蒺藜苜蓿、阿拉伯芥及抗菌素鏈黴菌中葡萄糖轉醣酶之分子選殖、蛋白質表現及其功能性分析

Abstract

皂苷是廣泛分布於生物體內的一種分子，其由皂苷原及其上修飾的醣基所組成，大多具有廣泛的生物活性，包括：抗微生物、抗真菌、降膽固醇、抗癌、溶血活性並可作為疫苗輔劑以增加免疫力等。文獻指出，皂苷的結構多樣性源自於其生合成的三個階段： (1) 由氧化鯊烯環化酵素家族產生的多樣性皂苷原碳骨架 (2) 氫氧化酶或氧化酶對於骨架上不同碳原子上的質子轉移、氫化及氧化作用 (3) 轉醣酵素於羥基上修飾醣鏈的轉醣作用。 皂苷原及其上修飾的醣基多樣性決定了皂苷的生物活性，且轉醣酶會將原本沒有生物活性的分子經由醣基的轉移而形成有活性的產物。因此，醣化作用不只影響皂苷的溶解度及穩定性，還可能對於皂苷的生物活性扮演關鍵的角色。在實驗室先前的研究中，已針對氧化鯊烯環化酵素的功能性胺基酸及其產物結構與催化機制進行研究，並且分離出多樣性的三萜類產物結構。為了更進一步提昇產物的多樣性與研究其生物活性，這些三萜類分子將作為轉醣酵素修飾的對象。在此研究中，四條轉醣酵素基因包括蒺藜苜蓿(Medicago truncatula) 中的UGT71G1及UGT73K1、抗生素鏈黴菌(streptomyces antibiotics) 中的OleD-ASP與阿拉伯芥(Arabidopsis thaliana) 中的UGT80A2用分子轉殖技術接合到表現載體上並於大腸桿菌(E. coli) 中進行蛋白質表現，其中三種轉醣酵素UGT71G1, UGT73K1與OleD-ASP可以經由親和性層析法進行純化。此外我們利用九種不同結構的分子作為醣基的接受者，尿嘧啶二磷酸葡萄糖(UDP-glucose) 作為醣基的提供者，並由薄層層析法做為快速且初步的活性篩檢，由其結果得知其中有五種分子有新產物產生，分別為雌二醇(beta-estradiol)、長春藤皂苷(hederagenin) 、順雄酯酮(cis-androsterone)、反雄酯酮(trans-androsterone)與一結構未知的分子X。產物的種類與結構可藉由高效液相層析法分離與電噴霧離子化質譜來鑑定，其中以OleD-ASP對compound-X的效率最佳，可產生一個雙葡萄糖苷與三個單葡萄糖苷產物。未來這些經由轉醣酵素催化形成的產物會進行生物活性的分析，以期可以應用於醫學研究上。另一方面，UGT80A2亦於酵母菌中進行蛋白表現，但並無明顯大量蛋白生成，之後將進行其DNA序列密碼子的修改後，再次進行蛋白表現的測試。並希望轉醣酵素於酵母菌突變株CBY57中表現，並利用酵母菌中氧化鯊烯環化酵素原生種與突變種所產生的多樣皂苷原產物進行轉醣的篩選並分析其活性。

Saponins are a group of natural products that are widely distributed in living organisms and possess a large variety of bioactivities, including antimicrobial activity, antifungal activity, anticholesterolemic activity, hemolytic activity, antitumor activity, as well as to being adjuvant to enhance immunity. The biological activities of triterpene saponins are attributed to their structural diversity, composed of various sapogenin and glycone moieties, and can be determined by three steps of biosynthesis: (1) cyclization of oxidosqualene into sapogenin backbone; (2) proton transformation, hydroxylation, or oxidation at different carbon atoms within the structure of sapogenin; and (3) glycosylation reaction to decorate sugar moieties at some hydoxyl groups. Glucosyltransferases (GTs) play a major role in turning inactive compounds into active triterpene saponins. Glycosylation not only enhances the solubility and stability of triterpene saponins but also prolongs their storage in organisms. Based on our previous investigations, Saccharomyces cerevisiae ERG7 is applied to examine structure-functional relationships. Diverse truncated and rearranged unnatural triterpene products are generated and isolated to proceed with further studies on acquiring new bioactivities via GTs-mediated sugar-decoration. In this thesis, four GTs are successfully cloned and expressed in E. coli system, including UGT71G1 (Medicago truncatula), UGT73K1 (Medicago truncatula), UGT80A2 (Arabidopsis thaliana), and OleDA242V/S132F/P67T (Streptomyces antibioticus). Three GTs (UGT71G1, UGT73K1 and OleD-ASP) were successfully purified by nickel affinity chromatography. The activities of these three GTs against nine compounds were screened with TLC and further characterized by HPLC/ESI-MS and ESI-MS/MS. UGT71G1 exhibit catalytic abilities toward hederagenin, -estradiol and one unknown structure compound X, while UGT73K1 catalyzes glucose transfer only to hederagenin. OleD-ASP with boardest substrates capacity that generate glycoside products from hederagenin, beta-estradiol, cis-androsterone, trans-androsterone and compound-X. In the future, products from the above GTs-mediated reactions with transferred glucose moiety will be characterized for various bioactivities, while triterpenes isolated from (ERG7) mutants will be subjected to glycosyltransferases tailoring reactions.