title: An extracellular polyhydroxybutyrate depolymerase in Thermus thermophilus HB8.
abstract: The thermophilic bacterium Thermus thermophilus HB8 has been characterized as a polyhydroxybutyrate (PHB)-degrading microorganism since it grows efficiently and forms clear zones on agar plates containing PHB as sole carbon source. T. thermophilus extracellular PHB depolymerase was purified to homogeneity using an affinity chromatography protocol. The purified enzyme was estimated to have an apparent molecular mass of 42 kDa. The extracellular PHB depolymerase gene was identified as the TTHA0199 gene product of T. thermophilus HB8. The amino acid sequence of the TTHA0199 gene product shared significant homologies to other carboxylesterases. A catalytic triad was identified consisting of S(183), E(310), and H(405). A pentapeptide sequence (GX(1)SX(2)G) exists within the molecule, characteristic for PHB depolymerases (lipase box) and for other serine hydrolases. Purified extracellular PHB depolymerase was stable at high temperatures with an optimum activity at pH 8.0. The apparent Km value of the purified enzyme for PHB was 53 microg/ml. As the main product of the enzymic hydrolysis of PHB, the monomer 3-hydroxybutyrate was identified, suggesting that the enzyme acts principally as an exo-type hydrolase.
authors.affiliation: Department of Chemistry, Aristotle University of Thessaloniki, Greece.
authors.forename: Christos P
authors.initials: CP
authors.lastname: Papaneophytou
authors.forename: Anastasia A
authors.initials: AA
authors.lastname: Pantazaki
authors.forename: Dimitrios A
authors.initials: DA
authors.lastname: Kyriakidis
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D020134:Catalytic Domain; D002846:Chromatography, Affinity / Q000379:methods; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D006885:Hydroxybutyrates / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011091:Polyesters / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D000094122:Polyhydroxybutyrates
chemical_list: D001426:Bacterial Proteins; D002265:Carboxylic Ester Hydrolases; D006885:Hydroxybutyrates; D011091:Polyesters; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C022417:poly-beta-hydroxybutyrate depolymerase