本文選題：桑葉Diels-Alderase + Diels-Alder反應(yīng)　； 參考：《浙江大學(xué)》2017年碩士論文

【摘要】：桑葉(MulberryLeaf),為�？粕偎幱弥参锷�(Morus alba L.)的葉片,具有降血壓、抑菌抗炎、抗病毒、抗腫瘤及改善糖尿病等眾多功效,富含多種生物活性物質(zhì)如黃銅類、生物堿、植物甾醇、γ-氨基丁酸及桑葉多糖等。課題組前期研究表明,桑葉經(jīng)光酶誘導(dǎo)后,產(chǎn)生了大量的Diels-Alder型加合物及其前體化合物如moracinN及morachalcone,并且相應(yīng)的酶活實驗已證實誘導(dǎo)桑葉中Diels-Alderase的存在。Diels-Alderase是催化有機合成領(lǐng)域廣泛應(yīng)用的Diels-Alder環(huán)加成反應(yīng)的酶,至今仍未有人分離得到確切的Diels-Alderase,其催化機制更是未知的。而本論文對桑葉中Diels-Alderase進行了初步分離與純化:將采摘的新鮮桑葉進行光酶誘導(dǎo)處理后,于25℃避光培養(yǎng)36h,用緩沖鹽溶液提取誘導(dǎo)桑葉的粗酶,并對粗酶液的提取工藝進行優(yōu)化,最后以Diels-Alderase酶活反應(yīng)作為監(jiān)測手段,對誘導(dǎo)桑葉的粗酶液進行分離純化。本論文的主要研究結(jié)果如下:1.考察了誘導(dǎo)桑葉中可溶性蛋白提取的最優(yōu)參數(shù)及粗酶中Diels-Alderase的生化特性。首先,對粗酶中Diels-Alderase的耐受溫度及pH范圍進行研究,發(fā)現(xiàn)該酶不耐熱,當(dāng)溫度高于40 ℃時活性急劇下降,當(dāng)溫度為70℃時基本失活,而pH耐受區(qū)間為5.0-10,最適pH為7.5。其次,以50mMPB-Na(pH8.0)為提取緩沖液,對液料比、浸提時間、浸提溫度和pH進行了單因素試驗,結(jié)果表明,對誘導(dǎo)桑葉中可溶性蛋白提取效率影響較大的因素是液料比、浸提時間與浸提溫度。由此設(shè)計中心組合試驗,以優(yōu)化誘導(dǎo)桑葉可溶性蛋白的提取條件,確定液料比、浸提溫度、浸提時間的零水平分別為8/1、20℃、4h。2.根據(jù)中心組合試驗,建立了以蛋白提取率為目標(biāo)函數(shù)的數(shù)學(xué)模型。結(jié)果表明,浸提溫度(A)、液料比(B)和浸提時間(C)是影響誘導(dǎo)桑葉中可溶性蛋白提取效率的主要因素。并且得到最優(yōu)提取條件:液料比6/1,浸提溫度30 ℃,浸提時間4.84h。經(jīng)實驗驗證,預(yù)測值和實測值的偏差為0.76%。響應(yīng)值和各因素的相關(guān)回歸模型為: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.探索了誘導(dǎo)桑葉中Diels-Alderase純化的條件。誘導(dǎo)桑葉粗酶液經(jīng)硫酸銨沉淀、PEG沉淀、離子交換層析、疏水相互作用層析及凝膠色譜層析等方法進行分離純化,通過優(yōu)化硫酸銨、疏水層析、凝膠色譜層析及離子交換層析的純化條件,確定純化流程。誘導(dǎo)桑葉的粗酶液依次經(jīng)硫酸銨沉淀、Phenyl-SepharoseHP疏水層析、Sephadex 200 pg凝膠層析和Q-Sepharose HP離子交換層析分離,其中經(jīng)Q-SepharoseHP離子交換層析得到的洗脫組分9,活性最高,經(jīng)SDS-PAGE鑒定,表明該組分純度最高,并且為近似單一條帶,說明該組分達到了電泳純。本論文研究了誘導(dǎo)桑葉可溶性蛋白提取的最佳工藝條件,考察了桑葉粗酶中Diels-Alderase的生化特性,經(jīng)蛋白分離純化過程,得到了具有活性的近似單一條帶,可能為桑葉中的Diels-Alderase,為今后桑葉中Diels-Alderase的進一步純化、驗證及異源表達提供了重要依據(jù)。
[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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R284

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