本文選題：CphA酶 + 亞胺培南　； 參考：《西北大學(xué)》2017年碩士論文

【摘要】：金屬β-內(nèi)酰胺酶(Metallo-beta lactamase,MβLs)通過水解β-內(nèi)酰胺抗生素的β-內(nèi)酰胺環(huán)而阻止抗生素對(duì)抗細(xì)菌感染的有益行為。來自于嗜水氣單胞菌的CphA酶是MβLs亞群的一員,其在活性位點(diǎn)只有一個(gè)Zn2+,并只特異性水解碳青霉烯類抗生素。然而對(duì)于CphA酶與碳青霉烯類抗生素之間的分子識(shí)別和相互作用知之甚少。本研究將在重組菌中表達(dá)并分離純化得到CphA酶的基礎(chǔ)上,采用熒光光譜法和分子對(duì)接模擬法相結(jié)合,以亞胺培南(imipenem,IMP)和比阿培南(biapenem,BIA)為研究對(duì)象,研究它們?cè)诮Y(jié)合過程中,酶的構(gòu)象變化、抗生素分子的空間分布取向、形成絡(luò)合物的本質(zhì)以及它們之間的作用力類型和作用位點(diǎn)數(shù)等。(1)將含有pET28b-CphA的大腸桿菌BL21(DE3)進(jìn)行誘導(dǎo)表達(dá),通過陽離子交換層析分離純化,然后用SDS-PAGE鑒定和Bradford測(cè)蛋白含量,最后對(duì)其進(jìn)行熒光表征。結(jié)果表明:得到有活性的純度較高的樣品,其分子量約為28 KDa,純化后產(chǎn)量約為0.18～0.21 mg/mL,當(dāng)激發(fā)波長為278 nm時(shí),發(fā)現(xiàn)在336.2 nm處熒光明顯。(2)在 277 K、pH 7.4 PBS 中,測(cè)得 CphA 酶對(duì) IMP 與 BIA 的 Km 分別為 356μM和286 μM;通過熒光發(fā)射光譜測(cè)得CphA酶與IMP和BIA的分子飽和比分別為0.95和0.98;同步熒光光譜結(jié)果表明:它們相互作用且Tyr殘基附近的微環(huán)境發(fā)生微弱的變化;測(cè)定277 K、281 K和285 K三個(gè)溫度下CphA酶分別對(duì)IMP和BIA結(jié)合過程的猝滅光譜,結(jié)果表明:它們之間發(fā)生的猝滅均為靜態(tài)猝滅;它們之間是依靠單一的結(jié)合位點(diǎn)結(jié)合,且均為自發(fā)的放熱過程。(3)分子對(duì)接結(jié)果顯示CphA酶與IMP和BIA在結(jié)合過程中存在一些相同之處:兩種抗生素β-內(nèi)酰胺環(huán)的C3羧基氧直接與Zn2+形成金屬配位鍵,促進(jìn)相互作用,且均使CphA酶loop環(huán)發(fā)生局部的構(gòu)象變化。不同之處為:IMP因側(cè)鏈空間位阻較小,使其全部進(jìn)入口袋,在相互作用過程中,共有3組靜電力和5組氫鍵形成;BIA因側(cè)鏈雙環(huán)三唑的位阻比較大,留部分側(cè)鏈位于口袋外側(cè),在相互作用過程中,共有4組靜電力和2組氫鍵,進(jìn)而使得CphA酶對(duì)BIA的親和力大于對(duì)IMP的,以及形成CphA-BIA復(fù)合物的穩(wěn)定性也大于CphA-IMP復(fù)合物的穩(wěn)定性。熒光光譜結(jié)果中CphA-BIA復(fù)合物體系的Ksv和Ka高于CphA-IMP復(fù)合物體系的,也證實(shí)這一推斷。另外,對(duì)接結(jié)果顯示復(fù)合物體系△G為負(fù)值,表明兩個(gè)復(fù)合物的形成均屬于自發(fā)的放熱過程,與前期熱力學(xué)結(jié)果一致。
[Abstract]:Metal beta lactamases (Metallo-beta lactamase, M beta Ls) inhibit the beneficial behavior of antibiotics against bacterial infection by hydrolysis of beta lactam beta lactam rings. The CphA enzyme from Aeromonas hydrophila is a member of the M beta Ls subgroup, with only one Zn2+ at the active site and only specifically hydrolyzed carbapenems. There is little knowledge about the molecular recognition and interaction between CphA enzyme and carbapenems. On the basis of the expression and separation and purification of CphA enzymes in the recombinant bacteria, the study is based on the combination of fluorescence spectroscopy and molecular docking simulation, and the research object is imipenem (imipenem, IMP) and apenem (biapenem, BIA). The conformation changes of the enzyme, the spatial distribution orientation of the antibiotic molecules, the nature of the complex and the number of action points between them, and so on. (1) the BL21 (DE3) containing pET28b-CphA was induced and expressed by the cation exchange chromatography, and then identified by SDS-PAGE and B The protein content was measured by Radford. The results showed that the high purity samples were obtained with a molecular weight of about 28 KDa and the yield was about 0.18 ~ 0.21 mg/mL after purification. When the excitation wavelength was 278 nm, the fluorescence was found at 336.2 nm. (2) the IMP and BIA Km were measured in 277 K and pH 7.4 PBS. The molecular saturation ratio of CphA enzyme to IMP and BIA was 0.95 and 0.98, respectively, by fluorescence emission spectra, respectively. The results of synchronous fluorescence spectra showed that they interact with the microenvironment of Tyr residues in the microenvironment slightly, and the CphA enzyme of 277 K, 281 K and 285 K three temperatures was sudden, and the CphA enzyme in the binding process of IMP and BIA, respectively. The quenching spectrum shows that the quenching between them is static quenching; they are dependent on a single binding site and are spontaneous exothermic processes. (3) molecular docking results show that there are some similarities between CphA enzyme and IMP and BIA in the process of binding: the C3 carboxyl oxygen of the two antibiotics beta lactam ring is directly and Zn2+ shaped. Metal coordination bonds promote interaction and make local conformation changes of the CphA loop ring. The difference is that IMP has 3 groups of static electricity and 5 groups of hydrogen bonds in the process of interaction, because the side chain space has smaller steric resistance, and the side chain of the side chain of double ring three azole is relatively large, and the remaining side chain is located outside the pocket. Side, in the process of interaction, there are 4 groups of static electricity and 2 groups of hydrogen bonds, which make the affinity of CphA enzyme to BIA greater than that of IMP, and the stability of the CphA-BIA complex is greater than the stability of CphA-IMP complex. The Ksv and Ka of the CphA-BIA complex system in the fluorescence spectrum is higher than the CphA-IMP complex system, and this also confirms this In addition, the docking results show that the complex system Delta G is negative, indicating that the formation of the two complexes all belong to the spontaneous exothermic process, which is in accordance with the previous thermodynamic results.

【學(xué)位授予單位】：西北大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R96

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