2009-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/681009摘要：本研究目的是從豆芽中分離出β葡聚醣&#37238;來發展電化學的生物感應器系統。此生物感應器可被用來即時監控靈芝發酵過程中的β葡聚醣。本研究將利用固定化豆芽中純化之酵素與不同的生物聚合物或酵素合併來當作生物感應器探針，同時也將瞭解這些β葡聚醣&#37238;的水解活性以及和與載體的結合性，另外，在不同固定化酵素系統下的產能也將會逕行比較。在動力學的研究方面，對於有無固定化的β葡聚醣&#37238;的Km和Vmax亦將測量，同時測定固定化酵素系統熱力學上、操作上和貯存上的安定性。 在最適生物感應器方面，通透膜和彈性膜將會被當作主要材質來期望得到感應器得最佳靈敏程度，將使用最適的固定化酵素系統來研發出一個簡易靈敏的浸獨檢驗條來測量水溶液中β葡聚醣。將利用傅立葉轉換紅外線光譜儀分析（FT-IR）、原子力顯微鏡（AFM）、接觸角測試等分析方法來測定不同載體之固定化酵素的光電幾何特性。 將使用其他的試劑來共同固定化β葡聚醣&#37238;來組成具有電化學或電導度學特性之生物感應器，而此酵素修飾電極將使用電化學或簡單滴擲方法來製備，也會使用循環伏安法（CV）、雙脈衝伏安法（DPV）、電導度學等方法來測定電極的特性與實用性。具有抗垢特性的電極、電化學表現、反應時間、操作循環、動力線性範圍、較低的偵測閾值等特性的電能變換器將被優先使用。酵素修飾探針也會在室溫及4℃和乾燥或濕潤的情況下確認其安定性及貯藏性。 最後此適當的電化學偵測器將裝配流動注入分析（FIA），這樣一來，發酵槽中樣品便可流經由β葡聚醣&#37238;和葡萄糖氧化&#37238;共同固定形成的微粒酵素反應器，這些酵素反應所產生的產物將被偶合光度計和電化學系統所記錄分析，本研究也會最適化此感應器的樣品注射、流速、接觸時間、質量擴散、反應靈敏程度等，像一般會逕行干擾測定的鹽類、多酚類、多醣和其他電活性物質也會嚴謹的確認以避免影響發酵過程中感應器的操作，這些系統的操作效能亦將在發酵過程中被即時的監控。最後期望能得到一個小型自動化多效的β葡聚醣生物感應器。 <br> Abstract: This research proposal is aiming at developing a new optical sensing platform as well as electrochemical biosensor system based on beta glucanase isolated from novel sprouts. The sensor will be used for online monitoring of beta glucan concentration during Ganoderma lucidum fermentation. The biosensor probes will be designed by immobilizing partially or completely purified beta glucanase from different sprouts along with other enzymes and reagents on selected biopolymers by different strategies. The immobilized beta glucanase will be characterized for hydrolytic activity and leakage from the matrix support. The yield of immobilization will be calculated for different immobilized systems with respect to leaching. The kinetic parameters Km and Vmax for free and immobilized beta glucanase will also be determined. The immobilized systems will be further checked for thermal, operational and storage stability. In case of optical biosensor, the transparent and flexible membranes will be the prime requirement to achieve maximum sensitivity of the sensor. A simple sensitive dip-and-read test strip for the determination of glucan in aqueous samples will be developed using best immobilized enzyme system. The optogeometric properties of immobilized enzymes along with the novel composite matrices will be studied using different techniques such as Fourier Transform Infrared (FT-IR) spectroscopy, Atomic Force microscopy (AFM), contact angle measurement, etc. Amperometric or conductometric biosensors will be constructed using co-immobilized beta glucanase along with other reagents. The enzyme modified electrode will be prepared by electrochemical or simple drop-cast method. The characteristics and performance of modified electrode will be checked using Cyclic Voltammetry (CV), double pulse voltammetry (DPV), conductometric, etc. Suitable transducer will be selected based on their anti-fouling property of probe, electrochemical behaviour, response time, operational cycles, linear dynamic range, lower detection limit of analyte, etc. Stability and storage of the enzyme modified probe will be checked critically at room temperature and 4 °C under both dry and aqueous conditions. Furthermore, the optical and electrochemical detectors will be assembled with the flow injection analysis (FIA). In this process, the samples from the fermentor unit will pass through an enzyme reactor containing co-immobilized β-glucanase and glucose oxidase in a form of granules. The products of the enzymatic reactions will be simultaneously analyzed by the coupled photometric and electrochemical system. The sensor systems will be standardized and optimized for sample injection, flow rate, contact time, mass diffusion, sensor response, etc. The interference of other salts, phenolic compounds, polysaccharides and other electroactive species will be also checked critically as it may affect the sensor performance during fermentation process. The operation efficiency of the both systems will be checked during online monitoring at the time of fermentation. Among the both, best biosensor will be automated and more efforts will be taken to miniaturize the beta glucan biosensor to make it user friendly.β葡聚醣β葡聚醣&#37238海帶聚醣固定化生物聚合物導電聚合物電化學探針生物感應器Beta glucanbeta glucanaselaminarinimmobilizationbiopolymersconducting polymerselectrochemicaloptic probebiosensor