海洋芽孢杆菌B-9987中Macrolactins糖基化后修饰步骤的分子机制研究

发布时间：2018-02-04 11:56

  本文关键词： 海洋芽孢杆菌B-9987 Macrolactins 糖基转移酶 酶学性质 次级代谢产物　出处：《中国海洋大学》2014年硕士论文　论文类型：学位论文

【摘要】：海洋芽孢杆菌B-9987 (Bacillus marinus)分离于我国渤海潮间带植物盐地碱蓬(Suaeda salsa)的根部,能够产生多种macrolactins化合物。Macrolactins是一系列24元大环内酯类化合物,主要分离自Actinomadura sp.和Bacillus sp.等海洋来源的微生物之中,具有广泛的抗菌、抗病毒和抗肿瘤等生物学活性。Macrolactins合成的最后阶段要经过一些后修饰步骤,如糖基化、酰基化等,这些后修饰步骤都发挥着重要的生物学功能。其中糖基化修饰能够增加化合物的水溶性,提高生物活性以及降低毒性,而负责糖基化的酶为糖基转移酶(Glycosyltransferases, GTs)。 GTs将活化的各种糖基通过O-、C-、N-或S-键连接到天然产物前体上,形成各种具有活性的天然产物。本论文以海洋芽孢杆菌B-9987为研究对象,采用比较基因组学的方法,从B-9987中鉴定了macrolactinsGT基因,对其进行了克隆、异源表达和体外酶学实验,具体研究工作如下：首先,对海洋芽孢杆菌B-9987的次级代谢产物进行了分离鉴定。运用正相硅胶柱层析、半制备HPLC等现代色谱技术,NMR、MS等现代波谱技术,从发酵物中分离鉴定了3个macrolactins化合物,经化合物的波谱学数据及与文献对照,这3个化合物的结构分别被鉴定为：macrolactin A、7-O-malonyl macrolactin A和macrolactin B。本研究以分离得到的macrolactins化合物作为糖基转移酶的底物,对macrolactins生物合成的糖基转移酶的基因进行功能鉴定,并进行下一步的酶学性质研究。其次,采用比较基因组学的方法,从B-9987中定位了macrolactins GT基因bmmGT1。构建了bmmGT1重组表达质粒并将其导入E.coli BL21中,在16℃,0.2mM IPTG诱导条件下,实现了可溶性表达,纯化后初步检测了其催化活性。再次,对分离纯化得到的糖基转移酶BmmGT1的酶学性质进行研究,主要包括影响酶活的pH、温度和金属离子。另外,本文还对BmmGT1的底物多样性进行了研究,包括糖基供体多样性、受体多样性以及催化糖基化逆反应的能力。在探索了对两种糖基受体macrolactinA和7-O-malonyl macrolactin A多样性的基础上,对BmmGT]催化这两种底物的动力学参数进行了比较,确定了酶的最适底物。最后,本文通过糖基化反应分离得到了3种新颖的糖基化产物。通过对macrolactins糖基化反应能够提高大环内酯类化合物的水溶性,进而可能改善其生物利用率,增强药效,降低毒副作用,为大环内酯类化合物的药物研制提供了一个新的策略。
[Abstract]:The marine bacillus B-9987 Bacillus marinus was isolated from the roots of Suaeda salsa, an intertidal plant in the Bohai Sea, China. It can produce a variety of macrolactins compounds. Macrolactins are a series of macrolides. Mainly isolated from marine microorganisms such as Actinomadura sp. and Bacillus sp., they have extensive antibacterial, antiviral and antitumor biological activities. Macrolactins undergo some post-modification steps, such as glycosylation, acylation, etc. These post-modification steps play an important biological role. Glycosylation can increase the solubility of the compounds, increase their biological activity and reduce their toxicity. The enzyme responsible for glycosylation is Glycosyltransferases. GTs5.The activated glycosyltransferases. GTs links the activated glycosyltransferases- or S- bonds to the precursors of natural products to form various active natural products. In this paper, Bacillus sp. B-9987 was used as the research object. MacrolactinsGT gene was identified from B-9987 by comparative genomics, cloned, heterologous expression and enzymatic experiments in vitro. The specific research work is as follows: first, The secondary metabolites of Bacillus sp. B-9987 were isolated and identified. Three macrolactins compounds were isolated and identified from the fermentation by normal phase silica gel column chromatography, HPLC and other modern chromatographic techniques. The structures of the three compounds were identified as: macrolactin A 7-O-malonyl macrolactin A and macrolactin B. the isolated macrolactins compounds were used as substrates of glycosyltransferase. The function of the glycosyltransferase gene of macrolactins biosynthesis was identified, and the enzymatic properties of the next step were studied. Secondly, the method of comparative genomics was used. The recombinant expression plasmid of macrolactins GT gene bmmGT1.The recombinant expression plasmid of bmmGT1 was constructed from B-9987. The recombinant plasmid was introduced into E.coli BL21. The soluble expression was achieved under the induction of 0.2mm IPTG at 16 鈩，

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