本文選題：金銀花 + 過氧化物酶　； 參考：《河南科技大學(xué)》2017年碩士論文

【摘要】：金銀花為忍冬屬植物干燥花蕾或初開的花,作為一種“藥食同源”原料,被廣泛應(yīng)用于食品加工。新鮮金銀花在加工過程中易發(fā)生褐變,導(dǎo)致功效成分損失和外觀劣變,酶促褐變是引起金銀花品質(zhì)劣變的一個重要原因,研究表明過氧化物酶可以參與酶促褐變反應(yīng)。本文采用勻漿浸提,三相分離法與離子交換層析相結(jié)合的方法提取純化金銀花中的過氧化物酶,并對酶學(xué)性質(zhì)及部分抑制劑抑制效應(yīng)進行研究,為控制酶促褐變強度及闡明酶促褐變代謝機理提供一定的理論基礎(chǔ)。主要實驗結(jié)論如下:采用勻漿法提取金銀花中過氧化物酶,通過單因素實驗及正交實驗設(shè)計,得到最優(yōu)提取條件為浸提時間1h,料液比1:7,緩沖液pH為6,在該條件下所得金銀花過氧化物酶比活力為173.27U/mg。影響金銀花過氧化物酶提取效果的因素依次是緩沖液pH、浸提時間、料液比。正交試驗的方差分析結(jié)果表明緩沖液pH對提取效果的影響達到顯著水平。在單因素實驗的基礎(chǔ)上經(jīng)響應(yīng)面優(yōu)化得出三相分離法各因素水平的最優(yōu)組合為pH為5.60硫酸銨質(zhì)量分數(shù)39.49%,叔丁醇與提取液體積比為1.38。在該條件下純化倍數(shù)為5.849,回收率為87.64%。該條件下金銀花過氧化物酶比活力為1021.6U/mg。420nm可作為金銀花褐變產(chǎn)生色素的測定波長。在最優(yōu)純化效果條件下色素去除率為92%。將三相法提取純化的金銀花過氧化物酶經(jīng)DEAE纖維素DE-52離子交換層析可以分離得到兩種金銀花過氧化物酶HSPⅠ和HSPⅡ,比活力為分別為3312.3U/mg和564.8U/mg,洗脫峰出現(xiàn)的NaCl濃度分別是0.215mol/L和0.291mol/L。對金銀花過氧化物酶酶學(xué)性質(zhì)研究表明,金銀花過氧化物酶最適溫度是30℃,該酶在10℃-40℃范圍內(nèi)穩(wěn)定性較好,最適pH和pH穩(wěn)定性研究表明酶的最適pH為5,酶在pH小于4的酸性環(huán)境中酶活力下降迅速,pH值在4-7范圍內(nèi)有較好的穩(wěn)定性。當反應(yīng)體系H2O2濃度為1 mmol/L時,酶促反應(yīng)速率趨于穩(wěn)定。當反應(yīng)體系愈創(chuàng)木酚濃度達到96 mmol/L時,酶促反應(yīng)速率趨于穩(wěn)定。對金銀花過氧化物酶的反應(yīng)動力學(xué)分析表明,金銀花過氧化物酶的雙底物酶促反應(yīng)類型為乒乓機制。當H2O2濃度一定時,酶對愈創(chuàng)木酚的Km值為8.12mmol/L,Vmax值為1.71mmol/L·min。當愈創(chuàng)木酚濃度一定時,H2O2的Km值為0.822mmol/L,Vmax值為1.38mmol/L·min。部分抑制劑及金屬離子對金銀花過氧化物酶的作用研究表明,Ca2+、Cu2+、Zn2+對金銀花過氧化物酶有一定激活作用,Mg2+、Mn2+、檸檬酸、抗壞血酸、-半胱氨酸、亞硫酸鈉、焦亞硫酸鈉對金銀花過氧化物酶均有一定抑制作用。L-半胱氨酸,檸檬酸,焦亞硫酸鈉,SDS對金銀花過氧化物酶的抑制效應(yīng)及抑制動力學(xué)研究表明,L-半胱氨酸,檸檬酸,焦亞硫酸鈉,SDS對金銀花過氧化物酶均有一定程度抑制作用,抑制能力從強到弱依次是焦亞硫酸鈉,L-半胱氨酸,檸檬酸,SDS。檸檬酸,焦亞硫酸鈉對金銀花過氧化物酶的抑制類型為不可逆抑制。L-半胱氨酸和SDS對金銀花過氧化物酶的抑制類型為可逆抑制,其中L-半胱氨酸的可逆抑制類型為競爭性可逆抑制,SDS的可逆抑制類型為非競爭性可逆抑制。L-半胱氨酸抑制常數(shù)KI為0.053mmol/L,SDS抑制常數(shù)KI為13.4mmol/L,KIS為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.
【學(xué)位授予單位】：河南科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q946.5

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