, and PHB synthase) are present and active in isolated nPHB granules in vitro. nPHB granules also catalyzed thiolytic cleavage of PHB in the presence of added CoA, resulting in synthesis of 3-hydroxybutyryl-CoA (3HB-CoA) from PHB. Synthesis of 3HB-CoA was also shown by incubation of artificial (proteinfree) PHB with CoA and PhaZa1, confirming that PhaZa1 is a PHB depolymerase catalyzing the thiolysis reaction. Acetyl-CoA was the major product detectable after incubation of nPHB granules in the presence of NAD ؉ , indicating that downstream mobilizing enzyme activities were also present and active in isolated nPHB granules. We propose that intracellular concentrations of key metabolites (CoA, acetyl-CoA, 3HB-CoA, NAD ؉ / NADH) determine whether a cell accumulates or degrades PHB. Since the degradation product of PHB is 3HB-CoA, the cells do not waste energy by synthesis and degradation of PHB. Thus, our results explain the frequent finding of simultaneous synthesis and breakdown of PHB.Poly(3-hydroxybutyrate) (PHB) is the most prominent member of the bacterial polyhydroxyalkanoates (PHA). PHA are osmotic neutral reservoirs of carbon and energy and are synthesized when more carbon sources are available than can be consumed by the bacteria. PHA are reutilized (mobilized) during times of starvation and greatly enhance the survival of bacteria in the absence of a suitable exogenous carbon source.PHB is synthesized by condensation of two molecules of acetyl coenzyme A (CoA) to acetoacetyl-CoA (with a thiolase encoded by phaA), subsequent reduction to 3-hydroxybutyrylCoA (with a reductase encoded by phaB), and polymerization to PHB (with a synthase encoded by phaC). The polymer can be hydrolyzed to 3-hydroxybutyrate by PHB depolymerases (PhaZs). Biosynthesis and biodegradation of PHA have been investigated by many research groups for about three decades, and a series of books and reviews have been published (11,13,30,34,(39)(40)(41)(42). PHB exists in two different forms. In vivo PHB granules consist of an amorphous polymer and are covered by a dense layer consisting of mainly proteins (phasins, PHB synthase, PHB depolymerases, and other proteins) (14,22,31,38). Such granules are called native PHB (nPHB) granules and can be isolated in the native form by glycerol density gradient centrifugation (12,23,45). Isolated PHB granules that have been treated with solvents or with compounds that remove the surface layer rapidly crystallize and are referred to as denatured PHB granules (23,24). (For more details on the impact of the biophysical state of the polymer granules on their susceptibility to enzymatic hydrolysis see references 5, 8, and 12.) Despite the progress made in understanding the function of individual proteins involved in PHA metabolism, the molecular tools and mechanisms with which a cell decides whether it should synthesize or degrade (mobilize) PHB are not known. Several reports have indicated that PHB synthesis and PHB degradation can happen simultaneously in Ralstonia eutropha, the model organism for PHB...

Two methods for accurate poly(3-hydroxybutyrate) (PHB) depolymerase activity determination and quantitative and qualitative hydrolysis product determination are described. The first method is based on online determination of NaOH consumption rates necessary to neutralize 3-hydroxybutyric acid (3HB) and/or 3HB oligomers produced during the hydrolysis reaction and requires a pH-stat apparatus equipped with a softwarecontrolled microliter pump for rapid and accurate titration. The method is universally suitable for hydrolysis of any type of polyhydroxyalkanoate or other molecules with hydrolyzable ester bonds, allows the determination of hydrolysis rates of as low as 1 nmol/min, and has a dynamic capacity of at least 6 orders of magnitude. By applying this method, specific hydrolysis rates of native PHB granules isolated from Ralstonia eutropha H16 were determined for the first time. The second method was developed for hydrolysis product identification and is based on the derivatization of 3HB oligomers into bromophenacyl derivates and separation by highperformance liquid chromatography. The method allows the separation and quantification of 3HB and 3HB oligomers up to the octamer. The two methods were applied to investigate the hydrolysis of different types of PHB by selected PHB depolymerases.Polyhydroxyalkanoates (PHAs) are typical storage compounds of carbon and energy and are widely found in prokaryotes. The most common PHA is poly(3-hydroxybutyrate) (PHB), and this polymer can be accumulated at up to 90% of the cellular dry weight during unbalanced growth in some bacteria (2a, 37, 49). PHAs are thermoplasts, and despite the relatively high production costs, PHB and copolymers of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate are industrially produced on a scale of a few hundred tons per year.One reason for the interest in biologically produced PHAs lies in the environmentally friendly production from renewable resources and in the biodegradability of PHAs. The ester bonds of the PHAs are the Achilles' heel of the polymer that can be hydrolyzed by a large variety of hydrolytic enzymes (PHA depolymerases). In vivo, PHAs can be hydrolyzed by the accumulating strain itself during periods of starvation (intracellular PHA hydrolysis by intracellular PHA depolymerases) (41). PHAs that were produced by humans or that were released from PHA-accumulating microorganisms (e.g., after death) can be cleaved by extracellular PHB depolymerases, and many examples of extracellular PHA depolymerases were described during the last two decades (14, 21). A differentiation of extracellular degradation from intracellular degradation is necessary because PHA exists in two biophysical conformations: in vivo, PHB is completely amorphous (native) and is covered by a surface layer that is about half the size of a cytoplasmic membrane (4, 5, 31) but apparently consists mainly of proteins, so-called phasins (48). After the release of the polymer from the cell (e.g., after cell lysis or by solvent extraction) or after damage of the surface l...

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