本文關(guān)鍵詞： 海洋芽孢桿菌B-9987 Macrolactins 糖基轉(zhuǎn)移酶 酶學(xué)性質(zhì) 次級(jí)代謝產(chǎn)物　出處：《中國海洋大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：海洋芽孢桿菌B-9987 (Bacillus marinus)分離于我國渤海潮間帶植物鹽地堿蓬(Suaeda salsa)的根部,能夠產(chǎn)生多種macrolactins化合物。Macrolactins是一系列24元大環(huán)內(nèi)酯類化合物,主要分離自Actinomadura sp.和Bacillus sp.等海洋來源的微生物之中,具有廣泛的抗菌、抗病毒和抗腫瘤等生物學(xué)活性。Macrolactins合成的最后階段要經(jīng)過一些后修飾步驟,如糖基化、酰基化等,這些后修飾步驟都發(fā)揮著重要的生物學(xué)功能。其中糖基化修飾能夠增加化合物的水溶性,提高生物活性以及降低毒性,而負(fù)責(zé)糖基化的酶為糖基轉(zhuǎn)移酶(Glycosyltransferases, GTs)。 GTs將活化的各種糖基通過O-、C-、N-或S-鍵連接到天然產(chǎn)物前體上,形成各種具有活性的天然產(chǎn)物。本論文以海洋芽孢桿菌B-9987為研究對(duì)象,采用比較基因組學(xué)的方法,從B-9987中鑒定了macrolactinsGT基因,對(duì)其進(jìn)行了克隆、異源表達(dá)和體外酶學(xué)實(shí)驗(yàn),具體研究工作如下：首先,對(duì)海洋芽孢桿菌B-9987的次級(jí)代謝產(chǎn)物進(jìn)行了分離鑒定。運(yùn)用正相硅膠柱層析、半制備HPLC等現(xiàn)代色譜技術(shù),NMR、MS等現(xiàn)代波譜技術(shù),從發(fā)酵物中分離鑒定了3個(gè)macrolactins化合物,經(jīng)化合物的波譜學(xué)數(shù)據(jù)及與文獻(xiàn)對(duì)照,這3個(gè)化合物的結(jié)構(gòu)分別被鑒定為：macrolactin A、7-O-malonyl macrolactin A和macrolactin B。本研究以分離得到的macrolactins化合物作為糖基轉(zhuǎn)移酶的底物,對(duì)macrolactins生物合成的糖基轉(zhuǎn)移酶的基因進(jìn)行功能鑒定,并進(jìn)行下一步的酶學(xué)性質(zhì)研究。其次,采用比較基因組學(xué)的方法,從B-9987中定位了macrolactins GT基因bmmGT1。構(gòu)建了bmmGT1重組表達(dá)質(zhì)粒并將其導(dǎo)入E.coli BL21中,在16℃,0.2mM IPTG誘導(dǎo)條件下,實(shí)現(xiàn)了可溶性表達(dá),純化后初步檢測(cè)了其催化活性。再次,對(duì)分離純化得到的糖基轉(zhuǎn)移酶BmmGT1的酶學(xué)性質(zhì)進(jìn)行研究,主要包括影響酶活的pH、溫度和金屬離子。另外,本文還對(duì)BmmGT1的底物多樣性進(jìn)行了研究,包括糖基供體多樣性、受體多樣性以及催化糖基化逆反應(yīng)的能力。在探索了對(duì)兩種糖基受體macrolactinA和7-O-malonyl macrolactin A多樣性的基礎(chǔ)上,對(duì)BmmGT]催化這兩種底物的動(dòng)力學(xué)參數(shù)進(jìn)行了比較,確定了酶的最適底物。最后,本文通過糖基化反應(yīng)分離得到了3種新穎的糖基化產(chǎn)物。通過對(duì)macrolactins糖基化反應(yīng)能夠提高大環(huán)內(nèi)酯類化合物的水溶性,進(jìn)而可能改善其生物利用率,增強(qiáng)藥效,降低毒副作用,為大環(huán)內(nèi)酯類化合物的藥物研制提供了一個(gè)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，

本文編號(hào)：1490147