利用氧化葡糖酸桿菌胞內活性蛋白結合葡萄糖氧化酵素應用於葡萄糖電催化之研究

Abstract

酵素型葡萄糖燃料電池（enzymatic glucose fuel cell）是直接利用酵素作為催化劑，氧化葡萄糖（glucose）釋放電子，從而獲取電能的一種功能設備，有望於作為醫療植入物、微型感測器及生物芯片的內置電源。在理論上，一分子葡萄糖完全氧化可産生24個電子，约產生5.50 W•h的能量。但目前成功研發出的酵素型葡萄糖燃料電池均為不完全氧化型，大多為使用葡萄糖氧化酵素（glucose oxidase，GOx）將葡萄糖經一步氧化成為葡萄糖酸-δ-内酯(glucono-δ-lactone)，同時釋放2個電子。而氧化葡糖酸桿菌（Gluconobacter oxydans）具備不完全氧化葡萄糖的代謝途徑，與現有的葡萄糖燃料電池氧化利用葡萄糖途徑相容，可將葡萄糖進一步氧化成二酮葡糖酸（diketogluconic acid）並額外釋放6個電子。 本研究嘗試使用以甘露醇為主要碳源的基礎培養基和以葡萄糖為主要碳源的誘導培養基培養氧化葡糖酸桿菌，通過測定其生長曲線以及所得酵素群的葡萄糖氧化酵素活性和葡糖酸脫氫酵素活性，獲得較佳培養方案。嘗試在蛋白緩衝液中添加1%葡萄糖或1%葡糖酸（gluconic acid），發現添加1%葡糖酸有助於維持酵素群的活性。 對於沉澱部分，使用10% Triton X-100溶解細胞膜脂質並以酸性丙酮沉澱獲得溶解下來的膜聯蛋白JAM。生化檢測表明，JAM組分具有葡萄糖氧化酵素活性和葡糖酸脫氫酵素活性；電化學檢測表明，JAM組分能以黃素腺嘌呤二核苷酸（FAD）作為電子傳遞介質，在含有5 mM葡萄糖的模擬陽極溶液系統中氧化葡萄糖產生22.1 μA/cm2氧化電流訊號；能與GOx共同作用，使所產生的氧化電流訊號上升至38.3 μA/cm2，表明該提取純化所得的酵素群或能催化葡萄糖的進一步氧化。 對於可溶性蛋白部分，採用硫酸銨鹽析將其劃分為JAS35 與 JSA70兩組。生化檢測表明，JAS35與 JSA70兩個組分均具有葡萄糖氧化酵素活性和葡糖酸脫氫酵素活性，但JAS35組分的活性更強；電化學檢測表明，JAS35組分能以磷酸酰胺腺嘌呤二核苷酸（NADP）作為電子傳遞介質，在含有5 mM葡萄糖的模擬陽極溶液系統中氧化葡萄糖產生19.7 μA/cm2氧化電流訊號；能與GOx共同作用，使所產生的氧化電流訊號上升至36.3 μA/cm2，表明該提取純化所得的酵素群或能催化葡萄糖的進一步氧化。使用superdex 75凝膠過濾層析對JAS35組分進行提純獲得具有較高葡萄糖氧化效能的新酵素群JAS35（LC），其在含有5 mM葡萄糖的模擬陽極溶液系統中氧化葡萄糖所產生的氧化電流訊號提升至59.2 μA/cm2，為JAS35組分的6.63倍。JAS35（LC）組分與GOx共同作用產生的氧化電流訊號達到99.5 μA/cm2，經20天冷藏後仍保留一定催化活性，可氧化葡萄糖酸內酯、葡糖酸產生電流訊號。 最後，使用Synapt G2 HDMS蛋白質譜鑑定獲得JAM和JAS35酵素群中可能的有效組分。

Enzymatic glucose fuel cell, a device that converts the glucose chemical energy into electricity through a chemical reaction with oxygen, is a potential power source for implantable devices, sensors and micro-chips. In theory, complete oxidation of one molecule glucose can produce 24 electrons and 5.50 W•h energy. However, glucose oxidation of enzymatic glucose fuel cells recently constructed can only convert glucose into glucono-δ-lactone by glucose oxidase (GOx) and produce 2 electrons. Gluconobacter oxydans is a kind of bacteria which can metabolize glucono-δ-lactone into diketogluconic acid without phosphorylation and release another 6 electrons. In this paper, we cultured Gluconobacter oxydans in D-mannitol culture medium or in glucose culture medium for about one to week waiting for the optical density (A600) reaching maximum, measured and investigated the growth curves in order to finding the better culture method. We broke Gluconobacter oxydans by french press and dissolved the proteins in 0.1 M PBS containing 1% glucose or 1% gluconic acid to keep the enzymatic activity and investigated which one is better. For the precipitate, we used 10% Triton X-100 to dissolve the lipid of cytomembrane releasing the membrane-bound proteins and precipitate them with acidic acetone solution. The membrane-bound proteins we obtained are named JAM. By biochemical examining, we found that JAM has GOx activity and gluconic acid dehydrogenase (Gdh) activity. By electrochenical examining, we found that JAM can convert the glucose chemical energy into electricity with Flavin Adenine Dinucleotide (FAD) as electron media and produce about 22.1 μA/cm2 current density at 0.8 V in 5 mM Glucose solution. JAM can also catalyze glucose oxidation coefficiently with GOx and enlarge the current density into 38.3 μA/cm2. For the soluble proteins, we added 35% or 70% ammonium sulfate to salt out them into two parts, which are called JAS35 and JSA70 respectively. By biochemical examining, we found the GOx activity and Gdh activity of JAS35 are better than JAS 70. By electrochenical examining, we found that JAS35 can convert the glucose chemical energy into electricity with Nicotinamide Adenine Dinucleotide Phosphate (NADP) as electron media and produce about 19.7 μA/cm2 current density at 0.8 V in 5 mM Glucose solution. JAS35 can also catalyze glucose oxidation coefficiently with GOx and enlarge the current density into 36.3 μA/cm2. In order to obtaining enzymes which have higher glucose oxidation activity, we used Superdex 75 column for the JAS35 gel filtration chromatography and partially purified JAS35 to a new group of enzymes named JAS35(LC). By electrochenical examining, we found that JAS35(LC) can produce higher current density at 0.8 V in 5 mM Glucose solution than JAS35. It reached 59.2 μA/cm2 and becomes 6.63 times of the current density JAS35 produced. JAS35(LC) can also catalyze glucose oxidation with GOx more effectively, whose current density reached 99.5 μA/cm2. After storing in 4 ℃, the catalytic activity of JAS35(LC) retained. JAS35(LC) can convert the glucono-δ-lactone chemical energy and gluconic acid chemical energy into electricity too. Finally, we identificated the proteins in JAM and JAS35 by LC-MS/MS using Synapt G2 HDMS and found that effective enzyme constituent in JAM and JAS35.