桑叶中Diels-Alderase的提取与初步纯化

发布时间：2018-04-22 13:10

本文选题：桑叶Diels-Alderase + Diels-Alder反应　；参考：《浙江大学》2017年硕士论文

【摘要】：桑叶(MulberryLeaf),为桑科桑属药用植物桑(Morus alba L.)的叶片,具有降血压、抑菌抗炎、抗病毒、抗肿瘤及改善糖尿病等众多功效,富含多种生物活性物质如黄铜类、生物碱、植物甾醇、γ-氨基丁酸及桑叶多糖等。课题组前期研究表明,桑叶经光酶诱导后,产生了大量的Diels-Alder型加合物及其前体化合物如moracinN及morachalcone,并且相应的酶活实验已证实诱导桑叶中Diels-Alderase的存在。Diels-Alderase是催化有机合成领域广泛应用的Diels-Alder环加成反应的酶,至今仍未有人分离得到确切的Diels-Alderase,其催化机制更是未知的。而本论文对桑叶中Diels-Alderase进行了初步分离与纯化:将采摘的新鲜桑叶进行光酶诱导处理后,于25℃避光培养36h,用缓冲盐溶液提取诱导桑叶的粗酶,并对粗酶液的提取工艺进行优化,最后以Diels-Alderase酶活反应作为监测手段,对诱导桑叶的粗酶液进行分离纯化。本论文的主要研究结果如下:1.考察了诱导桑叶中可溶性蛋白提取的最优参数及粗酶中Diels-Alderase的生化特性。首先,对粗酶中Diels-Alderase的耐受温度及pH范围进行研究,发现该酶不耐热,当温度高于40 ℃时活性急剧下降,当温度为70℃时基本失活,而pH耐受区间为5.0-10,最适pH为7.5。其次,以50mMPB-Na(pH8.0)为提取缓冲液,对液料比、浸提时间、浸提温度和pH进行了单因素试验,结果表明,对诱导桑叶中可溶性蛋白提取效率影响较大的因素是液料比、浸提时间与浸提温度。由此设计中心组合试验,以优化诱导桑叶可溶性蛋白的提取条件,确定液料比、浸提温度、浸提时间的零水平分别为8/1、20℃、4h。2.根据中心组合试验,建立了以蛋白提取率为目标函数的数学模型。结果表明,浸提温度(A)、液料比(B)和浸提时间(C)是影响诱导桑叶中可溶性蛋白提取效率的主要因素。并且得到最优提取条件:液料比6/1,浸提温度30 ℃,浸提时间4.84h。经实验验证,预测值和实测值的偏差为0.76%。响应值和各因素的相关回归模型为:Y=-0.211+(1.74E-03)A+0.079B+0.422C-(9.35E-04)AB+(2.07E-03)AC-0.020BC-(1.62E-05)A2+(5.37E-04)B2-0.033C23.探索了诱导桑叶中Diels-Alderase纯化的条件。诱导桑叶粗酶液经硫酸铵沉淀、PEG沉淀、离子交换层析、疏水相互作用层析及凝胶色谱层析等方法进行分离纯化,通过优化硫酸铵、疏水层析、凝胶色谱层析及离子交换层析的纯化条件,确定纯化流程。诱导桑叶的粗酶液依次经硫酸铵沉淀、Phenyl-SepharoseHP疏水层析、Sephadex 200 pg凝胶层析和Q-Sepharose HP离子交换层析分离,其中经Q-SepharoseHP离子交换层析得到的洗脱组分9,活性最高,经SDS-PAGE鉴定,表明该组分纯度最高,并且为近似单一条带,说明该组分达到了电泳纯。本论文研究了诱导桑叶可溶性蛋白提取的最佳工艺条件,考察了桑叶粗酶中Diels-Alderase的生化特性,经蛋白分离纯化过程,得到了具有活性的近似单一条带,可能为桑叶中的Diels-Alderase,为今后桑叶中Diels-Alderase的进一步纯化、验证及异源表达提供了重要依据。
[Abstract]:MulberryLeaf, a leaf of mulberry (Morus alba L.), a medicinal plant of the genus Morus, which has many functions such as lowering blood pressure, inhibiting anti-inflammatory, antiviral, antitumor and improving diabetes. It is rich in a variety of bioactive substances such as brass, alkaloids, phytosterols, gamma aminobutyric acid and mulberry leaves. After the enzyme induction, a large number of Diels-Alder adducts and their precursors such as moracinN and morachalcone have been produced, and the corresponding enzyme activity experiments have proved that the existence of.Diels-Alderase in the mulberry leaves is an enzyme that catalyzes the Diels-Alder ring addition reaction widely used in the field of organic synthesis. So far, no human separation has been obtained. The catalytic mechanism of the cut Diels-Alderase is more unknown. In this paper, the preliminary separation and purification of Diels-Alderase in mulberry leaves was preliminarily separated and purified: after the pickled fresh mulberry leaves were induced by photoperiod, 36h was cultured at 25 degrees C, and the crude enzyme was extracted from the mulberry leaves by the buffer salt solution, and the extraction process of the crude enzyme solution was optimized. Finally, Diel S-Alderase enzyme activity was used as a monitoring method to separate and purify the crude enzyme solution of mulberry leaves. The main research results of this paper are as follows: 1. the optimal parameters of the extraction of soluble protein in mulberry leaves and the biochemical characteristics of Diels-Alderase in the crude enzyme were investigated. First, the tolerance temperature and the pH range of Diels-Alderase in the crude enzyme were studied. It was found that the enzyme was not heat-resistant, when the temperature was higher than 40 C, the activity decreased sharply, when the temperature was 70, and the pH tolerance interval was 5.0-10, the optimum pH was 7.5., and 50mMPB-Na (pH8.0) was used as the buffer solution. The liquid material ratio, the extraction time, the extraction temperature and pH were tested. The results showed that the soluble eggs were induced in the mulberry leaves. The effect of the white extraction efficiency is the ratio of liquid to material, the extraction time and the extraction temperature. Therefore, the design center combined test to optimize the extraction conditions of the soluble protein of the mulberry leaves, determine the liquid ratio, the extraction temperature, and the zero level of the extraction time, respectively, at 8/1,20 C. 4h.2. based on the central combination test, the target protein extraction rate is set up as the target. The mathematical model of the function shows that the extraction temperature (A), liquid ratio (B) and extraction time (C) are the main factors affecting the extraction efficiency of soluble protein in the mulberry leaves. And the optimum extraction conditions are obtained: the liquid material ratio is 6/1, the extraction temperature is 30, and the extraction time 4.84h. is verified by the experiment. The deviation of the predicted value and the measured value is 0.76%. response value and each of the measured values. The correlation regression model of the factors was: Y=-0.211+ (1.74E-03) A+0.079B+0.422C- (9.35E-04) AB+ (2.07E-03) AC-0.020BC- (1.62E-05) A2+ (5.37E-04) B2-0.033C23. explored the conditions for inducing the purification of Diels-Alderase in the mulberry leaves. The induction of the mulberry leaf crude enzyme solution by ammonium sulfate precipitation, precipitation, ion exchange chromatography, hydrophobic interaction chromatography and gel chromatography The purification process of ammonium sulfate, hydrophobic chromatography, gel chromatography and ion exchange chromatography was optimized to determine the purification process. The crude enzyme solution was induced by ammonium sulfate precipitation, Phenyl-SepharoseHP hydrophobic chromatography, Sephadex 200 PG gel chromatography and Q-Sepharose HP ion exchange chromatography. The elution component obtained by Q-SepharoseHP ion exchange chromatography was 9 and the activity was the highest. The highest purity of the component was identified by SDS-PAGE, which showed that the purity of the component was the highest, and it was an approximate single strip, indicating that the component reached the purity of electrophoresis. This paper studied the optimum technological conditions for the extraction of soluble protein from mulberry leaves and examined the biochemistry of Diels-Alderase in the coarse mulberry leaves. In the process of protein separation and purification, an approximate single band with active activity is obtained, which may be Diels-Alderase in mulberry leaves. It provides an important basis for further purification, verification and heterologous expression of Diels-Alderase in mulberry leaves.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R284

【参考文献】