IN-VITRO BIOSYNTHESIS OF POLY(3-HYDROXYBUTYRIC ACID) BY USING PURIFIED POLY(HYDROXYALKANOIC ACID) SYNTHASE OF CHROMATIUM-VINOSUM

Purified recombinant poly(hydroxyalkanoic acid) (PHA) synthase from Ch romatium vinosum (PhaEC(Cv)) was used to examine in vitro the specific synthase activity. turnover of R-(-)-3 -hydroxybutyryl coenzyme A (3H B-CoA) and poly(3-hydroxybutyric acid) formation under various conditi ons, The 3HB-CoA consumption was terminated by a reaction-dependent in activation of the PHA synthase. Salts (MgCl2, CaCl2, NaCl), proteins ( bovine serum albumin, lysozyme phasine) or detergent (Tween 20) increa sed the 3HB-CoA turnover to 2.5-fold. Specific PHA synthase activity w as on only partially affected bq the added components. In general, a h igher concentration of salt often inhibited the activity of PhaEC(Cv), without affecting the yield according to 3HB-CoA turnover. NAD(+) and NADP(+) (2 mM) inhibited PhaEC(Cv) completely, whereas NADH and NADPH did not. Macroscopic poly(3HB) granules were formed in vitro if PhaEC (Cv) was incubated in the presence of sufficient amounts of 3HB-CoA an d if MgCl2 was present. The form and size of the granules synthesized in vitro were affected by the concentration of the PHA synthase protei n as well as by bovine serum albumin and the GA24 protein, a poly(3HB) -granule-associated protein of Alcaligenes eutrophus. Scanning electro n micrographs from the synthesized granules were obtained. The granule s consisted of poly(3HB) that had a molar mass in the range (1-2) X 10 (6) g/mol.