| Abstract: | Application of enzymes to industrial processes has been limited to degradative reactions, since many enzymatic syntheses require hydrolysis of stoichiometric amounts of ATP (adenosine triphosphate) to drive the reactions. Since ATP is expensive, it is necessary to regenerate ATP from ADP during the course of the reaction. Enzymatic regeneration from chemically synthesized high-energy phosphate donors, the best scheme currently available, has the major disadvantage in that the product produced from the phosphate donor accumulates and is not easily removed. In this research, we investigated whether the thermophilic ATP synthase (TF[0]F[1]) from the bacterium PS3 was suitable for ATP regeneration. This enzyme can synthesize ATP without producing waste products (i.e., spent donors) since the reaction is driven by a pH gradient provided by an acid-base change. PS3 synthase was selected since it is stable compared to other ATP synthases. TF[0]F[1] was purified from the thermophilic bacterium PS3 and reconstituted into liposomes by the cholate-dialysis method. The reconstituted TF[0]F[1] liposomes showed synthetic activity of 10-20 nmol ATP/mg TF[0]F[1] by an acid-base change when ATP was captured with hexokinase. Higher amounts of ATP (maximum of 60 nmol ATP/mg TF[0]F[1] could be synthesized when ATP was captured with glycerokinase. The fluorescence quenching assay after repeated acid-base changes showed that TF[0]F[1] liposomes did not lose proton-pumping activity even after six acid-base changes performed over a 2-week period. These preliminary results indicate that ATP can be synthesized by repeated acid-base changes. A modeling study of a large-scale ATP-regeneration scheme showed that significantly less energy is required if the liposomes are immobilized on glass spheres rather than used in aqueous suspension. In this scheme, the KOH and HCl required to produce the pH gradient are regenerated from KCl by water-splitting electrodialysis technology. Calculations show that about 200 g of ATP can be synthesized per kWh of electricity (DC) with this scheme. Assuming electricity can be purchased for $0.07/kWh, a kilogram of ATP could be regenerated using $0.35 worth of electricity. This compares very favorably with the purchase price of $370/kg of ATP. |
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