作用提高了果膠酶的催化活性,使酶促反應更易進行;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;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 ℃溫度范圍內的反應穩(wěn)定性;果膠酶在40-60 ℃溫度范圍內的失活動力學可通過兩相模型進行擬合,超聲波處理可提高果膠酶的熱穩(wěn)定性;超聲波處理后,果膠酶蛋白熒光強度下降,二級結構中β-折疊含量上升、無規(guī)卷曲含量下降,說明酶蛋白的空間結構變得更加整齊有序、其活性中心可能受到一定影響,超聲改性作用在24h內具有不可逆性。通過研究超聲波在果膠酶改性中的自由基效應和機械效應發(fā)現(xiàn):超聲波強度越強、作用時間越長、操作溫度越低,反應體系中羥自由基含量越高,超聲波自由基效應越強;濃度為4 mM的硫脲可有效屏蔽超聲波空化產生的自由基效應,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,果膠酶在超聲波場中的改性效果越顯著;在果膠酶的超聲輻射改性過程中,超聲波的自由基效應和機械效應都起到了一定作用,其中在酶的活化階段,超聲波的機械效應起主導作用;在酶的失活階段,超聲波的自由基效應起主導作用。(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包埋交聯(lián)法進行,固定化率在海藻酸鈉濃度為2%、氯化鈣溶液濃度為0.15 M的條件下達到最大值;當超聲波作用于果膠酶的固定化過程中時,果膠酶的固定化率在強度為9.0 W mL-1、作用時間為20 min的超聲波條件下取得最大值;當超聲波作用于固定化酶改性時,果膠酶的活性在強度為4.5WmL-1、作用時間為10min的超聲波條件下取得最大值。固定化載體的結構在超聲波作用下變得膨脹松散、凝膠基質孔徑增大,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,但固定化果膠酶對高溫和高pH的耐受性增強,而超聲波作用可增強果膠酶在每個溫度和pH下的反應活性;固定化顯著增強了果膠酶的熱穩(wěn)定性和反應穩(wěn)定性,同時使其獲得了重復利用性;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)超聲波預處理果膠底物對酶促反應及其產物的影響研究選擇強度為18.0 WmL-1、處理時間為30 min的超聲波條件作為果膠的預處理條件;經超聲波預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;超聲波預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提供更適宜的底物;果膠經預處理后,其酶解產物基本結構不變,主鏈降解更完全,半乳糖含量提高,抗癌活性明顯增強。(4)超聲波和果膠酶在果膠降解中的協(xié)同效應及機理研究強度為4.5 W mL-1、處理時間為10 min的超聲波條件對果膠的酶促降解促進效果最強;超聲波作用于酶促反應可提高果膠酶的催化效率及其與底物之間的親和力,并對果膠酶的結構進行了有利改性;果膠分子量的超聲降解、酶促降解和超聲波-果膠酶聯(lián)用降解過程均符合二級動力學模型,表明三個反應中果膠的降解是隨機進行的;通過定義協(xié)同性系數(shù)的概念,分別從還原糖產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)二者在果膠降解過程中產生協(xié)同效應,且溫度越低、協(xié)同效應越強;超聲波和果膠酶的聯(lián)用可顯著降低果膠的甲酯化度,但可保持其乙酰化度;酶解反應和聲酶解反應均通過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構中的聚合體和多分支結構,降解產物以短的線性單片段和單分支結構的形式存在。本研究的結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各個環(huán)節(jié)中的應用提供了理論基礎,對于擴大超聲波在食品工業(yè)中的應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.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：TS201.25
本文編號：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.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：TS201.25
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