| 英文摘要
| 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. |
