本文選題：低共熔溶劑 + 非水相酶催化�。� 參考：《山西大學(xué)》2017年碩士論文

【摘要】：糖苷及苷元類化合物廣泛分布于自然界中,具有抗腫瘤、抗氧化、抗疲勞以及防治心腦血管疾病等顯著藥理活性,在藥物化學(xué)領(lǐng)域是一類重要的藥用資源化合物。然而,由于植物資源的匱乏以及自身含量的低微,糖苷及苷元類化合物的需求不得不依靠于人工制備,如有機(jī)合成、組織培養(yǎng)以及酶催化生物轉(zhuǎn)化等。眾所周知,化學(xué)合成法及組織懸浮培養(yǎng)法均存在或多或少的缺陷(周期長且產(chǎn)率低等),制約了其進(jìn)一步發(fā)展應(yīng)用。不可置否,酶催化生物轉(zhuǎn)化過程具有反應(yīng)條件溫和、高效簡便且環(huán)境友好的顯著優(yōu)勢(shì),是糖苷及苷元類活性化合物制備的理想途徑,極具發(fā)展?jié)摿ΑＣ复呋磻?yīng)過程大多選擇在傳統(tǒng)緩沖液中進(jìn)行,但受制于某些反應(yīng)類型特性以及底物溶解性的差異,水相并不是適宜的反應(yīng)溶劑體系。鑒于上述情況,非水相酶催化應(yīng)用逐步發(fā)展,可以促進(jìn)傳統(tǒng)水相中難以進(jìn)行或效率極低的催化反應(yīng)過程的發(fā)生,極大順應(yīng)了酶工程學(xué)領(lǐng)域的應(yīng)用要求。低共熔溶劑(Deep Eutectic Solvents,DESs)是一類新型綠色溶劑,自2003年問世以來便憑借其獨(dú)特的理化性質(zhì)及優(yōu)良特性而廣受關(guān)注,并在非水相酶催化反應(yīng)的應(yīng)用中顯示出了獨(dú)特的優(yōu)越性�；诖�,本研究選用DESs含水溶液為酶催化反應(yīng)溶劑,首次以β-D-葡萄糖苷酶為研究對(duì)象,初步探討了不同DESs介質(zhì)對(duì)于糖苷酶催化特性的作用規(guī)律及影響機(jī)制;選用不同催化機(jī)制對(duì)應(yīng)的模型反應(yīng),通過最佳工藝的優(yōu)化及放大制備研究確定了該反應(yīng)體系在酶催化制備糖苷及苷元類活性物質(zhì)的可行性及先進(jìn)性。在本論文中,首先,我們合成了16種氯化膽堿型DESs,考察了苦杏仁來源β-D-葡萄糖苷酶在上述DESs介質(zhì)中的酶催化活性、穩(wěn)定性(熱穩(wěn)定性及存儲(chǔ)穩(wěn)定性),并篩選出了整體表現(xiàn)較優(yōu)的候選DESs。在上述候選DESs中,進(jìn)一步測(cè)定了酶的最適反應(yīng)溫度、pH值及米氏常數(shù)Km等酶學(xué)性質(zhì)參數(shù)。實(shí)驗(yàn)結(jié)果表明,DESs介質(zhì)對(duì)于苦杏仁來源β-D-葡萄糖苷酶催化特性具有顯著影響,酶催化活性略有降低,但穩(wěn)定性卻顯著提升,不同DESs類型的影響主要取決于氫鍵供體種類以及組成成分的摩爾比例。在維持酶的相對(duì)活力方面,多元醇類DESs表現(xiàn)明顯優(yōu)于酰胺類;當(dāng)氫鍵受體與供體摩爾比例為1:1時(shí),酶的相對(duì)活力為不同比例中最低值,而組成為其它比例(2:1,1:1,1:3)時(shí)則表現(xiàn)相當(dāng)。低濃度DESs體系中,酶的最適反應(yīng)溫度及pH值較緩沖液中無明顯改變,而酶促動(dòng)力學(xué)參數(shù)Km以及Vmax均存在一定程度下降,表明底物與酶分子的誘導(dǎo)契合作用增強(qiáng),但底物的傳質(zhì)擴(kuò)散卻受到抑制。第二,將所選DESs介質(zhì)ChCl/G(1:2)作為反應(yīng)體系,研究了酶催化糖基化合成紅景天苷的可行性。利用單因素試驗(yàn)與響應(yīng)面優(yōu)化法,確定了殼聚糖固定化的β-D-葡萄糖苷酶催化糖基化合成紅景天苷反應(yīng)最佳工藝條件:DESs濃度為80 vol%,反應(yīng)溫度50°C,體系pH值為5.8,反應(yīng)時(shí)間100 h,酶添加量為45 U/m L,底物酪醇/葡萄糖摩爾比例為10;在此條件下,底物最大轉(zhuǎn)化率可達(dá)31.6%,經(jīng)大孔樹脂層析初步提純,紅景天苷粗產(chǎn)品純度可達(dá)70%以上。此外,所采用的固定化酶也可循環(huán)利用,具有較高的操作穩(wěn)定性,保障了反應(yīng)的可行性以及可持續(xù)性。第三,評(píng)價(jià)了DESs介質(zhì)中酶催化水解制備大豆苷元的可行性與適用性。經(jīng)單因素試驗(yàn)與響應(yīng)面優(yōu)化法確定最佳工藝條件為:DESs濃度為30 vol%,反應(yīng)時(shí)間為100min,反應(yīng)溫度為55°C,pH值為5.4,酶用量為1.0 U/mL,底物濃度為300μg/m L;在此條件下,底物最大轉(zhuǎn)化率高達(dá)97.53%。按上述工藝條件進(jìn)行放大制備,產(chǎn)物大豆苷元在反應(yīng)過程中可以不斷析出,簡單過濾即可實(shí)現(xiàn)產(chǎn)物分離,而體系可繼續(xù)投料直接進(jìn)行下一步反應(yīng),可重復(fù)利用5次以上。此外,對(duì)于酶催化大豆苷水解反應(yīng)而言,我們發(fā)現(xiàn)DESs介質(zhì)對(duì)于底物大豆苷具有優(yōu)良的溶解性能,在大規(guī)模生產(chǎn)中具有更高的適用價(jià)值。第四,系統(tǒng)考察了不同DESs體系對(duì)于大豆苷及大豆苷元的溶解特性規(guī)律。結(jié)果顯示,在室溫下,大豆苷及大豆苷元分別在ChCl/EG(1:2,50 vol%)和ChCl/U(1:2,50vol%)體系中溶解效果最優(yōu),各自平衡溶解度分別達(dá)475.4μg/m L和993.6μg/m L。溶解過程中,我們發(fā)現(xiàn)DESs濃度對(duì)于溶質(zhì)溶解度影響較為顯著,但二者呈現(xiàn)出不同規(guī)律;在測(cè)定溶質(zhì)共存溶解度時(shí),研究表明大豆苷在一定程度上可以促進(jìn)大豆苷元的溶解,推測(cè)當(dāng)聯(lián)合使用時(shí)可以提高其體內(nèi)的溶解吸收。經(jīng)綜合篩選,最終選用ChCl/U(1:2)/ChCl/EG(1:2)(v/v,1:7;80,100 vol%)體系作為混合溶劑,采用不同熱力學(xué)方程進(jìn)行關(guān)聯(lián)擬合,其中Apelblat方程關(guān)聯(lián)度最優(yōu),相關(guān)系數(shù)R2均達(dá)到0.999以上。最后,我們利用紫外吸收以及熒光發(fā)射光譜技術(shù),初步研究了不同種類DESs介質(zhì)對(duì)于β-D-葡萄糖苷酶結(jié)構(gòu)的影響規(guī)律,從蛋白分子構(gòu)象角度分析了DESs體系影響酶催化特性的基礎(chǔ)作用機(jī)理。在DESs體系中,酶的紫外以及熒光光譜均發(fā)生了最大波長位移以及強(qiáng)度的改變,主要原因可能是由于溶劑效應(yīng)引起的酶蛋白分子中氨基酸側(cè)鏈的暴露,從而引起構(gòu)象改變所致。研究表明,DESs介質(zhì)中酶蛋白光譜的變化與其催化活性及穩(wěn)定性呈現(xiàn)一定關(guān)聯(lián)性,DESs介質(zhì)對(duì)于酶催化特性的影響主要是由于酶分子的蛋白結(jié)構(gòu)發(fā)生了改變�？偠灾�,本論文首次研究了β-D-葡萄糖苷酶在DESs介質(zhì)中的作用規(guī)律及可行性,成功構(gòu)建了酶催化制備糖苷以及苷元類化合物的新型綠色體系,具有高效簡便、環(huán)境友好等顯著優(yōu)勢(shì),展現(xiàn)出一定的實(shí)用價(jià)值,同時(shí)也可為此領(lǐng)域的相關(guān)學(xué)術(shù)研究提供必不可少的參考思路和理論依據(jù)。
[Abstract]:Glycosides and glycosides are widely distributed in nature, with significant pharmacological activities such as anti-tumor, antioxidation, anti fatigue and prevention and treatment of cardiovascular and cerebrovascular diseases. It is a kind of important medicinal resource compound in the field of drug chemistry. However, due to the lack of plant resources and the low content of its own, the need for glycosides and glucoside compounds We have to rely on artificial preparation, such as organic synthesis, tissue culture and enzyme catalyzed biotransformation. It is well known that chemical synthesis and tissue suspension culture have more or less defects (long period and low yield), which restricts its further development. The remarkable advantage of high efficiency, simple and environmentally friendly is an ideal way for the preparation of glycosides and active compounds of glycosides. The catalytic reaction process is mostly selected in the traditional buffer solution, but it is subject to some types of reaction characteristics and the difference of substrate solubility. The aqueous phase is not a suitable reaction solvent system. In this case, the non-aqueous enzymatic catalytic application is progressively developed, which can promote the development of the catalytic reaction process in the traditional water phase which is difficult to carry out or with very low efficiency. It has greatly complied with the application requirements of the field of enzyme engineering. Deep Eutectic Solvents (DESs) is a new type of green solvent, which has been unique since its advent in 2003. In this study, DESs aqueous solution was used as a solvent for enzymatic reaction. The effect of different DESs media on the catalytic properties of glucosidase was preliminarily studied in this study. In this paper, 16 kinds of choline chloride type DESs were combined into 16 kinds of choline chloride type. In this paper, we studied the bitter almond. The enzyme catalytic activity of the source beta -D- glucosidase in the DESs medium, stability (thermal stability and storage stability), and screening the candidate DESs. with better overall performance in the above candidate DESs, and further measuring the enzyme's optimum reaction temperature, pH value and Michaelis constant Km and other enzymology properties. The experimental results show that DESs medium is used. The catalytic properties of amygdala derived beta -D- glucosidase had a significant influence. The catalytic activity of the enzyme decreased slightly, but the stability was significantly improved. The effects of different DESs types mainly depend on the type of hydrogen bond and the molar ratio of the components. The DESs performance of the polyols is obviously superior to the amides in maintaining the relative activity of the enzyme. When the ratio of the bond receptor to the donor molar ratio is 1:1, the relative activity of the enzyme is the lowest in the different proportion, while the composition is the same as the other proportion (2:1,1:1,1:3). In the low concentration DESs system, the optimum reaction temperature and the pH value of the enzyme are not significantly changed in the buffer solution, but the enzyme kinetic parameters Km and Vmax are all decreased to a certain extent, indicating that the enzyme kinetic parameters, Km and Vmax, have been reduced to a certain extent. The interaction between the substrate and the enzyme molecule is enhanced, but the mass transfer and diffusion of the substrate is inhibited. Second, the feasibility of enzyme catalyzed glycosylation of salidroside was studied by using the selected DESs medium ChCl/G (1:2) as a reaction system. The immobilized beta -D- glucosidase catalysis of chitosan was determined by single factor test and response surface optimization. The optimum process conditions for glycosylation of salidroside are: DESs concentration is 80 vol%, reaction temperature is 50 degree C, pH value is 5.8, reaction time is 100 h, enzyme addition is 45 U/m L, and the molar ratio of substrate Tyrol / glucose is 10. Under this condition, the maximum conversion rate of substrate can reach 31.6%, and the crude product of salidroside is pure and pure by macroporous resin chromatography In addition, the immobilized enzyme can also be recycled, with high operating stability, the feasibility and sustainability of the reaction. Third, the feasibility and applicability of the enzyme catalyzed hydrolysis of Daidzein in DESs medium was evaluated. The optimum process conditions were determined by single factor test and response surface optimization. The DESs concentration is 30 vol%, the reaction time is 100min, the reaction temperature is 55 degree C, the pH value is 5.4, the enzyme dosage is 1 U/mL, the substrate concentration is 300 mu g/m L. Under this condition, the maximum conversion rate of the substrate is up to 97.53%. according to the above process conditions, and the product daidzein can be continuously precipitated during the reaction process, and the product can be realized by simple filtration. In addition, we found that the DESs medium has excellent solubility in the daidzein, and has a higher suitable value in the large-scale production. Fourth, the system has been examined by different DESs systems for the enzyme catalyzed daidzein hydrolysis. The dissolution characteristics of daidzein and daidzein showed that, at room temperature, daidzein and daidzein were best dissolved in ChCl/EG (1:2,50 vol%) and ChCl/U (1:2,50vol%) system respectively. The equilibrium solubility of daidzein and ChCl/U (1:2,50vol%) was 475.4 u g/m L and 993.6 micron g/m L. dissolution process respectively. We found that the concentration of DESs was affected by the solubility of the solute. More significant, but the two showed different rules. In determining the solubility of solute, the study showed that daidzein could promote the dissolution of daidzein to a certain extent. It is presumed that the dissolution and absorption of daidzein can be improved when combined. After comprehensive screening, the ChCl/ U (1:2) /ChCl/EG (1:2) (v/v, 1:7; 80100 vol%) system is selected as the final selection. In the mixed solvent, the correlation of the Apelblat equation was optimal and the correlation coefficient R2 reached 0.999. Finally, we studied the effect of different kinds of DESs media on the structure of beta -D- glucosidase by UV absorption and fluorescence emission spectroscopy. The basic mechanism of the effect of DESs system on the catalytic properties of enzymes is analyzed. In the DESs system, the maximum wavelength shift and the intensity change of the enzyme in the UV and fluorescence spectra are caused by the exposure of the amino acid side chain in the enzyme protein molecules caused by the solvent effect, and the result of the conformation changes. It is clear that the changes of the enzyme protein spectrum in the DESs medium are related to its catalytic activity and stability. The effect of DESs medium on the enzyme catalytic properties is mainly due to the changes in the protein structure of the enzyme molecules. In a word, the role and feasibility of the beta -D- glucosidase in the DESs medium is first studied in this paper. A new green system with enzyme catalyzed preparation of glycosides and glucoside compounds has been built. It has significant advantages, such as high efficiency, simplicity, environment friendly and so on. It shows a certain practical value. At the same time, it can also provide a necessary reference and theoretical basis for the related academic research in this field.
【學(xué)位授予單位】：山西大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R914

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 鮑會(huì)梅;;豆粕中大豆異黃酮含量快速測(cè)定方法的研究[J];食品研究與開發(fā);2016年20期

2 劉雅晶;;綠色溶劑中有機(jī)化學(xué)反應(yīng)的研究[J];工業(yè)設(shè)計(jì);2016年03期

3 何麗妃;薛原楷;石賢愛;;特殊反應(yīng)體系中交聯(lián)β-葡萄糖苷酶聚集體催化合成紅景天苷[J];過程工程學(xué)報(bào);2015年02期

4 初旭明;賈儒;楊姣;黃和;胡q，

本文編號(hào)：2073865