枯草桿菌UDP-glycosyltransferase之受質專一性研究與快速活性偵測法建立

Abstract

醣基化對於生物體的生化合成反應相當重要，在近期的疾病治療上扮演重要的角色，如能快速並高通量的偵測醣基轉移酶酵素活性，便可開啟此類酵素的應用。本研究旨於探討Bacillus cereus之UDP-glycosyltransferase（BcGT-1）的催化反應，我們藉由基因工程將BcGT-1建構在大腸桿菌之BL21(DE3)的表現系統上，並經過DEAE管柱純化，得到大量酵素。本研究因酵素受質的特性，得以利用BcGT-1對4-nitrophenol的轉醣能力，使用一便利的光學測定法，以直接偵測酵素BcGT-1的活性，此法具備快速、方便且能即時偵測等特色。

首先，我們觀察不同硝基苯酚(醣基接受者)的親核性與酵素反應的關係。研究顯示硝基苯酚之pKa對於BcGT-1的催化反應有密切關聯性，酵素的轉醣能力會隨醣基接受者親核力之下降而減弱。

此外，我們亦研究BcGT-1在合成應用之潛力，利用純化後的酵素與不同醣基提供者進行轉醣反應，發現酵素對於UDP-glucose具有高度專一性，而酵素對不同的醣基接受者，如：苯酚、苯硫醇、苯胺類進行轉醣反應，發現能對其成功接上葡萄醣基，此研究顯示此BcGT-1能用來合成各種新穎葡萄醣苷化合物，如O-glucoside、S-glucoside及N-glucoside等，未來將具有廣泛的應用性。

Glycosyltransferases have many important biological functions in all species and in the use of disease treatment. Thus we need a real time and high-throughput method to analyze glycosyltransferase activity. In this research, we focus on the UDP-glycosyltransferase from Bacillus cereus (BcGT-1) catalytic reaction. BcGT-1 was successfully over-expressed in E. coli BL21 (DE3) and purified by DEAE column. In this research, we use a quick colorimetric activity assay for glycosyltransferase BcGT-1. The assay principle is based on the glycosylation activity of BcGT-1 on 4-nitrophenol. It provides a timesaving, simple and high-throughput for analyzing glycosyltransferase activity.

First, the nitrophenolic compounds with different nucleophilicity were used to be the glucose acceptors in BcGT-1 glycosylation reaction. The nucleophilicity of nitrophenolic compounds was observed to play an important role in enzymatic reaction. The activity of BcGT-1 was decreased when nucleophilicity of acceptor was reduced.

In addition, we also investigate the potential of BcGT-1 to be used in synthesis. Using the purified BcGT-1 to catalyze the glycosylation with different donors shows that this enzyme has high specificity to UDP-glucose. However, this enzyme can convert phenolic, thiolphenolic and aromatic amine to their respective glucosides successfully. It means that BcGT-1, has high potential to be used to synthesize novel glycosides like O-glucoside、S-glucoside and N-glucoside.