發(fā)布時間：2018-05-12 09:15

  本文選題：右旋糖酐 + 親水相互作用色譜��； 參考：《蘇州大學》2016年碩士論文

【摘要】：右旋糖酐(Dextran)是葡聚糖的一種,為一線型結(jié)構(gòu)的中性多糖,廣泛存在于微生物中,構(gòu)成右旋糖酐主鏈的糖酐鍵為α-1,6糖酐鍵,并含有少量以α-1,3連接的支鏈結(jié)構(gòu)。右旋糖酐是應用最廣泛的葡聚糖,在醫(yī)藥、食品、化工等行業(yè)都有不同的用途。目前,右旋糖酐的生產(chǎn)是以蔗糖為底物,經(jīng)鏈球菌、乳酸桿菌、明串珠菌等微生物發(fā)酵而生成的高分子葡聚糖,再經(jīng)酸水解和乙醇分級得到具有不同相對分子質(zhì)量大小的右旋糖酐產(chǎn)品。但不同種類微生物發(fā)酵產(chǎn)生的右旋糖酐分子量差異很大,且支鏈形式和支鏈度上也存在很大差異。右旋糖酐分子量大小決定了其應用范疇,如低分子右旋糖酐臨床上應用于休克早期,中分子量的右旋糖酐其大小與人體血漿蛋白及球蛋白分子相近,可吸收進入體內(nèi)然后水解為葡萄糖而作為能量補充劑。同時,右旋糖酐支鏈形式和支鏈度對右旋糖酐的實際應用影響重大,但目前對右旋糖酐支鏈形式和支鏈度的判定缺乏有效的方法。在以往的文獻報導中,對右旋糖酐支鏈形式及支鏈度的研究主要有氣質(zhì)聯(lián)用和核磁共振兩種方式。氣質(zhì)聯(lián)用方法分析的前提是對樣品進行甲基化、水解、乙酰化等一系列衍生化,在此過程中無可避免地導致了樣品部分結(jié)構(gòu)信息的丟失。并且由于不同連接方式寡糖的離子化效率不同,氣質(zhì)聯(lián)用方法不能準確判斷支鏈度,只能判斷支鏈形式。核磁共振(NMR)是判斷支鏈形式和支鏈度的有效方式,但是NMR樣品耗量較大,且樣品的核磁譜信息會疊加在一起,費時費力很難解析。本文以右旋糖酐的右旋糖酐酶(dextranase)水解產(chǎn)物為研究對象,旨在建立超高效親水相互作用色譜-飛行時間質(zhì)譜聯(lián)用(UP-HILIC-Q/TOF)方法,通過優(yōu)化色譜方法對不同聚合度以及同一聚合度不同連接方式寡糖同分異構(gòu)體進行分離,可以有效消除寡糖α,β異構(gòu)影響且不需要衍生化。利用二級質(zhì)譜MS/MS對酶解終產(chǎn)物進行結(jié)構(gòu)解析,確證酶解終產(chǎn)物中寡糖聚合度及其連接方式。基于dextranase特異性水解葡聚糖α-1,6連接的作用,根據(jù)酶不識別片段的結(jié)構(gòu)即可推斷出右旋糖酐的支鏈形式。但同一聚合度不同連接方式的寡糖同分異構(gòu)體在此液相方法上的分離有限,且不同結(jié)構(gòu)的寡糖質(zhì)譜響應各異,不能很好地進行定量分析。因此在此研究工作的基礎上,鑒于離子色譜(HPAEC,簡稱IC)對糖類化合物很好的分離效果,以及安培脈沖檢測器(PAD)對糖類化合物高靈敏度的特異性檢測作用,我們改進儀器設置,在離子色譜和質(zhì)譜間安裝陽離子樹脂交換抑制器(MSM),對離子色譜得流動想進行在線脫鹽處理,克服了離子色譜高鹽濃度洗脫劑無法直接進入質(zhì)譜來分析的難點,使在線安培檢測器和在線質(zhì)譜檢測平行存在成為可能。該方法成功搭建了HPAEC-PAD-QTOF聯(lián)用平臺,很好地結(jié)合了離子色譜和QTOF高選擇性、高靈敏度、高準確度等特性,能很好地應用于右旋糖酐支鏈形式支鏈度的確定,該方法簡便、高效、樣品耗量少,是糖類化合物支鏈定性定量分析的有效方法。、基于多糖羧甲基化能提高多糖的溶解度、電負性,改變多糖分子伸展方向及立體結(jié)構(gòu),能讓多糖具備新的生物活性,本文主要研究了羧甲基化右旋糖酐寡糖在氧糖剝奪損傷模型中對神經(jīng)元細胞缺糖缺氧保護作用,并探索羧甲基數(shù)量、羧甲基取代度與抗氧糖剝奪活性的密切關(guān)系。
[Abstract]:Dextran (Dextran) is a kind of glucan. It is a linear structure of neutral polysaccharide, which is widely found in microbes. The sugar anhydride key of the main chain of dextran is a -1,6 sugar anhydride bond, and contains a small amount of branched chain structure connected by alpha -1,3. Dextran is the most widely used glucan and has different use in pharmaceutical, food, chemical and other industries. At present, the production of dextran is a high molecular weight glucan produced by fermentation of Streptococcus, Lactobacillus, and Streptococcus mutans with sucrose as the substrate. The dextran products with different molecular weight are obtained by acid hydrolysis and ethanol classification. However, the molecular weight difference of dextran produced by different kinds of microorganism fermentation The molecular weight of dextran determines its application, such as low molecular dextran in the early stage of shock, and the size of the middle molecular weight dextran is close to the human plasma protein and globulin molecule, and can be absorbed into the body and hydrolyzed into glucose. As an energy supplement. At the same time, the form of dextran branched chain and the degree of branch chain have great influence on the practical application of dextran, but there is no effective method to determine the form of dextran branched chain and the degree of branched chain. In the previous literature, the study of the form of dextran branched chain and the chain degree of the branched chain mainly include GC-MS and NMR The two methods. The precondition of GC-MS analysis is a series of derivatization, such as methylation, hydrolysis and acetylation of samples, which can not avoid the loss of some structural information in the sample. And because of the different ionization efficiency of the oligosaccharides in different connections, the GC-MS method can not accurately determine the degree of the branched chain. NMR (NMR) is an effective way to judge the form of the branched chain and the degree of branched chain. However, the consumption of NMR samples is larger, and the information of the samples will be superposed together. The time-consuming and hard work is difficult to analyze. This paper is aimed at establishing super efficient hydrophilic interaction with dextran dextran enzyme (dextranase) hydrolysate. Chromatography - time of flight mass spectrometry (UP-HILIC-Q/TOF) was used to separate the oligosaccharides and isomers of oligosaccharides with different polymerization degrees and the same degree of polymerization with the same degree of polymerization. The effects of oligosaccharide, beta isomerism and no derivatization were eliminated. Structure analysis of the final products of the enzyme hydrolysis was determined by two stage mass spectrometry MS/MS. The degree of oligosaccharide polymerization and its connection in the final product of enzymatic hydrolysis. Based on the role of dextranase specific hydrolysate glucan alpha -1,6 connection, the branch form of dextran can be deduced according to the structure of the unidentified fragment. However, the separation of oligosaccharide isomers from the same degree of polymerization with the same degree of polymerization is limited and different in this liquid phase method. The structure of the oligosaccharide mass spectrum response is different and can not be quantified well. Therefore, on the basis of this research work, in view of the good separation effect of HPAEC, IC, and the specific detection effect of the ampere pulse detector (PAD) on the high sensitivity of saccharides, we have improved the instrument setting. The cation resin exchange suppressor (MSM) is installed between the chromatograph and the mass spectrum. The flow of ion chromatography is to be desalted online, which overcomes the difficulty that the high salt concentration eluant can not enter the mass spectrometry directly. It is possible that the on-line ampere detector is parallel to the on-line mass spectrometry detection. The method has successfully built the HPAEC. -PAD-QTOF combined with high selectivity of ion chromatography and QTOF, high sensitivity and high accuracy can be used to determine the degree of branched chain in the form of dextran branch. This method is simple, efficient, and less sample consumption. It is an effective method for the quantitative analysis of carboxymethyl groups based on Carboxymethylation of polysaccharides. The solubility and electronegativity of high polysaccharide can change the stretching direction and stereostructure of polysaccharide molecules, which can make the polysaccharide have new biological activity. In this paper, the protective effect of carboxymethylation dextran oligosaccharide on oxygen glucose deprivation in the oxygen glucose deprivation model is mainly studied, and the number of carboxymethyl, the degree of carboxymethyl substitution and the stripping of oxygen glucose are explored. The close relationship between the activity and the activity.

【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R917

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