開發抑制劑為主的親和性層析方法以快速純化岩藻糖水解酶

Abstract

岩藻糖水解酶屬於醣苷酶，首先於溶酶體內發現，負責切除醣複合物非還原端的岩藻糖。岩藻糖水解酶不正常地自細胞內分泌至細胞外液，與許多疾病進程相關，例如結腸癌、子宮頸癌和肝癌。血清岩藻糖水解酶活性在肝癌病患顯著高於健康正常組，酵素活性被認為是肝癌早期診斷的生物標誌物候選蛋白質。 先前本實驗室報導1-amino-1-deoxymethyl fuconojirimycin (FNJ) 和其衍生物是岩藻糖水解酶的有效抑制劑，抑制常數在μM 至 pM 範圍。本論文建構了簡單快速的方法，用於純化鑑定肝癌血清中的岩藻糖水解酶。主要將 FNJ 與活化載體上的 carbonyl diimidazole 耦合形成醯胺鍵，我們使用兩種載體，分別為瓊脂醣和磁性奈米粒子。以高效液相層析儀定量耦合釋出的 imidazole，計算出耦合產率約 70%。珠體的酵素結合能力，由結合後剩餘的岩藻糖水解酶活性決定，最佳結合能力為磁性奈米粒子每微莫耳活化位，結合約 326.5 微克的岩藻糖水解酶。含 0.5% SDS 的溶離液具有岩藻糖水解酶最高回收效率約 90%。這個純化方法應用在純化大腸桿菌粗抽液中的岩藻糖水解酶，以及人類血清中的酵素。在肝癌病患血清，鑑定出其內岩藻糖水解酶活性異常增高的主要酵素為溶酶體的岩藻糖水解酶。根據凝集素微陣列分析，初步純化之病患血清岩藻糖水解酶，可能具有 Fuc 相關的 N- 醣基化後修飾。抑制劑為主的親和性層析方法，同時應用在闡述幽門螺旋桿菌感染胃腺癌細胞後，共培養液中活性大量表現的葡萄糖苷酶種類。本研究證實抑制劑親和性層析方法為快速經濟的純化方法，有效應用於不同的複雜生物樣本。

α-L-fucosidase (AFU) is a glycosidase first found in lysosomes. This enzyme catalyzes the hydrolysis of fucose-containing glycoconjugates. The release of abnormal AFU from cells to extracellular fluid is linked to several disease processes, including colorectal cancer, ovarian cancer and hepatocellular carcinoma (HCC). The level of AFU activity from the serum of HCC patients is higher than that of normal people, indicating that the serum AFU is a potential biomarker for early diagnosis. We previously demonstrated that 1-amino-1-deoxymethyl fuconojirimycin (FNJ) and derivatives are potent inhibitors of AFU (Ki values in the range of micro- to pico- molar). Herein in this thesis study, a facile method was developed for purification and identification of the serum AFU. The key step was to immobilize FNJ to carbonyl diimidazole-activated agarose matrix or magnetic nanoparticle via amide bond formation. The optimal immobilization yield was about 70%. The binding capacity was optimized to be 326.5 μg AFU/μmol on magnetic nanoparticle. Furthermore, the 0.5% SDS-containing eluent was found to offer the highest recovery yield of AFU about 90%. The method is applicable for purifying the AFU from human sera. For patients of HCC, the purified AFU is the lysosomal enzyme (FucA1), suggesting that FucA1 could be highly upregulated. It is of interest that the FucA1 of HCC contains fucosylated N-glycosylation. Additionally, the culture medium of H. pylori-infected gastric cancer cells was subjected to one step, deoxynojirimycin-based affinity chromatographic purification, leading to identification of a lysosomal α-glucosidase.