摘要：β-苯乙醇是一种用于化妆品和食品行业的高级芳香醇。全球市场对于β-苯乙醇的需求产量急速扩增，目前大多数文献报道使用酵母菌进行生物转化产天然的β-苯乙醇，但细菌菌株相较于真核菌株更容易获取，可通过更加简便的路径来合成，并且培养条件更为简单，因此获得产β-苯乙醇的细菌将对工业生产有着极其深远的意义。本课题通过采集玫瑰园、植物园等地的土壤样品若干，旨在获得一株对β-苯乙醇有一定耐受，且能利用L-苯丙氨酸或从头合成β-苯乙醇的细菌，并鉴定其种属。研究结果显示，通过镜检观察所筛选菌株的形态结构并进行肠杆菌科鉴定的生理生化实验，结合分子方法克隆细菌16S rDNA特异性序列，并将测序结果在GenBank数据库中进行BLAST比对，确定其为肠杆菌属细菌。经过生物发酵的实验表明该菌可以利用L-苯丙氨酸为唯一氮源生成β-苯乙醇，通过GC、MS、IR三种检测方法对发酵产物鉴定，结果表明该菌株确实具有生产β-苯乙醇的特性。

关键词： β-苯乙醇；细菌；菌种鉴定；气相色谱

Isolation and identification of a β- phenethyl alcohol producing bacterial strain

Abstract: β-phenylethyl is a kind of alcohol used widely in cosmetics and food industry. The demand for β-phenylethyl in global market is increasing rapidly. Currently, the majority of the literature and reports usually used yeast for biotransformation to produce natural β-phenylethyl. But the bacterial strain is easier to obtain. The β-phenylethyl can be synthesized by a more convenient pathway, and the cultural conditions are simpler. Therefore, the isolation of the β-phenylethyl -producing bacteria will be of great significance to the research of relevant subjects and industrial production. In this study, soil samples were obtained from rose garden and botanic garden. The purpose for collecting these samples were to isolate a β-phenylethyl producing and tolerance bacteria which can utilize L-Phe or glucose to synthesisβ-phenylethyl, and figure out the genus of this bacterial strain. It was found that the bacteria that was isolated belonged to enterobacter bacteria by microscopic examination, physiological and biochemical experiment, and the 16S rRNA sequencing. After the first stage fermentation and secondary fermentation, this bacteria produced β-phenylethyl using L-Phe as the only nitrogen source. The product was further identified by GC, MS and IR detection.It was determined that the strain had the characteristic to synthesize β-phenylethyl.

Keywords: β-phenylethyl alcohol; bacteria; Species identification; Gas Chromatography

目录

摘要 i

Abstract i

目录 iv

1 绪论 1

1.1 Β-苯乙醇的基本性质及其应用 1

1.1.1 β-苯乙醇的基本性质 1

1.1.2 β-苯乙醇的应用 2

1.2 国内外研究现状 2

1.2.1 β-苯乙醇的生产方法 3

1.2.1.1 物理方法来提取β-苯乙醇 3

1.2.1.2 化学方法来合成β-苯乙醇 3

1.2.1.2 微生物法来合成β-苯乙醇 4

1.2.2 β-苯乙醇微生物中的代谢途径 5

1.2.2.1 Ehrlich途径 5

1.2.2.2 β-苯乙醇从头合成途径 5

1.2.2.3 苯丙酮酸途径 6

1.2.3 β-苯乙醇细菌的分离鉴定研究目的和意义 6

2 材料与方法 7

2.1 实验材料及试剂 7

2.1.1 菌种来源及试剂 7

2.1.2 培养基 7

2.2 仪器设备 7

2.2.1 实验仪器与设备 7

2.3 菌种筛选实验方法 8

2.4 菌种鉴定实验方法 8

2.4.1 电子显微镜镜检 8

2.4.2 16s rRNA鉴定 8

2.5 菌种生理生化 10

2.6 物质鉴定 11

2.6.1 生物转化 11

2.6.2 GC检测 11

2.6.3 MS检测 12

2.6.4 IR检测 12

3 结果及分析 13

3.1 菌种筛选分离结果及分析 13

3.1.1 筛选纯化 13

3.2 菌种鉴定结果及分析 13

3.2.1 显微镜镜检法 13