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摘要从校园中多处采集土样，通过富集培养、平板初筛，再检测其转化能力和酶活力，筛选出能够催化氧化苯乙醇为苯乙酮的菌株，对其中活性较高的 5 号菌株进行简单的生理生化鉴定，此菌为革兰氏阳性、长杆菌。通过超声波破碎、硫酸铵分段盐析和透析脱盐，得到苯乙醇脱氢酶，蛋白质含量为 4.85 mg/mL，其中最适超声破碎全程时间为 80min,超声工作与间歇时间比为 1:2，硫酸铵最佳盐析范围为 60%～80%饱和度。对苯乙醇脱氢酶的酶学性质进行研究表明，酶反应最适温度为 40℃，在 30℃和 40℃之间酶具有一定的耐热性，该酶不能耐受高温；酶反应最适 pH 为 10.0，酶在 pH10.0～12.0 内比较稳定，具有很好的耐碱性，对酸较为敏感。8105
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关键词 苯乙醇 醇脱氢酶 提取纯化 酶学性质 Title Extraction and purification of alcohol dehydrogenase
Abstract
Some bacteria strains which can catalyze the oxidation of phenylethanol
to acetophenone are screened out from many soil samples collected from the
campus.This screening process can be achieved by enrichment culture, flat
screening and detecting the conversion and the enzyme activity.Strain
No.5 ,in which the activity is higher, is Gram-positive bacteria and
belongs to the long bacilli with the simple physiological and biochemical
identification.By sonication, ammonium sulfate segment salting out and
dialysis desalination, the phenylethanol dehydrogenase is extracted and
purified. the protein content is 4.85 mg / mL.In the research, the optimum
ultrasonication time is 80 min, the ultrasonic working with intermittent
time ratio is 1:2, and the optimum ammonium sulfate salting range is 60%
to 80% saturation. Studying the enzymatic properties of phenylethanol
dehydrogenase, it has been shown that the optimum temperature of the enzyme
reaction is 40℃ and the enzyme has heat resistance between 30℃ and 40
℃. The enzyme can’t tolerate high temperatures. The optimum pH is 10.0,
and the enzyme is relatively stable at pH 10.0～12.0.It is known that the
phenylethanol dehydrogenase has a very good alkali resistance and is more
sensitive to acid.
Keywords phenylethanol alcohol dehydrogenase extraction and
purification enzymatic properties 目 次
1 引言· 1
1.1 生物催化技术研究概况 1
1.1.1 生物催化技术· 1
1.1.2 生物催化在有机合成中的应用· 1
1.1.3 生物催化的主要方式· 1
1.2 醇脱氢酶研究概况 2
1.2.1 醇脱氢酶简介· 2
1.2.2 醇脱氢酶催化氧化还原反应的机理 3
1.2.3 醇脱氢酶应用研究现状· 3
1.2.4 醇脱氢酶的制备· 4
1.3 本课题的主要研究内容与研究意义 5
1.3.1 主要研究内容· 5
1.3.2 研究意义· 6
2 产苯乙醇脱氢酶菌株的筛选 7
2.1 引言· 7
2.2 材料· 7
2.2.1 菌种来源· 7
2.2.2 培养基配方· 7
2.2.3 主要试剂· 8
2.2.4 仪器与设备· 8
2.2.5 常用储存液及配制方法· 9
2.3 实验方法 9
2.3.1 配制筛选培养基· 9
2.3.2 菌株初筛方法· 9
2.3.3 检测菌株转化能力· 10
2.3.4 检测菌株酶活力· 10
2.3.5 菌株生理生化特征测定11
2.4 结果与讨论·11
2.4.1 菌株筛选结果11
2.4.2 菌株生理生化特征测定结果· 13
3 苯乙醇脱氢酶的制备 14
3.1 引言· 14
3.2 材料· 14