生物酶催化不饱和脂肪酸的硝化反应

发布时间：2018-05-12 18:03

  本文选题：生物催化 + 大豆过氧化物酶　； 参考：《大连工业大学》2015年硕士论文

【摘要】：生物催化因其高效、专一性强及环境污染少等优点成为绿色合成化学品的理想催化剂。然而,天然酶常因其在工业催化条件下的活性和稳定性较低而难以使用。纳米技术为构建高效酶催化剂提供了新的可能性。如何通过简便、高效、低成本的方法制备出具有高催化活性和高稳定性的纳米酶催化剂,并将其应用于化学合成是其中的关键问题。本课题以大豆过氧化物酶为基础,研究了一种新型的以磷酸铜为载体形成的有机-无机结合的纳米花固定化酶方法,并初步探索了合成的纳米花固定化酶对硝基油酸生成的催化作用,同时与化学法合成的硝基油酸进行了比较。得到以下结论:(1)以大豆皮为原料,对大豆过氧化物酶的提取及初步纯化进行了研究,通过酶液提取的单因素实验、正交实验,得到酶的最佳提取工艺条件。采用硫酸铵分级沉淀和丙酮分级沉淀的方法对大豆过氧化物酶进行了初步纯化。结果表明:酶的最适提取条件为料液比1:8,提取温度35℃,提取时间24 h;硫酸铵沉淀下限饱和度:30%,上限饱和度:80%;丙酮沉淀下限体积倍数:0.4,上限体积倍数:1.2。(2)利用实验室自提的大豆过氧化物酶作为研究对象,采用一种新型的固定化酶方法合成了具有花形结构的纳米花固定化酶。通过SEM、TEM图像展示了该纳米花固定化酶的形貌特征,研究了时间和初始酶浓度对该结构产生的影响。通过红外光谱、X射线衍射、能谱分析手段确定了此种纳米花固定化酶的载体为磷酸铜。通过愈创木酚法测定了纳米花固定化酶的活性,与游离酶相比纳米花固定化酶表现出增强的催化活性,最高酶活能够提高到446%。重复性试验证实了该纳米花固定化酶的循环性良好,经过5次循环后依然保持80%的活性。(3)以油酸为原料,采用化学法和生物法合成硝基油酸,得到的产物经过红外光谱,核磁共振及液质联用分析手段加以佐证,考察纳米花固定化酶对硝基油酸生成的影响。结果表明:生物法合成硝基油酸起到了抑制副产物生成的作用。生物法合成硝基油酸是一种新的尝试,对未来固定化酶与化学合成结合的研究奠定了基础。
[Abstract]:Biocatalysis is an ideal catalyst for green synthetic chemicals because of its high efficiency, high specificity and less environmental pollution. However, natural enzymes are often difficult to use because of their low activity and stability in industrial catalysis. Nanotechnology provides a new possibility for the construction of efficient enzyme catalysts. It is a key problem that how to prepare high catalytic activity and high stability nanometer enzyme catalyst by simple, high efficiency and low cost method, and apply it to chemical synthesis. Based on soybean peroxidase, a novel organic-inorganic binding enzyme immobilization method based on copper phosphate was studied in this paper. The catalytic effect of the immobilized enzyme on the formation of nitrooleic acid was preliminarily explored and compared with that of nitrooleic acid synthesized by chemical method. Conclusion: (1) the extraction and purification of soybean peroxidase were studied by using soybean skin as raw material. The optimum extraction conditions were obtained by single factor experiment and orthogonal experiment. Soybean peroxidase was preliminarily purified by ammonium sulfate fractionation and acetone fractionation. The results showed that the optimum extraction conditions were as follows: the ratio of material to liquid was 1: 8, the extraction temperature was 35 鈩，

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