摘要由于传统的蛋白酶催化 knovenagel 反应，所用的酶活性不够强，完成反应所需时间较长， 且有热稳定性差，易失活等缺点，所以本文采用固定化后的酶晶体进行催化。固定化后的酶 晶体既能克服游离酶的不足，还增加其活性，提高其产率等特点。本实验通过在磷酸盐缓冲 液中加入硫酸铜溶液并室温培养 3 天后得到磷酸铜酶晶体，然后用此酶晶体进行 knoevenagel 反应，测得酶晶体的活性为游离酶的 15 倍，且苯甲醛与乙酰丙酮在 150mg 的酶量下，于 60℃， DMSO:Water=3。75:1。25 的最优条件下反应 24h，游离酶和酶晶体的最高产率分别为 26。03%和 32。67%，同时在最优条件下还进行了底物的拓展，并发现当苯乙醛和苯甲酰丙酮反应时，酶 晶体的催化效果明显，产率达 46%，而游离酶却未发现。我们的研究发现酶晶体催化效果优 于游离酶，这一发现不仅扩大蛋白酶的应用范围，还将为有机合成领域提供更有潜在价值的 构建碳碳双键的制备方法。88804

Due to the traditional protease catalytic method for knovenagel reaction, enzyme activity is not so robust that it takes long time to complete the reaction time。 At the same time, thermal stability of enzyme is poor and easy to loss its activity。 In this paper, the inorganics-protease hybrid crystals are successfully prepared and used to catalyze the knovenagel reaction。 The crystallized inorganics-protease can overcome the deficiency of free enzyme, and increase its activity and approach the yield of product in the catalytic reaction。 The crystallized inorganics-protease was prepared through the copper sulfate and phosphate buffer solution for three days at room temperature。 The hydrolytic of the crystallized inorganics-protease presented 15 times activity than that of free

enzyme。  In  knovenagel  reaction, under  the  optimum  conditions of  reaction  such  as  60   ℃ and

DMSO: Water = 3。75:1。25 for 24 h, the yield was 26。03% and 32。67% for free enzyme and inorganics-protease hybrid crystals, respectively。 For the expansion of the substrate such as benzene acetaldehyde and benzoyl acetone, no product was obtained for free enzyme。 However, the yield was 46% for the inorganics-protease hybrid crystals。 The results show that the inorganics-protease hybrid crystals presents better than free enzyme in some reactions。 The discovery not only enlarges the application range of protease, but also provide more potential value in organic synthesis field construction method of preparation of C=C bond。

毕业论文关键词：固定化酶； 绿色 ； 混乱催化； 木瓜蛋白酶

Keywords: Immobilized enzyme; green； promiscuous catalysis； Papain

目 录

摘 要 2

目 录 3

1 引言： 4

2 正文： 5

2。1 研究目标 5

2。2 研究源Q于D优G尔X论V文Y网wwW.yOueRw.com 原文+QQ75201`8766 内容 6

2。3 实验部分 6

2。3。1 实验材料与器材 6

2。3。2 木瓜蛋白酶的固定化及活性测试 8

2。3。3 混乱催化