苯并噁唑酮降解关键基因cnbCαCβ的表达及酶学性质表征(论文10000字)
摘要
苯并噁唑酮(BOA)是一类重要的含氮稠杂环化合物，具有杀菌、除草等作用，所以在农药开发中具有举足轻重的作用，但由于此类化合物具有对动植物有很大的毒害性且很容易残留在土壤中，合理降解就显得格外重要。本文根据前期工作筛选到BOA高效降解菌株Pigmentiphagasp. DL-8，并通过生物信息学方法鉴定了参与BOA降解过程的关键酶BOA水解酶CbaA和2-氨基苯酚（2AP）降解基因簇cnb为基础，在本研究中进一步以菌株DL-8基因组为模板，成功克隆并异源表达了2AP双加氧酶编码基因cnbCαCβ，并对其酶学性质进行表征。结果表明：cnbCαCβ由684bp和945 bp的α、β两个亚基组成，将其在大肠杆菌BL21(DE3)中成功实现异源表达并通过亲和层析进行纯化，SDS-PAGE显示获得电泳纯重组rCnbCαCβ；rCnbCαCβ的米氏常数(Km)为0.92 μM，最大反应速率(Vmax)为38.7 μmol•min-1•mg-1，最适反应温度为40℃，最适反应pH为7.5，但该酶对环境条件（温度、pH、化学试剂等）稳定性较差。
关键词：苯并噁唑酮微生物降解Pigmentiphagasp.DL-8酶学性质
Cloning and expression of 2-aminophenol dioxygenase gene involved in benzoxazolonedegradation
Abstract
Benzo-oxazolone (BOA) is a kind of important nitrogen-containing fused heterocyclic compounds, with sterilization, weeding and so on, so in the development of pesticides plays a decisive role, but because of such compounds have a great animal and plant Toxic and easy to remain in the soil, a reasonable degradation is particularly important. Based on the preliminary work, the BOA-degrading strain, Pigmentiphagasp. DL-8, was screened by bioinformatics method. The key enzymes involved in the BOA degradation process were identified. The CABA and 2-aminophenol (2AP). In this study, the gene of the DL-8 genome was used as template to successfully clone and express the 2AP double oxygenase gene cnbCαCβ, and its enzymatic characterization was carried out. The results showed that cnbCαCβconsisted of α and β subunits of 684 bp and 945 bp.They were successfully expressed in Escherichia coli BL21 (DE3) and purified by affinity chromatography (Km) was 0.92 μM, the maximum reaction rate (Vmax) was 38.7 μmol•min-1•mg-1, the optimum reaction temperature was 40℃, and the maximum reaction rate (Vmax) was 38.7 μmol•min-1•mg-1, the optimum reaction temperature was 40℃, The optimum pH was 7.5, but the enzyme had poor stability to environmental conditions (temperature, pH, chemical reagents, etc.).
Key Words:Benzoxazolone; Microbial degradation; Pigmentiphagasp.DL-8; Enzymatic properties
目录
摘要 I
ABSTRACT II
第一章前言 1
第二章文献综述 2
2.1苯并噁唑酮简介 2
2.1.1苯并噁唑酮的理化性质 2
2.1.2苯并噁唑酮的作用 2
2.2苯并噁唑酮及其衍生物的危害 2
2.3苯并噁唑酮的微生物降解机制 3
第三章材料与方法 5
3.1材料 5
3.1.1 菌株、质粒以及引物 5
3.1.2培养基以及试剂 5
3.2 方法 6
3.2.1Pigmentiphaga sp. DL-8中的2AP双加氧酶基因挖掘 6
3.2.2 2AP双加氧酶基因cnbCαCβ的克隆和基因重组表达菌株的构建 6
3.2.3重组酶CnbCαCβ的诱导表达 9
3.2.4CnbCαCβ粗酶的亲和层析纯化 9
3.2.5 2AP双加氧酶CnbCαCβ的米氏常数(Km)和最大反应速率(Vmax) 10
3.2.6温度和pH值对重组酶CnbCαCβ活性和稳定性的影响 10
第四章结果与讨论 11
4.1 2AP代谢基因簇的挖掘 11
4.2 2AP代谢的关键基因cnbCαCβ的克隆表达和重组表达菌株的构建 11
4.3重组酶CnbCαCβ的诱导表达和纯化 12
4.4 2AP双加氧酶CnbCαCβ的米氏常数和最大反应速率 12
4.5 温度和pH对2AP双加氧酶CnbCαCβ活性的影响 13
4.6金属离子对2AP双加氧酶CnbCαCβ活性的影响 14
4.7 表面活性剂、螯合剂和有机溶剂对2AP双加氧酶重组酶rCnbCαCβ活性的影响 15
4.8讨论 15
致谢 16
参考文献 17 |
