脆弱拟杆菌CcrA酶与头孢菌素类抗生素之间的分子识别和相互作用

发布时间：2018-02-15 14:34

本文关键词： CcrA酶头孢他啶头孢噻肟分子识别相互作用　出处：《西北大学》2017年硕士论文　论文类型：学位论文

【摘要】：超级细菌的出现使得临床上使用的多数β-内酰胺类抗生素失效,这主要是因为其可以产生一种水解抗生素的金属β-内酰胺酶,脆弱拟杆菌产生的金属β-内酰胺酶CcrA是一种典型的金属酶,但目前对其与该类抗生素之间的结合过程研究比较少,对此,本研究采用实验测定和理论预测相结合的方法,进行对金属β-内酰胺酶CcrA与头孢噻肟(CTX)和头孢他啶(CAZ)之间分子识别和相互作用机理的研究,主要内容如下:1.在大肠杆菌中对金属β-内酰酶CcrA进行诱导表达,通过镍离子亲和层析法实现对目的蛋白的分离纯化,采用聚丙烯酰胺凝胶电泳法(SDS-PAGE)对蛋白进行鉴定,最后通过Bradford法测定蛋白的含量。结果表明,该过程实现了对目的蛋白CcrA的高效表达,且纯化后CcrA酶的纯度较高,蛋白含量为0.26-0.28 mg/mL。2.利用荧光光谱法对CcrA酶与两种头孢菌素类抗生素(CTX和CAZ)之间的分子识别和相互作用进行研究。测定了三个温度下(277K、281K、285K)抗生素和CcrA酶相互作用的荧光光谱,获得了二者结合过程中的荧光猝灭常数Ksv和猝灭速率常数Kq、结合常数Ka、结合位点数n以及结合过程中的热力学参数等。实验结果表明,两种抗生素与CcrA酶之间发生的猝灭均为静态猝灭;在相同温度下,CcrA酶与CTX结合体系的Ksv和Kq均大于CcrA酶与CAZ结合体系,且CcrA酶与CTX结合体系的结合常数Ka和结合位点数n也比较大,这表明CcrA酶与CTX的结合能力大于CcrA酶与CAZ,同时也表明,结合过程生成的CcrA-CTX复合物稳定性比较高。实验结果得出,CcrA酶与两种抗生素在结合过程中的吉布斯自由能、焓变等均为负值,表明CcrA酶与CTX、CAZ之间的相互作用是一个自发、放热的过程。3.通过分子对接模拟方法研究了在CcrA酶与CTX、CAZ之间相互识别和作用过程中,酶分子内氨基酸残基和小分子配体中官能团的相互作用模式。分析发现位于酶分子活性口袋附近的loop环柔性变化较大,表明二者在相互作用过程中,诱导契合效应使得抗生素进入酶分子活性口袋,loop环的柔性变化更有利于酶分子与抗生素结合,同时也佐证了以上实验得出的结论。两个结合体系对接结果得到的吉布斯自由能变均为负值,表明CcrA酶与两种抗生素之间的结合过程均可以自发进行,与实验结果相一致。
[Abstract]:The emergence of superbacteria invalidates most of the 尾-lactams used in clinical use, mainly because they produce a metallo- 尾-lactamase, a hydrolytic antibiotic. Metallo-beta-lactamase (CcrA) produced by Bacteroides fragilis is a typical metalloenzyme, but its binding process with this kind of antibiotics is less studied at present. The molecular recognition and interaction mechanism of metal 尾 -lactamases CcrA with cefotaxime and ceftazidime cazo (ceftazidime) were studied. The main contents were as follows: 1. The expression of metal 尾 -lactamase CcrA was induced in E. coli. The target protein was separated and purified by nickel ion affinity chromatography. The protein was identified by polyacrylamide gel electrophoresis (SDS-PAGE). Finally, the content of the protein was determined by Bradford. The high expression of the target protein CcrA was achieved in this process, and the purity of the purified CcrA enzyme was high. The protein content was 0.26-0.28 mg / mL 路2.The molecular recognition and interaction between CcrA enzyme and two cephalosporins were studied by fluorescence spectrometry. The fluorescence spectra of the interaction between CcrA enzyme and CcrA enzyme were determined at three temperatures. The fluorescence quenching constants (Ksv) and quenching rate constants (K _ Q), binding constants (K _ Q), binding sites (n) and thermodynamic parameters in the binding process were obtained. The quenching between the two antibiotics and the CcrA enzyme was static quenching, and the Ksv and KQ of the system were higher than that of the combination system of CcrA and CAZ at the same temperature. The binding constant Ka and binding site number n of CcrA enzyme to CTX system are also relatively large, which indicates that the binding ability of CcrA enzyme to CTX is higher than that of CcrA enzyme and cazase, and it also shows that the binding capacity of CcrA enzyme to CTX is higher than that of CcrA enzyme. The results show that the Gibbs free energy and enthalpy change of the binding process are all negative, which indicates that the interaction between the CcrA enzyme and the CCAZ is spontaneous. The process of exothermic. 3. Molecular docking simulation was used to study the process of mutual recognition and interaction between CcrA enzyme and CTX caz. The interaction patterns of amino acid residues in enzyme molecules and functional groups in small molecular ligands were analyzed. It was found that the flexibility of the loop ring located near the active pockets of enzyme molecules changed greatly, which indicated that the interaction between the two molecules occurred in the process of interaction. The effect of induced binding makes the flexibility change of antibiotics into the loop loop of enzyme active pocket more favorable to the binding of enzyme molecules to antibiotics. The Gibbs free energy of the two binding systems is negative, which indicates that the binding process between CcrA enzyme and two antibiotics can be carried out spontaneously, which is consistent with the experimental results.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

【相似文献】