金银花中过氧化物酶的纯化及性质研究

发布时间：2018-06-09 11:30

本文选题：金银花 + 过氧化物酶　；参考：《河南科技大学》2017年硕士论文

【摘要】：金银花为忍冬属植物干燥花蕾或初开的花,作为一种“药食同源”原料,被广泛应用于食品加工。新鲜金银花在加工过程中易发生褐变,导致功效成分损失和外观劣变,酶促褐变是引起金银花品质劣变的一个重要原因,研究表明过氧化物酶可以参与酶促褐变反应。本文采用匀浆浸提,三相分离法与离子交换层析相结合的方法提取纯化金银花中的过氧化物酶,并对酶学性质及部分抑制剂抑制效应进行研究,为控制酶促褐变强度及阐明酶促褐变代谢机理提供一定的理论基础。主要实验结论如下:采用匀浆法提取金银花中过氧化物酶,通过单因素实验及正交实验设计,得到最优提取条件为浸提时间1h,料液比1:7,缓冲液pH为6,在该条件下所得金银花过氧化物酶比活力为173.27U/mg。影响金银花过氧化物酶提取效果的因素依次是缓冲液pH、浸提时间、料液比。正交试验的方差分析结果表明缓冲液pH对提取效果的影响达到显著水平。在单因素实验的基础上经响应面优化得出三相分离法各因素水平的最优组合为pH为5.60硫酸铵质量分数39.49%,叔丁醇与提取液体积比为1.38。在该条件下纯化倍数为5.849,回收率为87.64%。该条件下金银花过氧化物酶比活力为1021.6U/mg。420nm可作为金银花褐变产生色素的测定波长。在最优纯化效果条件下色素去除率为92%。将三相法提取纯化的金银花过氧化物酶经DEAE纤维素DE-52离子交换层析可以分离得到两种金银花过氧化物酶HSPⅠ和HSPⅡ,比活力为分别为3312.3U/mg和564.8U/mg,洗脱峰出现的NaCl浓度分别是0.215mol/L和0.291mol/L。对金银花过氧化物酶酶学性质研究表明,金银花过氧化物酶最适温度是30℃,该酶在10℃-40℃范围内稳定性较好,最适pH和pH稳定性研究表明酶的最适pH为5,酶在pH小于4的酸性环境中酶活力下降迅速,pH值在4-7范围内有较好的稳定性。当反应体系H2O2浓度为1 mmol/L时,酶促反应速率趋于稳定。当反应体系愈创木酚浓度达到96 mmol/L时,酶促反应速率趋于稳定。对金银花过氧化物酶的反应动力学分析表明,金银花过氧化物酶的双底物酶促反应类型为乒乓机制。当H2O2浓度一定时,酶对愈创木酚的Km值为8.12mmol/L,Vmax值为1.71mmol/L·min。当愈创木酚浓度一定时,H2O2的Km值为0.822mmol/L,Vmax值为1.38mmol/L·min。部分抑制剂及金属离子对金银花过氧化物酶的作用研究表明,Ca2+、Cu2+、Zn2+对金银花过氧化物酶有一定激活作用,Mg2+、Mn2+、柠檬酸、抗坏血酸、-半胱氨酸、亚硫酸钠、焦亚硫酸钠对金银花过氧化物酶均有一定抑制作用。L-半胱氨酸,柠檬酸,焦亚硫酸钠,SDS对金银花过氧化物酶的抑制效应及抑制动力学研究表明,L-半胱氨酸,柠檬酸,焦亚硫酸钠,SDS对金银花过氧化物酶均有一定程度抑制作用,抑制能力从强到弱依次是焦亚硫酸钠,L-半胱氨酸,柠檬酸,SDS。柠檬酸,焦亚硫酸钠对金银花过氧化物酶的抑制类型为不可逆抑制。L-半胱氨酸和SDS对金银花过氧化物酶的抑制类型为可逆抑制,其中L-半胱氨酸的可逆抑制类型为竞争性可逆抑制,SDS的可逆抑制类型为非竞争性可逆抑制。L-半胱氨酸抑制常数KI为0.053mmol/L,SDS抑制常数KI为13.4mmol/L,KIS为11.5mmol/L。
[Abstract]:Honeysuckle is a kind of dry flower bud or first flower of the genus Lonicera. As a kind of "medicine and food homologous" raw material, it is widely used in food processing. Fresh honeysuckle is easily browning during processing, resulting in loss of functional components and deterioration of appearance. Enzymatic browning is an important reason for the deterioration of the quality of honeysuckle. Enzyme can be involved in enzymatic browning reaction. In this paper, the peroxidase in honeysuckle was extracted and purified by homogenate extraction, three phase separation method and ion exchange chromatography, and the enzymatic properties and inhibition effect of some inhibitors were studied to provide a certain reason for controlling the enzymatic browning intensity and clarifying the mechanism of enzymatic browning metabolism. The main experimental conclusions are as follows: using homogenate method to extract peroxidase in honeysuckle, through single factor experiment and orthogonal design, the optimum extraction conditions are the extraction time 1H, the ratio of material to liquid to 1:7, and the buffer solution pH is 6. Under this condition, the activity of the peroxidase of honeysuckle is 173.27U/mg. affecting the peroxidase extraction of honeysuckle. The effect factors are the buffer solution pH, the extraction time and the ratio of material to liquid. The orthogonal test analysis of variance shows that the effect of pH on the extraction effect reaches a significant level. On the basis of the single factor experiment, the optimum combination of each factor level of the three-phase separation method by the response surface is that pH is 5.60 ammonium sulfate mass fraction 39.49%, The volume ratio of butanol and extract was 1.38. under the condition of 5.849, and the recovery rate was 87.64%.. The specific activity of honeysuckle peroxidase was 1021.6U/mg.420nm, which could be used as the determination wavelength of the browning pigment of honeysuckle. Under the optimal purification effect, the pigment removal rate was 92%., which was extracted and purified by the three-phase method. Two kinds of honeysuckle peroxidase HSP I and HSP II can be separated by DEAE cellulose DE-52 ion exchange chromatography. The specific activity of the enzyme is 3312.3U/mg and 564.8U/mg respectively. The concentration of NaCl in the elution peak is 0.215mol/L and 0.291mol/L., respectively. The temperature is 30 C, the enzyme is stable in the range of 10 -40 C. The optimum pH and pH stability studies show that the optimum pH is 5, the enzyme activity decreases rapidly in the acidic environment with pH less than 4, and the pH value has a good stability in the 4-7 range. When the reaction system H2O2 concentration is 1 mmol /L, the enzyme reaction rate tends to be stable. When the reaction system is reacted, the reaction system is stable. When the concentration of guaiacol reached 96 mmol/L, the rate of enzymatic reaction tended to be stable. The kinetic analysis of the reaction kinetics of honeysuckle peroxidase showed that the double substrate enzyme reaction type of honeysuckle peroxidase was ping-pong mechanism. When the concentration of H2O2 was fixed, the Km value of the enzyme to guaiacol was 8.12mmol/L, and the Vmax value was 1.71mmol/L min. as the guaiaci. When the concentration of phenol is certain, the Km value of H2O2 is 0.822mmol/L, the value of Vmax value is 1.38mmol/L. Min. and the effect of metal ions on honeysuckle peroxidase show that Ca2+, Cu2+, Zn2+ have certain activation effect on honeysuckle peroxidase, Mg2+, Mn2+, citric acid, anti blood acid, cysteine, sodium sulfite and sodium pyrosulfite on gold and silver The inhibitory effects and inhibition kinetics of.L- cysteine, citric acid, sodium pyrosulfite and SDS on Flos Lonicerae peroxidase showed that L- cysteine, citric acid, sodium pyrosulfite and SDS had a definite inhibition effect on the peroxidase of honeysuckle, and the inhibition ability from strong to weak was in turn from strong to weak. Sodium sulfite, L- cysteine, citric acid, SDS. citric acid, and sodium pyrosulfite on the inhibition type of honeysuckle peroxidase are irreversible inhibition of the inhibitory type of.L- cysteine and SDS on honeysuckle peroxidase, in which the reversible inhibitory type of L- cysteine is a competitive reversible inhibition, and the reversible inhibitory type of SDS is the type of inhibition. Non competitive reversible inhibition of.L- cysteine inhibition constant KI was 0.053mmol/L, SDS inhibition constant KI was 13.4mmol/L, KIS was 11.5mmol/L.
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q946.5

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