【作者】 朱少娟；

【导师】 施用晖；

【作者基本信息】 江南大学 ， 粮食、油脂及植物蛋白工程， 2004， 硕士

【摘要】 酶法对食品蛋白质进行改性，是有效改善蛋白质的物理、化学性质，提高适口性、贮存与营养特性的技术。近年来，声学技术向生物、食品技术领域不断交叉、渗透，应用超声技术在水溶液中提高酶活力、促进酶催化反应已有一些研究报道，但不同酶、底物作用效果不尽一致，作用机理尚不清楚。本论文以胰蛋白酶水解酪蛋白为模型，研究超声波对胰蛋白酶水解酪蛋白的酶反应条件、动力学的影响及其反应机理。 首先通过单因素实验确定了促进酶解反应的适宜超声参数。超声功率为225W、超声频率20KHz，超声时间与间歇时间比为1：1，超声时间10min。进一步研究了适宜超声条件下的胰蛋白酶反应动力学。测定了超声场下温度，pH，底物浓度，酶浓度对水解率的影响，计算胰蛋白酶米氏常数Km、最大反应速度Vm。结果表明，超声波不改变胰蛋白酶的最适pH；但影响胰蛋白酶的温度特性，在5℃，20℃超声波作用下，水解率分别达54％和73％，比同温度对照提高了4.4倍和2.33倍；而超声波不改变木瓜蛋白酶温度和pH特性。本试验超声条件下，胰蛋白酶的米氏常数Km减小，最大反应速度Vm增大；超声场下酶浓度为0.02％、0.04％时的水解率分别与对照酶浓度为0.08％、0.2％时的水解率相当。超声波提高胰蛋白酶水解效率，超声反应10min可达到常规反应40min时的水解率，并可节约酶用量，降低生产成本。 高效液相色谱排阻法测定了胰蛋白酶在超声10min、未超声10min＼40min条件下水解物的分子量分布。超声波不影响胰蛋白酶的特异性，但影响酶解产物的分子量分布。常规条件下水解40min产物肽总量(总峰面积)与超声水解10min接近，但200-2000分子量肽量高16％；常规条件下水解40min与10min相比，肽总量提高54.8％，小分子肽含量增加。 对超声波促进胰蛋白酶酶解反应的机理进行了初步的探讨。通过紫外及荧光光谱法测定，观察到超声波影响酶及底物分子构象，适宜的超声波改变了酶与底物的荧光吸收光谱和荧光强度，可能使酶的活性中心更暴露，底物结构更松散，促进酶与底物的结合。通过Arrehnius作图分析了超声波对酶反应活化能的影响，结果表明超声波降低了酶解反应的活化能，加速传质，降低了底物和产物对反应的抑制作用，适宜的超声波提高胰蛋白酶水解效率。更多还原

【Abstract】 The application of ultrasound to enzymatic reaction can increase enzyme activities and accelerate enzymatic reactions. In this thesis studied the effects of ultrasound treatment on the hydrolysis of casein by trypsin, the optimal parameters were determined: ultrasound power 225W, frequency 20KHz, ultrasound treatment time to pause time 1:1, total ultrasound time 10min. Under above-determined optimal conditions, the kinetics of enzymatic action were further studied. The temperature characteristics of trypsin-catalyzed reactions with ultrasound treatment was greatly changed. The hydrolysis rate were 54%and 73% at 5 and 20, increased about 4.4times and 3.3 times in treatment of ultrasound than the control (without ultrasound treatment). The optimal pH of trypsin was not affected by the ultrasound treatment. Km was decreased and Vm increased; the hydrolysis rate of different casein concentrate were all higher than the control. The hydrolysis rates with enzyme concentrations of 0.02% and 0.04% under ultrasound treatment were equal to those with enzyme concentrations of 0.08% and 0.2% without ultrasound treatment respectively.The molecular weight distribution of the hydrolysates were determined by high performance size exlusion chromatography( HPSEC). Three different samples were prepared under the reaction 10min with ultrasound, reaction 10min and 40min without ultrasound respectively. The results showed that ultrasound treatment didn’t affect the specific property of trypsin, but the molecular mass distribution of casein tryptic hydrolysis were changed. Compared the samples obtained with ultrasound for 10min, the total peptides amounts were almost equal, but peptides of MW between 200 and 2000 increased by 15% in reaction without ultrasound for 40min. The amount of total peptides increased by 54.8% in reaction 40min without ultrasound than those of reaction 10min without ultrasound treatment.Finally the hydrolysis mechanism of casein under ultrasound by trypsin was investigated. UV absorption spectrum and fluorescence emission spectrum of trypsin and casein pretreated by ultrasound were measured, and the activation energy (Ea) were calculated by Arrhnius plot. The results suggested that the ultrasound treatment could decrease the activation energy of reaction of trypsin, enhance mass transformation, decrease the effect of inhibition of substrate and products, the ultrasound under optimum condition could increase the hydrolysis rate.更多还原