Microbulbifer sp. A4B-17菌株的分支酸裂解酶基因研究
毕业论文关键词：分支酸裂解酶，对羟基苯甲酸，Microbulbifer
Characterization of a Chorismate Lyase Gene of Microbulbifer sp.A4B-17
Lv Danli Supervisor: Peng Xue
(School of Life Sciences, Jiangsu Normal University, Xuzhou,Jiangsu,221116,China)
Abstract: Microbulbifer sp. A4B - 17 strains isolated from Marine environment, and found that it can synthesize 4-hydroxybenzoate (4HBA) and 4HBA esters. 4 HBA is a kind of organic raw material,which can be used for synthesis of anti-corrosion, fungicide, bactericide, and the main liquid crystal intermediates .Nowadays, the production process of 4 HBA is normally chemical synthesis, the process has big energy consumption, low production efficiency, high cost and easy to pollute the environment. It was found that a gene with number GL000722 may be coding for chorismate lyase, which is the key enzyme for catalyzing the conversion of chorismate to 4HBA. In order to verify function of the GL000722 gene, which was cloned into the expression vector, and the assay for enzyme activity was carried out. The results prove that GL000722 coding product has chorismate lyase activity.
Key words: 4-Hydroxybenzoate; Microbulbifer; Chorismate lyase
目录
引言 1
1 材料与方法 1
1.1菌株和质粒 1
1.2普通试剂 2
1.3分子生物学相关试剂 2
1.4培养基及其配制 2
1.5筛选使用的试剂 2
1.6提取DNA时的溶液 3
1.7电泳检测时所用溶液 3
1.8实验仪器 3
1.9实验方法 4
1.9.1准备部分 4
1.9.2设计引物 5
1.9.3 PCR 6
1.9.4宿主菌和载体的选用 8
1.9.5 PCR产物和载体的酶切 8
1.9.6目的基因与载体连接并转化 8
1.9.7培养与筛选 9
1.9.8检测并大量培养目标重组菌 9
1.9.9 IPTG诱导 10
1.9.10粗酶提取 10
1.9.11酶活力测定 10
1.9.12 HPLC分析 11
2 结果与分析 11
2.1 GL000722基因分析 11
2.2酶切PCR产物和载体结果 12
2.3重组载体筛选 12
2.4酶活测定结果 13
3 讨论 14
4致谢 15
参考文献 15
引言
Microbulbifer sp.A4B-17 菌株是海洋环境中分离出来的菌，其生产的生理活性物质和海藻成分降解酶等正受到人们的重视【1】【2】【3】。A4B-17菌株具有合成4HBA和4HBA酯类的功能【4】，并且是已发现的在微生物中合成量最多的菌。4HBA是用途广泛的有机合成原料，性质稳定，不挥发，化学结构简单类似于芳香族氨基酸【5】，主要用于食品、化妆品、医药的防腐、防霉剂和杀菌剂，尤其是液晶的主要合成中间体，市场需求量越来越大。