本文選題：糖基轉(zhuǎn)移酶　切入點(diǎn)：右旋糖酐蔗糖酶　出處：《合肥工業(yè)大學(xué)》2017年碩士論文

【摘要】：糖基轉(zhuǎn)移酶(Glycosyltransferase,GT,EC 2.4.x.y)的轉(zhuǎn)糖基作用是催化葡萄糖基轉(zhuǎn)移到受體分子上,使受體分子糖基化,形成糖基化衍生物包括非天然糖類化合物和功能性低聚糖等。非天然糖類化合物可以改變?cè)瓉?lái)天然化合物的理化性質(zhì),功能性低聚糖作為調(diào)節(jié)腸道平衡的雙歧因子應(yīng)用在醫(yī)藥、食品、保健品等領(lǐng)域。本論文通過(guò)分子學(xué)手段對(duì)右旋糖酐蔗糖酶(Dextransucrase,DSR)進(jìn)行改造,構(gòu)建篩選得到具較高活性的突出轉(zhuǎn)糖基的DSR突變酶,揭示其轉(zhuǎn)糖基功能的關(guān)鍵區(qū)域;并對(duì)該突變酶的性質(zhì)進(jìn)行研究,同時(shí)利用改造后的轉(zhuǎn)糖基酶對(duì)不同糖類受體和維生素C(VC)進(jìn)行糖基化,與原始酶進(jìn)行分析比較,獲得該突變酶對(duì)受體糖基化的催化規(guī)律。首先通過(guò)分子截短方法,截短右旋糖蔗糖酶的C-端1494 bp片段,得到剩余1-3087 bp基因片段的DSR-S1-ΔA突變酶,并對(duì)突變酶的表達(dá)條件進(jìn)行優(yōu)化。酶學(xué)性質(zhì)研究發(fā)現(xiàn):該酶最適pH均為5.4,pH穩(wěn)定性均為4.4-7;最適溫度均為25℃;DSR-S1-ΔA酶在沒(méi)有受體情況下,酶活完全喪失;受體存在時(shí),DSR-S1-ΔA酶活是原始酶酶活的71.4%,其中糖基轉(zhuǎn)移活性是水解活性的5.3倍,DSR-S1-ΔA耐熱性比原始酶有所下降,在30℃保藏1 h酶活力只有20.8%。以麥芽糖為受體研究表明,受體濃度為200 mM時(shí)酶活最高,供體蔗糖對(duì)DSR-S1-ΔA酶的影響較大,酶活隨著蔗糖濃度提高而不斷增加。結(jié)果顯示DSR-S1-ΔA主要活性為轉(zhuǎn)糖基功能,多糖聚合能力低,從而明確了DSR的轉(zhuǎn)糖基功能區(qū)域。通過(guò)對(duì)麥芽糖、纖維二糖、棉子糖和水蘇糖等不同糖類受體的轉(zhuǎn)糖基研究,結(jié)果表明:麥芽糖是該酶轉(zhuǎn)糖基反應(yīng)的最佳受體,同時(shí)對(duì)纖維二糖、棉子三糖和水蘇糖都有活性,但轉(zhuǎn)糖基依次減弱;不同天然低聚糖的轉(zhuǎn)糖基反應(yīng)都趨向于生成低分子量的糖,轉(zhuǎn)移1-2個(gè)葡萄糖基到受體分子,并且受體分子量越小越易進(jìn)行轉(zhuǎn)糖基作用,α鍵連接糖類化合物比β鍵連接糖類化合物更易進(jìn)行轉(zhuǎn)糖基反應(yīng);說(shuō)明改造后的酶能利用更少的麥芽糖合成更多的低聚糖。論文最后利用DSR-S1-ΔA酶對(duì)VC進(jìn)行了轉(zhuǎn)糖基初步研究,獲取了VC的葡萄糖基衍生物。綜上,本研究明確了右旋糖酐蔗糖酶轉(zhuǎn)糖基功能的分子區(qū)域,有助于進(jìn)一步認(rèn)識(shí)右旋糖酐蔗糖酶的催化機(jī)制;同時(shí)對(duì)于功能低聚糖的制備和天然產(chǎn)物的糖基化研究具有重要意義。
[Abstract]:The transglycosylation of glycosyltransferase GTN EC 2.4.x.y) catalyzes the transfer of glucose to the receptor molecule and glycosylation of the receptor molecule. The formation of glycosylated derivatives includes non-natural carbohydrate compounds and functional oligosaccharides, which can change the physical and chemical properties of the original natural compounds. Functional oligosaccharides are used in medicine as bifidus factors regulating intestinal balance. In this paper, we modified the dextran sucrase (Dextransucrase) by molecular method to construct and screen the DSR mutant with high activity, and to reveal the key region of its transglycosyl function. At the same time, the modified transglycosylase was used to glycosylation of different carbohydrate receptors and vitamin C (C), and compared with the original enzyme. The catalytic effect of the mutant on the glycosylation of the receptor was obtained. Firstly, the C-terminal 1494 BP fragment of dextran sucrase was truncated by molecular truncation method, and the DSR-S1- 螖 A mutant of the remaining 1-3087bp gene fragment was obtained. The expression conditions of mutant enzyme were optimized. The results of enzymatic properties showed that the optimum pH value of the enzyme was 5.4 ~ 7, the optimum temperature was 25 鈩，

本文編號(hào)：1683358