比菲德氏嬰兒雙歧桿菌中氮-乙醯六碳糖激酶及大腸桿菌中半乳糖激酶之分子選殖、蛋白質表現及其功能性分析

Molecular Cloning, Protein Expression and Activity Assay of N-acetylhexosamine 1-kinase from Bifidobacterium infantis and Galactokinase from Escherichia coli

羅子傑(Tzu-Chieh Lo)

指導教授 ： 吳東昆

國立交通大學/生物科技學院/生物科技學系暨研究所/碩士(2015年)

https://doi.org/10.6842/NCTU.2015.00108

摘要

醣類，又稱作碳水化合物，廣佈於自然界。醣類為人體之重要的營養素，主要分成四大類：單醣、雙醣、寡醣和多醣。他們在生活中扮演著很重要的角色，多醣可作為儲存養分的物質(澱粉和醣原)，或作為動物外骨骼和植物細胞的細胞壁(甲殼素和纖維素)，另外，五碳醛醣的核醣是構成各種輔因子的不可或缺失之物質(ATP、FAD和NAD)，也是一些遺傳物質分子的骨幹(RNA)。據先前文獻報告指出，醣類的眾多衍生物同時也與免疫系統、受精、預防疾病、血液凝固和生長等有極大的關聯。
研究指出，醣化學的發展，受限無法有效率的合成出新的醣類化合物。對於製藥產業而言，醣隨機化技術(glycorandomization)提供醣類化合物的分子多樣化建構了新的技術平台，大幅度地加快了醣類化合物的合成速度，是獲得先導新型藥物分子的方法。基於此概念，在此研究中，兩條基因: 比菲德氏嬰兒雙歧桿菌(Bifidobacterium infantis)中的氮-乙醯六碳糖激酶(N-acetylhexosamine 1-kinase, NahK)及大腸桿菌(Escherichia coli)中的半乳糖激酶(Galactokinase, Galk)則作為主要的研究對象。為了增加實驗產物之多樣性及有效鑑定酵素對於各種不同單醣之活性，本實驗室目前以23個不同的單醣作為酵素修飾的測試對象。
先將基因以分子選殖的技術，接合至表現載體上，再轉型於大腸桿菌(E. coli) 之中，利用外源蛋白表現系統進行蛋白質表現及純化，之後再進行酵素之活性分析。經由薄層色層分析法(Thin Layer Chromatography, TLC)初步篩選出經酵素催化所產生的新產物，待將之進行分離、純化後，再以電噴灑離子化質譜儀(Electrospray Ionization-Mass Spectrometry, ESI-MS)做新產物的鑑定及其分子量的確認。我們希望在未來，能將這些篩選出來的單醣做為候選受質，進一步地針對我們後續的實驗進行測試及分析，以期能應用於未來醣類藥物學方面的研究。

關鍵字

糖隨機化 ； 激酶

並列摘要

Carbohydrates are distributed widely in nature. They are important nutrients for the human beings. It can be divided into four different categories: monosaccharides, disaccharides, oligosaccharides and polysaccharides. It plays a quite important role for lives. Polysaccharides can storage as energy substances, like starch and glycogen, or as animals’ exoskeleton and plants’ cell wall, like chitin and cellulose. In addition, the five-carbon aldose ribose is the material of diverse cofactor, including ATP, FAD and NAD. It is also related some genetic molecular materials. According to some literatures previously published, many sugars derivatives showed great relevance to the immune system, fertilization, disease prevention, blood clotting and growth.
There are many limitation and barriers about glycochemistry development. Glycorandomization, it provides a new approach to select sugars as possible candidates easily and quickly. It acceletate the speed of synthesis of new carbohydrates. Base on this concept, in this study, we selected two of the potential anomeric kinase genes, named NahK and GalK, respectively. NahK, its full name is N-acetylhexosamine 1-kinase, comes from the species Bifidobacterium infantis. GalK, galactokinase, comes from Escherichia coli. Our monosaccharide libraries are composed of 23 diverse monosaccharides, as the testifying substrates. In our study, two kinases were successfully cloned, functionally expressed in Eschericia coli system and sequently purified using Ni-NTA resin. Then, the enzymes were combined with different monosaccharides to do enzymatic activity assay. We checked the experimental results firstly by TLC analysis. After we isolated the products, the products was applied to ESI-MS to confirm the molecular weight and characterization. We hope that the selected monosaccharides can be potential candidates to new drug industry.

並列關鍵字

glycorandomization ； kinase

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