摘要： 本文以砂蚕作为实验材料，以砂蚕溶栓酶作为研究对象，研究了砂蚕溶栓酶提取工艺、砂蚕溶栓酶粗酶液活性测定、砂蚕溶栓酶纯化工艺以及砂蚕溶栓酶纯品测定。本文采用不同饱和度硫酸铵沉淀法、双水相液体萃取法为提取工艺。从试验成果显示：60%的硫酸铵饱和度，冰箱4℃辅助提取6h以上为最佳提取方式；以Folin-酚法和酪蛋白平板法组合的方法测定砂蚕溶栓酶粗酶活性测定，Folin-酚法通过测吸光度由标准曲线计算酶活性，平板法以酪蛋白为酶反应底物，观察酶活性反应形成的透明水解圈情况；砂蚕溶栓酶提取产物的分离纯化以凝胶层析法为主，砂蚕溶栓酶的提取后纯品的活性检测方法采用粗酶活性检测方法。结果表明：20g砂蚕粉末，200ml中性磷酸缓冲液（PBS）溶解，超声条件下浸提半小时后过滤，向滤液中缓慢的加入硫酸铵粉末到60%的饱和度，冰箱冷藏辅助沉淀6h以上，4000rmp离心20分钟，称量沉淀重量，计算得出砂蚕溶栓酶的最终提取率；随后取1g沉淀，用中性磷酸缓冲液将沉淀重新悬浮，用Folin-酚法测定酶活性。此方法得到的砂蚕溶栓酶含量相对较多且活性影响比较小。将剩余的沉淀用中性磷酸缓冲液悬浮，通过层析柱进行层析洗脱。得率在0。4%左右。81091

毕业论文关键词：砂蚕溶栓酶；提取工艺；酶活性检测；分离纯化

Absctract：In this paper, the silkworm rhizome was used as the experimental material, and the extraction technology of peptone was studied。 The results showed that the method was simple, Determination of pure enzyme。 The extraction method used in this paper is ammonium sulfate precipitation method, double water phase extraction method。From the experimental results, the optimum extraction conditions were obtained at 60 ℃ for 4 hours, and the optimum extraction conditions were as follows: the folin-phenolic and casein plate method combined with folin- Phenol method by measuring the absorbance from the standard curve to calculate the enzyme activity, plate method to casein as the substrate to observe the formation of the enzyme reaction of the transparent hydrolysis of the ring。 Silkworm peptone purification of chromatography-based, pure enzyme activity determination method Determination of crude enzyme activity。The results showed that 20 g of the silkworm powder was extracted with 200 ml of neutral phosphate buffer under ultrasonic conditions for 30 min and then filtered。 The ammonium sulfate powder was slowly added to 60% saturation to form a precipitate。 After centrifugation at 4000 rpm, And then the extraction rate was calculated, and then 1 g of the precipitate was resuspended with neutral phosphate buffer, and the enzyme activity was measured by the folin-phenol method。 The results showed that the content of thrombolytic enzyme was relatively high and the effect of activity was relatively small。 The remaining precipitate was suspended in neutral phosphate buffer and chromatographed on a chromatographic column。 The yield is about 0。4%。

Key words: Silkworm anthelmintin, extraction process, enzyme activity detection, isolation and purification

目录

1。绪论 １

  1。1 本课题国内外研究现状概述 １

  1。2 血栓性疾病以及其危害介绍 １

  1。3 血栓的主要形成过程 １

  1。4 溶栓酶的溶栓机制 ３

  1。5 最常见的血栓栓塞性疾病、以及主要临床治疗手段 ４

    1。5。1 常见的血栓性疾病类型