The Essential Residue Analysis and Application of UDP-glycosyltransferase from Bacillus Cereus

• Main Authors: Lu, Chia-Yu, 呂佳諭
• Other Authors: Li, Yaw-Kuen
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/61476715553553563915

碩士 === 國立交通大學 === 應用化學系碩博士班 === 99 === Quercetin is a kind of flavonol which attracts considerable interest due to its anti-oxidant, anti-bacterial, anti-cancer and blocking human platelet aggregation properties. Quercetin 3-o-β-D-glucoside is effective in oxidative stress defensing and free radical scavenging. Quercetin glucosides are known to have increased stability, solubility and bioavailability when compared with quercetin. In the past, the way to get secondary metabolites is either chemical synthesis or plants extraction, but the complicated procedures in them have led to an increasing interest in the use of enzyme catalysis. In this research, we use UDP-glycosyltransferases from Bacillus cereus (BcGT-1) to convert quercetin to quercetin glucoside. In expression and purification of BcGT-1, keeping high oxygen concentration in the culture solution and using phosphate buffer with dithiothreitol to avoid forming the intra- or inter- protein disulfide bonds are key points to purify protein. The quercetin has five potential glucosylation positions to synthesize quercetin monoglucosides. In order to confirm the detail structure of these different quercetin monoglucosides, they are produced and separated by HPLC and identified their structure by 1H-NMR and LC-MS. Surprisingly, our result shows that the major products are 3’-O (50.4%) and 4’-O (42.1%) glucosides which is different to Joong-Hoon, Ahn’s previous result (3-O and 7-O glucosides are major). On the other hand, in order to understand the essential groups of BcGT-1, we compare three UGTs (UGT71G1、VvGT1 and OleI) from different resources with BcGT-1 and speculate that the four amino acids, His-14, Phe-240, Glu-310 and Asp-326 of BcGT-1, which are highly related to catalytic activity. According to our mutation results and papers review, we suggest His-14 as catalytic base and Glu-310 as a key residue that may interact with O2* and O3* of the ribose ring are the essential amino acid groups of BcGT-1.