Microbulbifersp.A4B-17的羧酸酯酶研究
毕业论文关键词：微泡菌；对羟基苯甲酸酯类；合成生物学；羧酸酯酶
Characterization of A Carboxylesterase Gene of Microbulbifer sp. A4B-17
Abstract: Microbulbifer sp. A4B-17 isolated from Marine can synthesize and accumulate the 4-hydroxybenzoic acid (4HBA) and 4HBAesters. 4HBA esters have antimicrobial activity, which is used widely as preservatives in the current. 4HBAesters are synthesized by the esterification of 4HBA and corresponding alcohols. 4HBA is coming from the oil compound (benzene) by the chemical methods, which consume a lot of energy with low production efficiency and cause a serious pollution problem. Our study is to synthesize 4HBA esters from renewable sources using strain A4B-17. Genome analysis of strain A4B-17 indicated that a gene numbered GL004038, might encode a carboxylesterase responsible for the biosynthesis of 4HBA esters. In order to clarify the function of this gene, I cloned and expressed GL004038 using T vector in E. coli, and carried out an enzyme assay. Butyl 4HBA was detected by HPLC analysis in reaction mixture, which containing enzyme, 4HBA and butyl alcohol. This result indicated that the gene product of GL004038 is a carboxylesterase.
Keywords: Microbulbifer; 4HBA esters; synthetic biology; carboxylesterase
目录
引言 1
1. 材料与方法 2
1.1 材料 2
1.1.1 菌株和质粒 2
1.1.2 生化试剂与分子生物学试剂 2
1.1.3 普通试剂 2
1.1.4 溶液与缓冲液 2
1.1.5 仪器设备 3
1.2 方法 4
1.2.1 相关溶液制备 4
1.2.2 大肠杆菌感受态细胞制备 6
1.2.3 碱裂解法制备质粒 6
1.2.4 A4B-17 菌株的全基因组提取 7
1.2.5 GL004038基因分析 8
1.2.6 构建表达载体 8
1.2.7 酶反应检测 11
2. 结果与分析 13
2.1 GL004038基因分析 13
2.2 引物设计 15
2.3 PCR扩增 15
2.4 重组质粒筛选 16
2.5 重组载体正反向鉴定 17
2.6 基因测序 19
2.7 酶活测定 20
3.讨论 21
4.致谢 22
5.参考文献 22
引言
Microbulbifer sp. 是一种从海洋中分离出来的海洋细菌，中文名称为溶藻细菌，该菌生产的生理活动物质以及海藻降解酶引起了人们的关注，Microbulbifer sp. A4B-17在2006年被发现能够合成对羟基苯甲酸（4HBA）[1]和4HBA酯类，并且是已发现的在微生物中合成量最多的菌株，A4B-17菌株合成的4HBA酯类有4HBA丁酯、4HBA壬酯、4HBA庚酯，其中4HBA丁酯的含量最多[2]。4HBA酯类具有抑菌作用，帮助抑制周围微生物的生长，扩大自己的生存空间[3] [4]。该菌株拥有3条糖代谢途径，糖酵解途径、磷酸戊糖途径和ED途径。4HBA的合成是部分利用莽草酸途径首先合成分支酸，在分支酸裂解酶的催化下生成4HBA。
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