枯草桿菌UDP-glycosyltransferase之重要胺基酸殘基分析及其應用

Abstract

槲黃素 (quercetin) 屬於類黃酮 (flavonoids) 的黃酮醇 (flavonols)，具有抗氧化、抗癌、預防心臟疾病及保護胃部等有利人體之作用。Quercetin 3-o-β-D-glucoside 也具相同生物活性，且quercetin glucosides的溶解性、穩定性，生物可利用率比quercetin高，以往主要藉由植物萃取及繁複的有機合成方式取得quercetin glycosides，既費時又費工，而利用UDP glycosyltransferases (UGTs) 可有效簡化合成步驟，提升醣基化合物產率，是極具價值的轉醣酵素。 本研究利用醣基轉移酶─BcGT-1合成quercetin glucosides，旨於探討 quercetin的轉醣位向及BcGT-1重要催化胺基酸。於BcGT-1酵素表現與純化研究上，利用高轉速(170 rpm)使菌液保有高氧濃度，並用含5 mM DTT (dithiothreitol) 磷酸鈉緩衝溶液做為沖提液，防止蛋白質形成雙硫鍵而失活沉澱析出，再由弱陰離子交換樹脂Toyopearl DEAE-650M 得高純度BcGT-1酵素。quercetin 具五個可以轉單個葡萄糖的氫氧基，藉由HPLC (C18 column )、LC-MS 及1H-NMR 確認轉醣位向及合成產物比例分析。quercetin monoglucosides 之產物分析結果顯示，quercetin 主要轉醣位向為3’-O (50.4%)和4’-O (42.1%) glucoside與Joong-Hoon, A.等人所之提出主要轉醣位向為3-O 和7-O glucosides 的結果大不相同。經比對受質同為flavonoid 且為inverting β-form glycosyltransferases 的UGTs 酵素如UGT71G1、VvGT1和Ole I之胺基酸序列，發現BcGT-1之主要催化胺基酸可能為His-14、Phe-240、Glu-310及 Asp-326，根據產物比例分析顯示H14A 及E310A 均使酵素活性嚴重喪失，兩者產物量分別為野生種BcGT-1 的1/30倍及1/25倍，故推測His-14 可能為使基質去質子的催化鹼，而Glu-310 則可能與穩定ribose ring 上O2* 及O3* 有關。

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.