Paecilomyces Lilacinus 中外切型葡萄糖胺苷水解酵素之研究暨生醫感測技術之開發

Abstract

第一部分 本實驗室由自然界中篩選到具有昆蟲病原性的黴菌─淡紫擬青黴 (Paecilomyces lilacinus)。以 0.5% 幾丁聚醣為碳源、混合了 M9 buffer，pH 6.0 培養液進行馴化培養，可以得到大量表現的外切型葡萄糖胺苷水解酵素 (exo-□-D-glucosaminidase)，並以陽離子交換管柱液相層析可得純度大於 80% 的酵素。酵素的分子量約為 95 kD，其於 pH 6.0 左右活性與穩定性最佳，在 45 °C 下的催化活性最好。此葡萄糖胺苷水解酵素可以有效率的由非還原端水解 95% 去乙醯化的幾丁聚醣，生產葡萄糖胺 (2-amino-2-deoxy-D-glucopyranose，□-D-glucosamine，GlcN)，而當幾丁質為受質時則無法水解產生乙醯葡萄糖胺 (2-acetamido-2-deoxy-D-glucopyranos，□-D-N-acetylglucosamine，GlcNAc)。 根據質譜儀分析葡萄糖胺苷水解酵素對幾丁六糖的水解反應可以證實，此酵素是一外切型水解酵素。由 real-time 質譜儀分析酵素和不同幾丁寡糖的水解作用，可了解葡萄糖胺苷水解酵素對受質的特異性，以及利用本實驗室現有之幾丁質水解酵素 A (chitinase A) 的轉醣產物和葡萄糖胺苷水解酵素反應，可證實此酵素可以水解 GlcN-GlcN 和 GlcN-GlcNAc 糖苷鍵，但對 GlcNAc-GlcN 及 GlcNAc-GlcNAc 糖苷鍵水解效果不明顯。利用添加了 10% 不同醇類於 95% 去乙醯化幾丁聚糖溶液中與酵素反應，由不同烷基化的葡萄糖胺產物 (alkyl-□-D-glucosamines) 證明葡萄糖胺苷水解酵素具有轉醣功能，並以 NMR 光譜分析酵素轉醣後生成的甲基葡萄糖胺，可以證實此葡萄糖胺苷水解酵素是一位相保留 (retention) 的水解酵素。

第二部分 本研究分為兩個部分，第一部分是利用噬菌體展示庫 (phage display library)，以敗血症的指標蛋白質前降鈣素 (procalcitonin，PCT) 為抗原，篩選出可辨識 PCT 之人造抗體─PCTscFv。PCTscFv 修飾了 FITC (fluorescein isothiocyanate)，以及 PCT 修飾了 rhodamine，以螢光光譜儀分析兩者混合後的 FRET (fluorescence resonance energy transfer) 現象，證實兩者間具有辨識效果，並可以 Benesi-Hildebrand 公式計算出兩者間的平衡常數 KD = 16.4 (□M)。在石英晶體微天平 (quartz crystal microbalance，QCM) 的晶片上固定化了 PCTscFv 後，可檢測到 PCT 的最低濃度為 10-10 g/mL，並由 Langmuir isotherm 公式求得 KD = 10.8 (nM)。QCM 檢測血清中的 PCT 實驗中，利用 anti-PCT 抗體辨識結合在 PCTscFv 上的 PCT，我們可成功量測到含有 PCT 10-13 g/mL 稀釋 1000 倍的血清樣品 (等於無稀釋血清中含有 PCT 10-10 g/mL)。 另一部分是以噬菌體展示庫篩選出可辨識雙酚 A (BPA) 的 VH、VL，在其 N 端融合了麥芽糖水解酵素 (maltose binding protein，MBP)，形成 MBP_VH、MBP_VL 融合蛋白質，以開放型三明治免疫分析 (open-sandwich immune-assay，OSIA) 的技術於不同生物感測器上進行 BPA 的檢測。在以 OSIA 結合表面電漿共振光譜儀 (surface plasmon resonance，SPR) 進行檢測的實驗中，在 SPR 晶片上固定化 MBP_VL 後，再注入固定濃度的 MBP_VH 與不同濃度的 BPA 混合樣品，結果顯示因形成三明治結構 MBP_VL─BPA─MBP_VH 而偵測到 BPA 的最低濃度為 10-15 M，且對於結構相似於 BPA 的小分子 ethylphenol、tyramine、dopamine 辨識效果不佳，證明 VH、VL 對 BPA 具有專一性；另外發現在自組裝單分子膜 (self-assembly monolayers，SAM) 晶片上，修飾了含有 20% -COOH 的檢測效果最佳。在利用 QCM 結合了 OSIA 技術的實驗中，固定化 MBP_VL 於 QCM 晶片後，再注入固定濃度的 MBP_VH 與不同濃度的 BPA 混合樣品，結果顯示可偵測到的 BPA 最低濃度為 10-10 M，而且具有專一性。最後將 MBP_VH、MBP_VL 分別修飾於金奈米粒子 (Gold nanoparticles，AuNP) 以溶液顏色變化的方式檢測 BPA，將 AuNP-MBP_VH 與 AuNP-MBP_VL 混合後再加入 BPA，因為三明治結構的產生，使得 AuNP 聚集而有表面電漿共振產生吸收紅位移，導致顏色的改變。實驗結果顯示，除了具有專一性，可偵測到的 BPA 最低濃度達 10-9 ~ 10-10 M 之間，等於 0.23 ~ 0.023 ppb。 利用噬菌體展示庫篩選人造抗體，無論對大分子或是小分子均可有效的辨識，且專一性高；再者，篩選過程及生產人造抗體的耗時短，相信此技術對生物辨識、生物檢測提供了一個全新的方向。

Part 1 An entomopathogenic fungus, Paecilomyces lilacinus, was found to grow on chitosanase-detecting plates. Besides an endo-chitosanase, an exo-β-D-glucosaminidase was purified by cation-exchange chromatography from this microorganism cultivated in M9 minimal media containing 0.5% chitosan as the sole carbon source. The molecular weight of the enzyme is 95 kDa; the optimum pH and temperature for activity are 6.0 and 45°C, respectively. The purified exo-β-D-GlcNase promotes the hydrolysis of 95% deacetylated chitosan from its non-reducing end and liberates 2-amino-2-deoxy-D-glucopyranose (GlcN) as the sole product; however, 2-acetamido-2-deoxy-D-glucopyranose (GlcNAc) was not detected when chitin was used as the substrate. The cleavage pattern was confirmed by using real-time mass spectrometry shows that exo-β-D-glucosaminidase cleaves the glycosidic bonds between GlcN-GlcN and GlcN-GlcNAc but not between GlcNAc-GlcN or GlcNAc-GlcNAc. In the presence of a 10% solution of various alcohols, many alkyl-β-D-glucosaminides were obtained, indicating that exo-β-D-glucosaminidase is a retaining enzyme. Part 2 The study is separated into two sections. In the first section, we screened out the artificial antibody ─ PCTscFv from phage display library which was using procalcitonin (PCT) as antigen. PCT is an important bio-marker for diagnosing sepsis. After modifying fluorescein isothiocyanate (FITC) on PCTscFv and rhodamine on PCT, the recognition ability between PCTscFv and PCT was comfirmed by analyzing fluorescence spectra of fluorescent resonance energy transfer (FRET) phenomenon. And the equilibrium dissociation constant was calculated according to the fluorescenc spectra and Benesi-Hildebrand equation, KD = 16.4 (□M), respectively. After immobilized PCTscFv on quartz crystal microbalance (QCM) chip, the recognition ability between PCTscFv and PCT was comfirmed by QCM, and the detection limit of PCT concentration reached 10-10 g/mL. KD was calculated according to Langmuir isotherm equation, KD = 10.8 (nM). Furthermore, we successfully detected low concentration of PCT (10-10 g/mL) in human serum by adding anti-PCT antibody to amplify QCM signal. In the second section, we used VH (BPA) and VL (BPA) screened from phage display library, provided by Dr. Ueda, for detecting small molecule bisphenol A (BPA), which is one of the most common reagent for plastics producing. BPA may release into water or food plastic containers and cause different diseases. We fused maltose binding protein (MBP) to N terminal of VH or VL for better protein solubility, resulting in MBP_VH and MBP_VL. Then we used the open-sandwich immuno-assay (OSIA) technique to detect BPA on different platforms. After using OSIA technique combined with surface plasmon resonance spectrum (SPR) detection, we noticed that the 20% -COOH self-assembly monolayers (SAM) chip performenced the better detection range of BPA concentration, and the detection of BPA concentration could reach to 10-15 M, which is lower than other methods used nowadays. And this detection system also showed the specificity toward other molecules such as ethylphenol, tyramine and dopamine, which structures are similar to BPA. According to QCM combined with OSIA detections, the results comfirmed only when BPA exsisted that could form the sandwich structure MBP_VL-BPA-MBP_VH, and the detection limit of BPA is 10-10 M. This system also showed the specificity of recognizing BPA. Ultimatly, we immobilized MBP_VH and MBP_VL individually on gold nanoparticles (AuNP), forming AuNP-MBP_VH and AuNP-MBP_VL. After adding BPA into the mix solution of AuNP-MBP_VH and AuNP-MBP_VL, the color of AuNP was changed from red to dark purple according to the forming of sandwich structure leading AuNP to aggregate. AuNP method can detect BPA concentration to 10-9 ~ 10-10 M which is equal to 0.023 ~ 0.23 ppb. Besides, AuNP detection is a easy, quick and simple operation. The artificial antibody screened from phage display library can provide effiecient recognizations on either big molecule or small molecule antigens with high specificities. Furthermore, the phage display library screening and artificial antibody producing processes takes less time than traditional one. We firmly believe this system can provide a new direction for bio-recognization and bio-sensing.