表面活性劑與過氧化氫酶相互作用對酶活性和構(gòu)象的影響
發(fā)布時間：2018-09-06 09:04
【摘要】：與很多合成的催化劑相比,酶催化具有較高的選擇性和催化效率、較小的生物毒性及反應(yīng)條件溫和等優(yōu)點(diǎn),是生物體內(nèi)各種生物化學(xué)過程必不可少的。在酶的應(yīng)用中,總是要考慮兩個基本問題,即酶在反應(yīng)環(huán)境下的催化活性和活性的動力學(xué)穩(wěn)定性。表面活性劑是酶催化反應(yīng)體系中經(jīng)常使用的添加劑,夠調(diào)控酶的催化活性和穩(wěn)定性。本論文采用等溫滴定量熱(ITC)、熒光光譜、圓二色譜和紫外可見光譜等方法,研究了陰離子、陽離子和兩性離子表面活性劑與過氧化氫酶(BLC)的相互作用及對酶結(jié)構(gòu)和活性的影響,從熱力學(xué)與分子結(jié)構(gòu)的角度討論了表面活性劑對酶活性影響的機(jī)理。主要研究內(nèi)容和結(jié)果如下:1.通過紫外可見光譜(UV-vis)表征了表面活性劑對酶活性的影響。結(jié)果表明,陰離子型表面活性劑(SDS、SDSo)在低濃度下能抑制BLC活性,并且在它們各自的臨界膠束濃度(cmc)附近時能夠使酶變性。當(dāng)陽離子型表面活性劑(DTAB)逐漸加入到BLC溶液時,在DTAB濃度達(dá)到cmc以前,過氧化氫酶經(jīng)歷了聚集而后再分散的過程,酶的相對活性降低到80%左右,而動力學(xué)穩(wěn)定性明顯提高。兩性離子型甜菜堿表面活性劑(SB3-12)對酶的活性沒有明顯的影響,對酶的天然結(jié)構(gòu)也具有一定的保護(hù)作用。在SB3-12存在時,過量的SB3-12能抑制SDS對BLC活性的影響。發(fā)現(xiàn)當(dāng)xSB3-120.5時,BLC依然能夠保持較高的活性,當(dāng)xSB3-120.5時,BLC的活性迅速降低,BLC的活性受兩種表面活性劑的摩爾分?jǐn)?shù)控制。當(dāng)BLC在DTAB與SB3-12混合表面活性劑溶液中培養(yǎng)時,酶活性受DTAB濃度控制。2.采用等溫滴定量熱研究了表面活性劑與酶分子之間的相互作用。研究結(jié)果表明,表面活性劑與酶的相互作用的熱力學(xué)與極性頭基的電荷性質(zhì)有關(guān),離子型表面活性劑首先會與酶分子上的相反電荷位點(diǎn)結(jié)合。SDS與酶之間的靜電作用伴隨著烷基鏈與酶分子的疏水作用,過量的SDS在酶分子表面形成分子簇,使酶展開;當(dāng)DTAB與酶發(fā)生靜電作用時,由于酶表面的負(fù)電荷位點(diǎn)附近缺少疏水域,過量的DTAB在不同酶分子上形成的分子簇能夠聚集成共享膠束,促進(jìn)了BLC分子的靠近并發(fā)生聚沉。隨著DTAB濃度的增加,共享膠束解離成結(jié)合在酶表面上的半膠束,增加的靜電排斥效應(yīng)使聚集體開始重新分散;兩性離子的SB3-12與酶分子之間的相互作用要比DTAB或SDS與酶分子之間的相互作用都要弱。SB3-12和SDS形成的混合表面活性劑與酶的相互作用由它們的摩爾分?jǐn)?shù)控制,當(dāng)xSDS0.50時,SDS分子協(xié)同地與酶分子作用形成分子簇,同時也與SB3-12膠束作用形成混合膠束,在一定的組成區(qū)間能夠抑制SDS對BLC的變性效應(yīng)。當(dāng)xSDS0.50時,過量的SDS作用于BLC分子,使酶分子展開。對于SB3-12/DTAB/BLC混合體系,SB3-12不影響DTAB與BLC的靜電作用,但是能夠促進(jìn)BLC分子之間形成混合的或SB3-12和DTAB分別構(gòu)成的共享膠束,減小了使BLC聚沉的DTAB濃度。3.采用熒光光譜法和圓二色譜法研究了不同類型的表面活性劑對酶構(gòu)象的影響。在陰離子型表面活性劑(SDS、SDSo)存在時,BLC發(fā)生了三級結(jié)構(gòu)由松弛到展開的漸變過程,α-螺旋和β-折疊的分?jǐn)?shù)也同時發(fā)生變化,無規(guī)則卷曲分?jǐn)?shù)增加,直至酶分子完全展開。在含SB3-12的體系中,酶的二級和三級結(jié)構(gòu)沒有發(fā)生明顯的變化。DTAB使酶分子的三級結(jié)構(gòu)發(fā)生了明顯的變化,但是由于酶聚集體的生成,CD光譜不能檢測酶的二級結(jié)構(gòu)。在酶和SB3-12共存的溶液里加入SDS,在SDS過量以后,酶的二級和三級結(jié)構(gòu)的變化與沒有SB3-12時有類似的規(guī)律。結(jié)合這些酶活性、分子間相互作用熱力學(xué)及酶結(jié)構(gòu)的研究結(jié)果,發(fā)現(xiàn)各種性質(zhì)的變化與表面活性劑的濃度相關(guān),據(jù)此提出了所研究的表面活性劑與BLC相互作用的機(jī)理。4.本課題組前期工作研究表明α-糜蛋白酶(α-CT)在陽離子型Gemini表面活性劑存在下具有超活性。作為此研究工作的延伸進(jìn)一步研究了12-10-12與α-CT混合體系。通過等溫滴定量熱法系統(tǒng)地測定了在12-10-12濃度小于cmc時與α-CT的相互作用焓,討論了在12-10-12緩沖溶液中酶的超活性及低的穩(wěn)定性的原因。
[Abstract]:Compared with many synthetic catalysts, enzymatic catalysis has the advantages of high selectivity and catalytic efficiency, less toxicity and mild reaction conditions, and is essential for various biochemical processes in organisms. Mechanical stability. Surfactants are additives frequently used in enzyme catalytic reaction systems, which can regulate the catalytic activity and stability of enzymes. In this paper, anionic, cationic and amphoteric surfactants and catalase (BLC) were studied by means of isothermal titration calorimetry (ITC), fluorescence spectroscopy, circular dichroism and ultraviolet-visible spectroscopy. The main contents and results are as follows: 1. The effects of surfactants on enzyme activity were characterized by UV-vis spectroscopy. The results showed that anionic surfactants (SDS, SDS) had significant effects on enzyme activity. O) inhibited the activity of BLC at low concentrations, and denatured the enzyme near their respective critical micelle concentrations (cmc). When cationic surfactants (DTAB) were gradually added to the BLC solution, catalase undergone a process of aggregation and redispersion before the concentration of DTAB reached cmc, and the relative activity of catalase decreased to about 80%. Amphoteric betaine surfactant (SB3-12) has no obvious effect on the activity of the enzyme and has a certain protective effect on the natural structure of the enzyme. In the presence of SB3-12, excessive SB3-12 can inhibit the effect of SDS on the activity of BLC. It is found that when xSB3-120.5, BLC can still maintain high activity, while x SB3-12 exists. The activity of BLC was controlled by the molar fraction of two surfactants. When BLC was cultured in the mixed surfactant solution of DTAB and SB3-12, the activity of BLC was controlled by the concentration of DTAB. 2. The interaction between surfactants and enzyme molecules was studied by isothermal titration calorimetry. Thermodynamics of the interaction between sex agents and enzymes is related to the charge properties of the polar head groups. Ionic surfactants first bind to the opposite charge sites on the enzyme molecules. The electrostatic interaction between SDS and enzymes is accompanied by hydrophobic interaction between alkyl chains and enzymes. Excess SDS forms molecular clusters on the surface of enzymes, and enzymes unfold when DTAB and DTAB interact with each other. Due to the absence of hydrophobic domains near the negative charge sites on the enzyme surface, excessive DTAB clusters on different enzyme molecules can aggregate into shared micelles, which promotes the proximity and precipitation of BLC molecules. The interaction between SB3-12 and enzyme molecules is weaker than that between DTAB or SDS and enzyme molecules. The interaction between SB3-12 and enzyme molecules is controlled by their molar fraction. When xSDS is 0.50, SDS molecules cooperate with enzyme molecules. When xSDS is 0.50, excessive SDS acts on the BLC molecule to expand the enzyme molecule. For SB3-12/DTAB/BLC mixtures, SB3-12 does not affect the electrostatic interaction between DTAB and BLC, but can promote the denaturation of BLC molecule. Mixed or shared micelles of SB3-12 and DTAB were formed to reduce the concentration of DTAB which agglomerated the BLC. 3. The effects of different surfactants on the conformation of the enzyme were studied by fluorescence spectroscopy and circular dichroism spectroscopy. In the system containing SB3-12, the secondary and tertiary structures of the enzyme did not change significantly. DTAB changed the tertiary structure of the enzyme molecule significantly, but CD spectra did not change because of the formation of enzyme aggregates. It can be used to detect the secondary structure of enzymes. Adding SDS into the coexistence solution of enzymes and SB3-12, the changes of secondary and tertiary structure of enzymes are similar to those without SB3-12. Combining these enzymes, the thermodynamics of intermolecular interaction and the structure of enzymes, the changes of various properties and the concentration of surfactants are found. The mechanism of the interaction between the surfactant and BLC was proposed. 4. Previous work of our group showed that alpha-chymotrypsin (alpha-CT) had superactivity in the presence of cationic Gemini surfactant. As an extension of this work, the mixed system of 12-10-12 and alpha-CT was further studied. The enthalpy of interaction with alpha-CT at 12-10-12 concentration less than CMC was measured systematically. The reasons for the superactivity and low stability of the enzyme in 12-10-12 buffer solution were discussed.
【學(xué)位授予單位】：河南師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：O629.8
本文編號：2225861
[Abstract]:Compared with many synthetic catalysts, enzymatic catalysis has the advantages of high selectivity and catalytic efficiency, less toxicity and mild reaction conditions, and is essential for various biochemical processes in organisms. Mechanical stability. Surfactants are additives frequently used in enzyme catalytic reaction systems, which can regulate the catalytic activity and stability of enzymes. In this paper, anionic, cationic and amphoteric surfactants and catalase (BLC) were studied by means of isothermal titration calorimetry (ITC), fluorescence spectroscopy, circular dichroism and ultraviolet-visible spectroscopy. The main contents and results are as follows: 1. The effects of surfactants on enzyme activity were characterized by UV-vis spectroscopy. The results showed that anionic surfactants (SDS, SDS) had significant effects on enzyme activity. O) inhibited the activity of BLC at low concentrations, and denatured the enzyme near their respective critical micelle concentrations (cmc). When cationic surfactants (DTAB) were gradually added to the BLC solution, catalase undergone a process of aggregation and redispersion before the concentration of DTAB reached cmc, and the relative activity of catalase decreased to about 80%. Amphoteric betaine surfactant (SB3-12) has no obvious effect on the activity of the enzyme and has a certain protective effect on the natural structure of the enzyme. In the presence of SB3-12, excessive SB3-12 can inhibit the effect of SDS on the activity of BLC. It is found that when xSB3-120.5, BLC can still maintain high activity, while x SB3-12 exists. The activity of BLC was controlled by the molar fraction of two surfactants. When BLC was cultured in the mixed surfactant solution of DTAB and SB3-12, the activity of BLC was controlled by the concentration of DTAB. 2. The interaction between surfactants and enzyme molecules was studied by isothermal titration calorimetry. Thermodynamics of the interaction between sex agents and enzymes is related to the charge properties of the polar head groups. Ionic surfactants first bind to the opposite charge sites on the enzyme molecules. The electrostatic interaction between SDS and enzymes is accompanied by hydrophobic interaction between alkyl chains and enzymes. Excess SDS forms molecular clusters on the surface of enzymes, and enzymes unfold when DTAB and DTAB interact with each other. Due to the absence of hydrophobic domains near the negative charge sites on the enzyme surface, excessive DTAB clusters on different enzyme molecules can aggregate into shared micelles, which promotes the proximity and precipitation of BLC molecules. The interaction between SB3-12 and enzyme molecules is weaker than that between DTAB or SDS and enzyme molecules. The interaction between SB3-12 and enzyme molecules is controlled by their molar fraction. When xSDS is 0.50, SDS molecules cooperate with enzyme molecules. When xSDS is 0.50, excessive SDS acts on the BLC molecule to expand the enzyme molecule. For SB3-12/DTAB/BLC mixtures, SB3-12 does not affect the electrostatic interaction between DTAB and BLC, but can promote the denaturation of BLC molecule. Mixed or shared micelles of SB3-12 and DTAB were formed to reduce the concentration of DTAB which agglomerated the BLC. 3. The effects of different surfactants on the conformation of the enzyme were studied by fluorescence spectroscopy and circular dichroism spectroscopy. In the system containing SB3-12, the secondary and tertiary structures of the enzyme did not change significantly. DTAB changed the tertiary structure of the enzyme molecule significantly, but CD spectra did not change because of the formation of enzyme aggregates. It can be used to detect the secondary structure of enzymes. Adding SDS into the coexistence solution of enzymes and SB3-12, the changes of secondary and tertiary structure of enzymes are similar to those without SB3-12. Combining these enzymes, the thermodynamics of intermolecular interaction and the structure of enzymes, the changes of various properties and the concentration of surfactants are found. The mechanism of the interaction between the surfactant and BLC was proposed. 4. Previous work of our group showed that alpha-chymotrypsin (alpha-CT) had superactivity in the presence of cationic Gemini surfactant. As an extension of this work, the mixed system of 12-10-12 and alpha-CT was further studied. The enthalpy of interaction with alpha-CT at 12-10-12 concentration less than CMC was measured systematically. The reasons for the superactivity and low stability of the enzyme in 12-10-12 buffer solution were discussed.
【學(xué)位授予單位】：河南師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：O629.8
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