基于转糖基功能的右旋糖酐蔗糖酶分子改造、表达及催化性能的研究

发布时间：2018-03-29 23:28

  本文选题：糖基转移酶　切入点：右旋糖酐蔗糖酶　出处：《合肥工业大学》2017年硕士论文

【摘要】：糖基转移酶(Glycosyltransferase,GT,EC 2.4.x.y)的转糖基作用是催化葡萄糖基转移到受体分子上,使受体分子糖基化,形成糖基化衍生物包括非天然糖类化合物和功能性低聚糖等。非天然糖类化合物可以改变原来天然化合物的理化性质,功能性低聚糖作为调节肠道平衡的双歧因子应用在医药、食品、保健品等领域。本论文通过分子学手段对右旋糖酐蔗糖酶(Dextransucrase,DSR)进行改造,构建筛选得到具较高活性的突出转糖基的DSR突变酶,揭示其转糖基功能的关键区域;并对该突变酶的性质进行研究,同时利用改造后的转糖基酶对不同糖类受体和维生素C(VC)进行糖基化,与原始酶进行分析比较,获得该突变酶对受体糖基化的催化规律。首先通过分子截短方法,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突变酶,并对突变酶的表达条件进行优化。酶学性质研究发现:该酶最适pH均为5.4,pH稳定性均为4.4-7;最适温度均为25℃;DSR-S1-ΔA酶在没有受体情况下,酶活完全丧失;受体存在时,DSR-S1-ΔA酶活是原始酶酶活的71.4%,其中糖基转移活性是水解活性的5.3倍,DSR-S1-ΔA耐热性比原始酶有所下降,在30℃保藏1 h酶活力只有20.8%。以麦芽糖为受体研究表明,受体浓度为200 mM时酶活最高,供体蔗糖对DSR-S1-ΔA酶的影响较大,酶活随着蔗糖浓度提高而不断增加。结果显示DSR-S1-ΔA主要活性为转糖基功能,多糖聚合能力低,从而明确了DSR的转糖基功能区域。通过对麦芽糖、纤维二糖、棉子糖和水苏糖等不同糖类受体的转糖基研究,结果表明:麦芽糖是该酶转糖基反应的最佳受体,同时对纤维二糖、棉子三糖和水苏糖都有活性,但转糖基依次减弱;不同天然低聚糖的转糖基反应都趋向于生成低分子量的糖,转移1-2个葡萄糖基到受体分子,并且受体分子量越小越易进行转糖基作用,α键连接糖类化合物比β键连接糖类化合物更易进行转糖基反应;说明改造后的酶能利用更少的麦芽糖合成更多的低聚糖。论文最后利用DSR-S1-ΔA酶对VC进行了转糖基初步研究,获取了VC的葡萄糖基衍生物。综上,本研究明确了右旋糖酐蔗糖酶转糖基功能的分子区域,有助于进一步认识右旋糖酐蔗糖酶的催化机制;同时对于功能低聚糖的制备和天然产物的糖基化研究具有重要意义。
[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，

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