釀酒酵母、幽門螺桿菌及阿拉伯芥中轉醣酶之分子選殖、蛋白質表現及其功能性分析

Abstract

皂苷為一種廣泛分布於植物中的次級代謝產物；結構主要由疏水性的皂苷原及所鍵結之親水性醣基所構成。普遍的生物活性包括：抗感染性微生物、抗癌、抗發炎等，中草藥中常見的甘草及人蔘多半含有皂苷成分在內。皂苷的生合成路徑主要分為 三大步驟：(1) 氧化鯊烯環化酵素進行之環氧化產生初步的皂苷原碳骨架 (2) 氫氧化酶對於碳骨架上特定位置之碳原子進行氫氧化作用 (3) 轉醣酶於羥基上鍵結上醣基的轉醣作用。皂苷上的醣基對於皂苷本身的生物活性有重要影響。研究發現醣化作用可增加生物分子的多樣性、溶解性及穩定性。 在此研究中，因其中兩條轉醣酵素基因:釀酒酵母 (Saccharomyces cerevisiae) 中的UGT51及幽門螺桿菌 (Helicobacter pylori) 中的HP0421皆以膽固醇(cholesterol)及麥角固醇 (ergosterol) 作為其活體內 (in vivo) 受質。為進一步提升產物之多樣性及鑑定酵素對於受質之活性，實驗室目前以多種四環固醇類作為轉醣酵素的修飾的對象。將轉醣酵素基因以分子轉殖技術接合到表現載體上，並於大腸桿菌 (E.coli) 系統中進行蛋白質表現後，進行酵素之活性分析。經由薄層層析法初步篩選出有新產物產生的分子，以高效液相層析法做二次確認。其中UGT51及HP0421皆對反式雄酯酮(trans-androsterone)催化效率較佳，故對產物進行分離純化後，以電噴霧離子化質譜及核磁共振儀鑑定其分子量與結構。未來將藉由可被催化反應之受質的結構，以分子模擬的方式，更深入了解酵素催化機制，以期應用於未來之生物活性分析。 另一方面，蒺藜苜蓿 (Arabidopsis thaliana) 中的UGT72B1，因此酵素可同時在氮原子及氧原子上鍵結上葡萄糖，雖受質結構性質與另外兩者不同，仍將四環固醇作為受質進行活性分析。但並無新產物生成，未來將嘗試分子模擬後，修改基因序列密碼子，增加可作用之受質的多樣性。

Saponins are a group of secondary metabolites widely expressed in plant species; their structure contains a hydrophobic sapogenin and a hydrophilic sugar moiety. Saponins play biologically important roles in plant defense and are pharmacologically relevant compounds, showing detergent, anti-fungal, anti-inflammatory, and anti-tumor activities. Saponins are biosynthesized mainly via the following three steps: (1) cyclization of oxidosqualene into sapogenin backbone; (2) hydroxylation of different carbon atoms of sapogenin; and (3) glycosylation of sapogenins, resulting in the addition of a sugar moiety at the hydroxyl groups. Glycosylation determines chemical complexity and diversity and enhancement of solubility and stability (which improves their storage and accumulation in cells). Glycosyltransferase catalyzes the formation of glycosidic bonds with the help of a sugar donor (such a sugar donor contains a nucleoside phosphate or lipid phosphate leaving group). In our study, three glycosyltransferases were successfully cloned and expressed in an Eschericia coli system, including UGT51 (Saccharomyces cerevisiae), HP0421 (Helicobacter pylori), and UGT72B1 (Arabidopsis thaliana). These were successfully purified using Ni-NTA chromatography. The activities of two of three glycosyltransferases on several compounds, such as cholesterol, ergosterol, cis-androsterone, trans-androsterone, the intermediates and products of steroidogenesis pathway, etc., were determined ab initio and characterized using thin layer chromatography and high performance liquid chromatography. Subsequently, ESI-MS/MS was applied to determine the molecular weight of these enzymes. Moreover, using NMR 1D and 2D spectra, 1H, 13C, DEPT 90 & 135, HSQC, and HMBC, the structure of the glycosylated products could be analyzed correctly.