摘要本次本科设计的题目是“产氨基酸脱氢酶菌株的筛选与培养”，我们目标是能够筛选出产D-苯甘氨酸脱氢酶的菌株，然后对该菌株进行培养与保存。D-苯甘氨酸脱氢酶以FAD当成辅基，即其接受电子的不是NAD+。从学校附近的湖水以及土壤样品筛选产苯甘氨酸脱氢酶菌株，对于能够利用D-苯甘氨酸的菌株进行鉴定。先进行富集培养，然后通过TTC培养基进行初步筛选，筛选出符合条件的菌株，再通过划线分离等获得单菌落，进行纯培养，超速离心获得细菌，加入1% TritonX-100溶液，利用超声波破碎、离心得到粗酶液，用铁氰化钾还原法来剖断。用铁氰化钾和D-甘氨酸钠为底物，铁氰化钾在D-苯甘氨酸脱氢酶作用下，被还原为亚铁氰化钾，普鲁士蓝是亚铁氰化钾遇到了Fe3+会生成的产物，普鲁士蓝在700nm波长下可以测到它的吸光值，就可以检测之生成量，作为D-苯甘氨酸脱氢酶的还原力，用缓冲液作为对照，测该酶活性。经过不同浓度的硫酸铵，蛋白就会沉淀，从而可以分离纯化目的酶D-苯甘氨酸脱氢酶。73656

毕业论文关键词  D-苯甘氨酸  氨基酸脱氢酶  铁氰化钾 

毕业设计说明书外文摘要

Title Screening of the strains which can produce amino acid dehydrogenase

Abstract This topic of graduation project is "how to screen and culture the strains which can produce amino acid dehydrogenase ",our aim is to be able to screen the strains which can produce D- benzene glycine dehydrogenase , and then culture and preservate the strains 。 D- benzene glycine dehydrogenase use FAD as prosthetic group, rather than NAD+。 From the lake water and soil  around the school, to screen the strains which can produce D- benzene glycine dehydrogenase at the first ,then identify the strains。We can use culture medium to enrich the strains , and then through the TTC to carry out preliminary screening, screening out qualified strain which is single colony isolation from streaking, then pure culture it,use ultracentrifugation,break up soluble membrane with 1%TritonX-100, using ultrasonic to crush, speeding away from  ultracentrifugation to obtain crude enzyme solution,using potassium ferricyanide reduction method to identify, using potassium ferricyanide and D- phenylglycine sodium as the substrate, the catalytic reaction of D- glycine dehydrogenase will ferricyanide reduction for potassium ferrocyanide, potassium ferrocyanide and Fe3+ and generate Prussian blue, the absorption value was measured at the wavelength of 700nm, to detect the generation amount of Prussian blue, as the reducing power D- phenylglycine dehydrogenase。 With the buffer as control, the measurement of enzyme activity。 Ammonium sulfate precipitation, separation and purification of the active D- benzene glycine dehydrogenase。

Keywords  D- phenylglycine  amino acid dehydrogenase  potassium ferricyanide   

目   次

1 引言 1

1。1氨基酸脱氢酶的介绍 1

1。2 D-氨基酸脱氢酶的研究现状与前景 1

1。3 筛选产脱氢酶菌的方法 2

1。4研究课题内容与目的 3

2 实验材料与步骤 4

2。1实验材料与试剂 4

2。2实验仪器 4

2。3实验步骤 4

3 结果分析 12

结  论 18     

致  谢