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標題: | 選殖與大量表現大腸桿菌K1之N-乙醯基神經胺糖酸合成?及其酵素特性分析 Cloning, Overexpression, and Characterization of N-Acetyl-D-neuraminic Acid Synthetase from Escherichia coli K1 |
作者: | Jia-Yuan Chen 陳嘉元(冠廷) |
出版年 : | 2000 |
學位: | 碩士 |
摘要: | 位在細菌表面的菌鞘,屬於Kl抗原的α-2,8醣聚合體,扮演著細菌入侵人類或者其他動物體內時,決定對於宿主是否具有毒性的關鍵角色。這些多聚醣的合成,一般都是經由下列步驟所產生:唾液酸的合成、唾液酸經活化形成CMP-唾液酸以及運送唾液酸給適當的受器。目前已有兩種格蘭氏陰性菌唾液酸合成的酵素被研究出來,一種為唾液酸分解?,能夠進行可逆反應,以N-乙醯甘露糖胺酸和丙酮酸進行縮合反應得到唾液酸,一般情況下為代謝分解作用;另一種為唾液酸合成?,為一個不可逆反應,以N-乙醯甘露糖胺酸和磷酸烯醇丙酮酸進行縮合反應得到唾液酸。
本篇研究是利用分生技術選殖大腸桿菌Kl唾液酸合成?,進行大量表現和純化,也對其酵素特性作一番探討。雖然這個酵素在早先已經被表現純化出來,但是其活性非常地低,以致於必需使用缺乏唾液酸分解?的菌種,並加入磷解?抑制劑來改善唾液酸合成?的表現及活性;另外也得以C14同位素標定N-乙醯甘露糖才能檢測唾液酸的產生。在大量表現該蛋白質之後,我們應用Ni2+金屬離子親和性管柱層析法,高度純化N端帶有組氨酸標識之唾液酸合成?,得到較以前人所表現唾液酸合成?高的比活性,並藉由Enterokinase蛋白質水解?將不必要的N端胺基酸序列切除,得到完整的野生型唾液酸合成?。另一方面,我們發現這個酵素是以雙體的形式存在,並含有兩種不同分子量40 kDa和33 kDa單體存在,利用不同的陰離子交換樹脂和逆向層析法,並不能夠將其分離出。現階段我們無法得知為何會有33 kDa單體存在,前人也曾經觀察過這種現象,不過可以確定的是,33 kDa單體是從40 kDa單體上胺基酸K279及I280之間斷裂所得到,而且這種蛋白質水解現象在經過純化以後仍然可以觀察到。 唾液酸合成?最佳酸鹼值在8.5,而在7.0?9.0範圍有較穩定的特性;在溫度45℃下反應性最高,並需要二價 Mg2+或Mn2+離子參與才能有明顯的活性。另外我們以不同醣分子作為唾液酸合成?的受質,發現必須要以甘露糖胺酸為骨架和 N-取代基存在才能有反應性,而其他的醣分子均無反應,因此可能酵素活化區和結合部位與這兩者有密切的關係。而其中N-Acetylglycolyl-D-mannosamine (ManNGcAc)利用Periodate-Thiobarbituric Acid Assay、薄膜層析法以及核磁共振光譜,可以檢測到有相對於N-乙醯甘露糖胺酸的高活性存在;因此可以應用在合成唾液酸衍生物上,但是其低活性的特徵仍是必須克服的障礙。 這是第一篇有關大腸桿菌Kl唾液酸合成?,不需要藉由缺乏唾液酸分解?菌種的表現,就能夠拿到大量且比活性高的報導;另一方面,也觀察到44 kDa之唾液酸合成?會水解產生33 kDa之單體;及利用生物資訊(Bio-Infomatics)分析方式,與其他已知相關唾液酸合成的蛋白質序列比對的結果,推測到兩段具有高度保留性的胺基酸序列:GxSDHxxxxxxxxxAVxxGxxxIEKHF和xxxxxIxAExxxNHNGS,這兩個區域具有疏水性的特徵,以及特定酸鹼催化的胺基酸存在,因此可能在酵素活化區和受質結合區上扮演了重要的角色,這都有待日後作進一步的研究加以證實。 The K1 capsular polysaccharide, an α-2,8 linked polymer of N-acetyl-D-neuraminic acid (NeuAc), is an important virulence determinant of extra intestinal neuropathogenic Escherichia coil. The biosynthesis of this sugar polymer is composed of the following steps; synthesis of NeuAc, activation of NeuAC to CMP-NeuAc, and transfer of NeuAc to the acceptor. Two enzymes have been reported for the synthesis of NeuAc in Gram-negative bacteria. One is sialic acid aldolase that catalyzes the reversible reaction from N-acetylmannosamine (ManNAc) and pyruvate to NeuAc. The reaction equilibrium favors the catabolic direction. The other is sialic acid synthetase responsible for the aldol reaction of ManNAc and phospho(enol)pyruvate (PEP) to form NeuAc. In this thesis, the overexpression, purification and characterization of sialic acid synthetase from E.coil K1 are investigated. Although this enzyme has been cloned and expressed, its activity is so low that past studies had to use sialic acid lyase-deficient mutant strain in a combination with a phosphatase inhibitor to improve the activity of overexpressed synthetase. Additionally, the isotope labeled C14-ManNAc was required to detect the NeuAc activity. The recombinant N-terminal His-tagged sialic acid synthetase, purified by the Ni2+ affinity chromatography, was measured to have higher specific activity than that had been reported previously. The removal of the N-terminal His-tag by the enterokinase digestion led to isolation of the native sialic acid synthetase. This enzyme was determined to exist in heterogeneous dimeric forms. The two subunits of 40 kDa and 33 kDa to constitute these dimers were found difficult to be separated by anion exchange and reverse phase HPLC chromatography. The 33 kDa protein resulted from the cleavage of the 40 kDa protein at K279 of the C-terminal segment. The protein degradation was also observed after purification, similar to the reported previously. The enzyme is stable in the pH range of 7 to 9 with an optimum at 8.5 and thermostable up to 45 ℃. The metal ion, Mg2+ or Mn2+, is required for enzyme activity. Various monosaccharides have been tested for the substrate specificity. The result suggested that the N-substituent at the C-2 position and the D-manno configuration are both important for enzyme specificity. Among the different sugars tested, N-Acetylglycolyl-D-mannosamine (ManNGcAc) has been found to have 85% activity compared to ManNAc, as determined by periodate-thiobarbituric acid assay, thin layer chromatography, and 1H NMR. Therefore, it is possible to synthesize sialic acid derivates by this enzyme but the low reactivity remains as a problem to be overcome. This is the first report for the overproduction of E.coli sialic acid synthetase with high purity over 95% and good activity without using sialic acid lyase-deficient mutant. Finally, the protein sequence analysis suggests that two highly conserved amino acid sequences are rather hydrophobic, GxSDHxxxxxxxxxAVxxGxxxIEKHF and xxxxxIxAExxxNHNGS. They may be involved in either substrate binding or catalysis. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75147 |
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顯示於系所單位: | 生化科學研究所 |
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