1995. Ser42, Asp138, and His178 were identified as catalytic-triad amino acids, with Ser42 as the putative active site. Remarkably, the 1st 23 amino acids of the PHB depolymerase previously assumed to be intracellular revealed features of classical transmission peptides, and Edman sequencing of purified PhaZ1 confirmed the functionality of the expected cleavage site. Extracellular PHB depolymerase activity was absent, and analysis of cell fractions unequivocally showed that PhaZ1 is definitely a periplasm-located enzyme. The previously assumed intracellular activator/depolymerase system is unlikely to have a physiological function in PHB mobilization in vivo. A second gene, encoding the putative true intracellular PHB depolymerase (PhaZ2), was recognized in the genome sequence of (50) and has been classified as polythioesters (27, 28). Investigation of the biodegradation of PHA should distinguish between intracellular and extracellular degradation (for a recent review, see research 20). Intracellular degradation is the active mobilization (hydrolysis) of the polymer from the accumulating bacterium itself. In the case of extracellular degradation, PHA is utilized by means of extracellular enzymes that are secreted by PHA-degrading microorganisms. The source of extracellular polymers dmDNA31 is definitely PHA released by accumulating bacteria after death. PHA in vivo and outside of the bacteria are present in two different conformations. In vivo, polymer molecules are in the amorphous rubbery state (highly mobile chains inside a disordered conformation), and PHA granules are covered by a 4-nm-thick surface layer. The surface coating of isolated PHB granules consists of proteins and phospholipids (3, 26, 31, 44), which are damaged or lost upon extraction of the polymer from your cell (12, 13, 33), and the polyester chains tend to adopt an ordered helical conformation and to develop a crystalline phase. This polymer is referred to as denatured (crystalline) PHA (5, 6, 32). Extracellular PHB is definitely a partially crystalline polymer with an amorphous portion (glass transition temp [(PhaZ7), which was specific for nPHB and was dmDNA31 unable to hydrolyze dPHB, was explained (15). For H16 (42). Intracellular nPHB depolymerases of are not related to extracellular dPHB depolymerases with respect to amino acid sequence but share MAPK3 significant amino acid similarities with each other and with additional putative intracellular PHB depolymerases found in the databases (10, 38, 53). None of them of the currently known extracellular or intracellular PHB depolymerases requires any proteins as cofactors. However, appeared to be an exclusion. was the first bacterium in which degradation of nPHB granules had been intensively investigated (32): due to the high rate of in vitro self-hydrolysis of nPHB granules isolated from to investigate PHB hydrolysis by parts. They found that hydrolysis of nPHB to 3HB required three parts. The 1st component was a soluble (intracellular), heat-sensitive depolymerase that may be enriched from soluble cell components. However, efficient hydrolysis of nPHB granules in vitro by soluble PHB depolymerase required pretreatment dmDNA31 of PHB granules having a heat-stable second component called the activator that was also present in soluble cell components. The third component dmDNA31 was a dimer hydrolase responsible for hydrolysis of the primary degradation products of PHB (i.e., dimers and oligomers of 3HB) to 3HB. Interestingly, the action of the activator in the PHB depolymerase reaction could be replaced by slight trypsin treatment of nPHB. However, the activator was not a protease, and it triggered PHB granules by a mechanism different from that dmDNA31 of trypsin. Recently, the activator ApdA was purified (18) and its function was analyzed (17). It turned out that ApdA in in vivo is definitely a PHB-bound molecule with all the features of a phasin (44). With this study we continued our investigation of the depolymerase system by analysis of the soluble PHB depolymerase. MATERIALS AND METHODS Bacterial strains, plasmids, and tradition conditions. The bacterial strains and plasmids used in this study are outlined in Table ?Table1.1. was cultivated photoheterotrophically in PYI medium as explained recently (17, 18). Only small amounts of PHB were produced in this medium. For PHB production and isolation of PHB depolymerase, bacteria of a PYI culture were transferred (0.05 to 0.1 volumes).