Biochemical and molecular characterization of the Pseudomonas lemoignei polyhydroxyalkanoate depolymerase system

Abstract

Two types of C-terminal sequences apparently represent substrate-binding sites; the PHB type is present in the PHB depolymerases of A. faecalis and P. pickettii and in PhaZ2, PhaZ3, and PhaZ5, and the PHV type is present in the PHV-hydrolyzing depolymerases (PhaZ4 and PhaZ1). phaZ1 was transferred to A. eutrophus H16 and JMP222. All transconjugants of both strains were able to grow with extracellular PHB as a carbon source and produced translucent halos on PHB-containing solid media. PhaZ1, PhaZ2, PhaZ4, and PhaZ5 were purified from P. lemoignei and from recombinant E. coli; the processing sites of the precursors in E. coli were the same as in P. lemoignei, and similar substrate specificities were determined for the wild-type and the recombinant proteins. All PHA depolymerases hydrolyzed PHB at high specific activities. PhaZ1 and PhaZ4 additionally cleaved PHV, and PhaZ4 hydrolyzed poly(4-hydroxybutyrate). None of the depolymerases was able to hydrolyze polylactide or PHA consisting of monomers with more than five carbon atoms. While the wild-type depolymerase proteins were glycosylated and found to contain glucose and N-acetylglucosamine, none of the recombinant proteins was glycosylated. PHB hydrolysis was dependent on divalent cations such as Ca 2؉ and was inhibited by the presence of EDTA.

Degradation of polyesters by a novel marine Nocardiopsis aegyptia sp. nov.: Application of Plackett-Burman experimental design for the improvement of PHB depolymerase activity

“…This led to the suggestion that the N. aegyptia PHB depolymerase is constitutive. Our results are in contrast to all other known PHB bacterial depolymerases, which are repressed in the presence of a soluble carbon source (Jendrossek et al, 1995). In accordance with our results are those reported for Pseudomonas lemoignei (Nakayama et al, 1985;Steinbüchel and Hein, 2001;Tomasi et al, 1996), which produced polymerase maximally during growth on succinate.…”

Bacterial polyhydroxyalkanoates-eco-friendly next generation plastic: Production, biocompatibility, biodegradation, physical properties and applications

“…Whereas most PHA-degrading bacteria analyzed so far apparently produced only one PHA depolymerase, Pseudomonas lemoignei was found to have at least five PHA depolymerases (199). The structural genes of several PHA depolymerases have been cloned and sequenced (197,(200)(201)(202). Biochemical analysis of the purified depolymerase proteins and analysis of the DNA-deduced amino acid sequences revealed that PHA depolymerases apparently possess a catalytic triad consisting of serine, histidine and aspartate residues which have been demonstrated to form the active site in bacterial lipases (203).…”

Biochemical and molecular characterization of a periplasmic hydrolase for oxidized polyvinyl alcohol from Sphingomonas sp. strain 113P3

“…The amino acid sequences of the OPHs from strain 113P3 and strain VM15C were aligned by CLUSTAL W (Thompson et al, 1994) with the hypothetical PHB depolymerases from Mesorhizobium loti, Rhizobium sp. and Sinorhizobium meliloti strain 1021, to which the OPH from strain 113P3 showed homology (29-32 % identity) and the well-characterized PHB depolymerases from Pseudomonas lemoignei (PhaZ1 to PhaZ5; Jendrossek et al, 1995b) (Fig. 3).…”