IN-VITRO SYNTHESIS OF POLY(3-HYDROXYBUTYRIC ACID) BY USING AN ENZYMATIC COENZYME-A RECYCLING SYSTEM

Purified recombinant poly(hydroxyalkanoic acid), PHA, synthase from Ch romatium vinosum was used to examine in vitro poly(3-hydroxybutyric ac id) (P(3HB)) formation. In combination with purified propionyl-coenzym e A transferase of Clostridium propionicum a two-enzyme in vitro P(3HB ) biosynthesis system was established which allowed the synthesis of P (3HB) from free D-(-)-3-hydroxybutyric acid as substrate. The coenzyme ii residue for the activation of this hydroxyacid was provided by ace tyl-coenzyme A. By adding acetyl-coenzyme A synthetase to this system, a three-enzyme in vitro P(3HB) biosynthesis system was established. C oenzyme A that was released during the polymerization reaction was cou pled to acetate which again served as the coenzyme A donor for the act ivation of 3-hydroxybutyric acid. The energy for the in vitro P(3HB) s ynthesis was provided by ATP hydrolyses resulting in acetyl-coenzyme A synthesis catalyzed by the acetyl coenzyme A synthetase. In this way the in vitro synthesis of P(3HB) became independent of the consumption of the expensive coenzyme A. By this procedure a handy system is avai lable to produce in vitro PHA on a semipreparative scale. (C) 1998 Fed eration of European Microbiological Societies. Published by Elsevier S cience B.V. All rights reserved.