《红冬孢酶母中AHL降解酶的分离纯化》由会员分享，可在线阅读，更多相关《红冬孢酶母中AHL降解酶的分离纯化（45页珍藏版）》请在金锄头文库上搜索。
1、河北工业大学 硕士学位论文 红冬孢酶母中AHL降解酶的分离纯化 姓名：罗利龙 申请学位级别：硕士 专业：生物化工 指导教师：宋水山 20100501 河北工业大学硕士学位论文 i 红冬红冬孢孢酵母中酵母中 AHL 降解酶的分离纯化降解酶的分离纯化 摘摘 要要 群体感应（QS）广泛存在于细菌中，是细菌根据细胞密度变化调控基因表达的一种 机制。许多植物病原菌依赖 QS 调控致病基因和毒性因子的表达，导致植物发病，因此通 过抑制 QS 效应就为控制细菌病害提供了一种有效的方法。酰基高丝氨酸内酯 （N-acylhomoserine lactone，AHL）是细菌调控群体感应系统的关键信号分子。近年的研
2、 究表明， 不同生物体包括细菌和真核生物中都存在类别不同的能够降解酰基高丝氨酸内酯 的酶。 在 AHL 依赖型致病菌和转基因植物中 AHL 降解酶能有效的抑制 QS 信号分子的积 累， 从而阻断病原菌的发病机制， 提高植物的抗病性。 这些新颖的群体感应淬灭酶的发现， 不仅为防治细菌侵染提供了可行的途径， 也对研究它们在宿主中的作用和对生态系统的潜 在影响提出挑战。邱健等首次筛选到一株能够降解 AHL 的酵母菌，经鉴定为红冬孢酵母 （Rhodosporidium toruloides） 这一发现丰富了群体感应淬灭酶的来源。 本论文对红冬孢酵 母菌种 AHL 降解酶进行了分离纯化。红冬孢酵母在麦氏
3、培养基中培养好以后离心收集菌 体，通过超声破碎细胞得到总蛋白。用硫酸铵沉淀法得到 AHL 降解酶的粗制品。然后通 过离子交换、疏水层析以及胶回收方法实现了对 AHL 降解酶的分离纯化。获得了银染电 泳纯的单一条带。通过实验研究了 pH 以及盐浓度对 AHL 降解酶的影响，研究了 AHL 降 解酶的底物谱以及对 AHL 的作用位点。并通过双向电泳对 AHL 降解酶的分子量以及等 电点进行了测定。 关键词：关键词：群体感应淬灭酶，分离纯化，胶回收 河北工业大学硕士学位论文 iii THE SEPARATION AND PURIFICATION OF AHL-DEGRADATION ENZYME I
4、N RHODOSPORIDIUM TORULOIDES ABSTRACT Quorum-Sensing(QS), which regulates gene expression depending on population density, is universal in bacteria. QS is involved in the regulation of virulent factor and pathogenic gene expression, and plays a key role in causing plant disease by plant bacterial pat
5、hogens. Hense, targeting the bacterial QS seem to be a promising approach for control the disease caused by QS-mediated bacterial pathogens. N-acyl homoserine lactone (AHL) acts as a key singnal in bacterial Quorum-Sensing. Several groups of AHL-degradation enzymes have recently been identified in a
6、 rang of living organisms including bacteria and eukaryotes. Expression of these genes encoding AHL-dagrading enzyme in AHL-dependent pathogens and plants efficiently quench the acceleration of QS signal and blocks pathogenic infection.Discovery of these novel quorum quenching enzymes has not only p
7、rovided a promising means to control bacterial infections,but also presents new challenges to investigate their roles in host organisms and their potential impact on ecosystems. Qiu jian et al screened a yeast which can be able to degrade AHL and was identified as Rhodosporidium toruloides. The disc
8、overy riched the quorum quenching enzyme.In this paper we success in separation and purification of this enzyme. The R. toruloides was cultured in MacConkey medium and then we collected the bacterial by centrifugation. Through the ultrasonic we get the total proteins. The total proteins were concent
9、rated by ammonium sulphate fractionation and then purified by anion exchange 红冬孢酵母中 AHL 降解酶的分离纯化 iv chromatography, hydrophobic interaction and gel recovery and finally showed a single band on the SDS-PAGE. Through experiment we studied the pH and the salt concentration on the impact of the AHL-degr
10、ading enzyme and the AHL-degrading enzyme substrate spectrum as well as the degradation activity. We get the molecular weight and the isoelectric point of the enzyme by two-dimensional electrophoresis. KEY WORDS: quorum-quenching enzyme, separation and purification, gel recovery 河北工业大学硕士学位论文 v 符号说明符
11、号说明 AHL QS SAM MTA Acyl-ACP IPTG DE TEMED TCA KD acyl-homoserine lactone quorum sensing S-adenosylmethionine methythioadenosine acyl-acyl carrier protein isopropylthio-D-galactoside two-dimensional electrophoresis N,N,N,N-Tetramethylethylenediamine Trichloroacetic Acid kilodalton 酰基高丝氨酸内酯 群体感应 腺苷甲硫氨
12、酸 二维凝胶电泳 酰基-酰基载体蛋白 异丙基硫代-D-半乳糖苷 千道尔顿 四甲基乙二胺 三氯乙酸 甲基硫代腺苷 ? 1 ? 1-1 ? 1-1-1? ? ? ? ? ?Quorum Sensing?QS?1-3?QS? ?QS? ?QS? ?Agrobacterrium tumefaciens?Ti?Erwinia carotovora? ?Pseudomonas aeruginosa?P. aureofaciens? ?E. carotovora?4-11?QS? ? QS? ?QS?QS? ? 1-1-2? 1-1-2-1? ?Acyl-homoserine lactone?AHL?G-?
13、QS? ?AHLs?-?-?- ?AHLs12?1?AHLs? ? ?4?18?AHLs? ? ?1?AHLs?12?14? ?Bainton?13?E. carotovora?3-Oxo-C6-HSL?-?3-Oxo-C6-HSL?L ?AHLs?L?Chhabra?D?AHL? ?QS?12,14?AHL?1.1? ?QS? 15?AHLs?C 4-HSL? 3-Oxo-C4-HSL?P. aureofaciens16?Vibrio harveyi?17? ? AHL ? 2 ?1.1 ? Fig. 1.1 The AHLs and their hydrolysis products AHLs?G-?lux?lacZ?gfp?18-21?22? ?AHLs?G-? ? ? ?AHLs?AHLs?1.1?AHL? QS? ?1.1 ?AHL?QS? Table 1-1 Some examples
