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摘要R-(-)-扁桃酸脱氢酶能够以NAD为辅酶，催化底物R-扁桃酸生成苯乙酮酸，扁桃酸脱下来的氢传递给NAD。筛选出产扁桃酸脱氢酶的菌种，经16S rDNA测序鉴定菌种为恶臭假单胞菌FJ506，并对该菌种的发酵条件进行优化，确定恶臭假单胞菌FJ506的最适培养温度为30 ℃，培养时间为18 h。经过硫酸铵盐析、疏水层析等操作分离纯化获得纯的R-扁桃酸脱氢酶，SDS-聚丙烯酰胺凝胶电泳后测得亚基的相对分子质量为31 000。酶的回收率为9.4%，纯化后酶的比活力为6.67 U/mg，纯化倍数为24.70倍。18800
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酶学特性研究结果表明，R-（-）-扁桃酸脱氢酶的最适pH为8.5，最适温度为26 ℃。探究了金属离子对R-（-）-扁桃酸脱氢酶的影响，其中Zn2+、Ba2+、Ca2+对酶活有抑制作用，而Cu2+能使R-（-）-扁桃酸脱氢酶失活。
关键字 恶臭假单胞菌FJ506 扁桃酸脱氢酶 分离纯化
毕业设计说明书（毕业论文）外文摘要
Title Extraction and Purification of (R)-mandelate Dehydrogenase and Its Producing Strain.
Abstract
NAD(H)-dependent R-mandelate dehydrogenase can catalyze the oxidation of (R)-mandelic acid to benzoylformic acid, resulting in the reduction of NAD.
The strain, with high production rate of (R)-mandelate dehydrogenase (RMDH), were screened and identified. Based on 16S rDNA analysis,the strain was Pseudomonas putida FJ506. The time and temperature optimum of fermentation was found to be 18h and 30ºC. The molecular weight of the enzyme was 31 000. The specific enzyme activity after purification was 6.67 U/mg. The recovery of (R)-mandelate dehydrogenase activity was 9.4% , and the purification fold was 20.70.
The temperature and pH optimum of (R)-mandelate dehydrogenase is 26 ºC and pH 8.5. And Zn2+、Ba2+、Ca2+ were strong inhibitors of (R)-mandelate dehydrogenase while Cu2+ was lethal.
Keywords Pseudomonas putida mandelic acid dehydrogenase purification
目 次
1 绪论 1
1.1 手性扁桃酸研究概况 1
1.1.1 手性物质介绍 1
1.1.2 手性扁桃酸 1
1.2 R-(-)-扁桃酸脱氢酶 1
1.3 课题研究背景及意义 4
1.4 主要研究内容及任务 4
2 菌种筛选 6
2.1 菌体的培养 6
2.1.1 配制培养基 6
2.1.2 接种 6
2.2 酶活检测 6
2.3 本章小结 7
3 菌株的鉴定 8
3.1 16S rDNA鉴定 8
3.1.1 鉴定原理 8
3.1.2 具体操作步骤 8
3.2 质粒的提取 9
3.2.1 碱裂解法提取质粒 9
3.3 恶臭假单胞菌FJ506的革兰氏染色 10
3.3.2 实验材料和试剂 10
3.3.3 实验步骤 11
3.4 恶臭假单胞菌FJ506的形态结构的观察 11
3.4.1 实验原理 11
3.4.2 实验材料和试剂 11
3.4.3 验步骤 11
3.5 实验结果与分析 12
3.5.1 16S rDNA鉴定 12
3.5.2 质粒的提取 12
3.5.3 恶臭假单胞菌FJ506的革兰氏染色 13
3.5.4 恶臭假单胞菌FJ506的形态结构的观察 13
4 恶臭芽孢杆菌发酵条件优化 14