In vitro biosynthesis of poly(3-hydroxybutyric acid) by using purified poly(hydroxyalkanoic acid) synthase of Chromatium vinosum

被引:86
作者
Jossek, R
Reichelt, R
Steinbüchel, A
机构
[1] Univ Munster, Inst Mikrobiol, D-48149 Munster, Germany
[2] Univ Munster, Inst Med Phys & Biophys, D-48149 Munster, Germany
关键词
D O I
10.1007/s002530051166
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Purified recombinant poly(hydroxyalkanoic acid) (PHA) synthase from Chromatium vinosum (PhaEC(Cv)) was used to examine in vitro the specific synthase activity. turnover of R-(-)-3 -hydroxybutyryl coenzyme A (3HB-CoA) and poly(3-hydroxybutyric acid) formation under various conditions, The 3HB-CoA consumption was terminated by a reaction-dependent inactivation of the PHA synthase. Salts (MgCl2, CaCl2, NaCl), proteins (bovine serum albumin, lysozyme phasine) or detergent (Tween 20) increased the 3HB-CoA turnover to 2.5-fold. Specific PHA synthase activity was on only partially affected bq the added components. In general, a higher 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 and if MgCl2 was present. The form and size of the granules synthesized in vitro were affected by the concentration of the PHA synthase protein as well as by bovine serum albumin and the GA24 protein, a poly(3HB)-granule-associated protein of Alcaligenes eutrophus. Scanning electron micrographs from the synthesized granules were obtained. The granules consisted of poly(3HB) that had a molar mass in the range (1-2) X 10(6) g/mol.
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页码:258 / 266
页数:9
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