Purification and properties of an intracellular 3-hydroxybutyrate-oligomer hydrolase (PhaZ2) in Ralstonia eutropha H16 and its identification as a novel intracellular poly(3-hydroxybutyrate) depolymerase.
An intracellular 3-hydroxybutyrate (3HB)-oligomer hydrolase (PhaZ2(Reu)) of Ralstonia eutropha was purified from Escherichia coli harboring a plasmid containing phaZ2(Reu). The purified enzyme hydrolyzed linear and cyclic 3HB-oligomers. Although it did not degrade crystalline poly(3-hydroxybutyrate) (PHB), the purified enzyme degraded artificial amorphous PHB at a rate similar to that of the previously identified intracellular PHB (iPHB) depolymerase (PhaZ1(Reu)). The enzyme appeared to be an endo-type hydrolase, since it actively hydrolyzed cyclic 3HB-oligomers. However, it degraded various linear 3HB-oligomers and amorphous PHB in the fashion of an exo-type hydrolase, releasing one monomer unit at a time. PhaZ2 was found to bind to PHB inclusion bodies and as a soluble enzyme to cell-free supernatant fractions in R. eutropha; in contrast, PhaZ1 bound exclusively to the inclusion bodies. When R. eutropha H16 was cultivated in a nutrient-rich medium, the transient deposition of PHB was observed: the content of PHB was maximized in the log growth phase (12 h, ca. 14% PHB of dry cell weight) and decreased to a very low level in the stationary phase (ca. 1% of dry cell weight). In each phaZ1-null mutant and phaZ2-null mutant, the PHB content in the cell increased to ca. 5% in the stationary phase. A double mutant lacking both phaZ1 and phaZ2 showed increased PHB content in the log phase (ca. 20%) and also an elevated PHB level (ca. 8%) in the stationary phase. These results indicate that PhaZ2 is a novel iPHB depolymerase, which participates in the mobilization of PHB in R. eutropha along with PhaZ1.
Kobayashi
Teruyuki
T
Laboratory of Molecular Microbiology, Department of Biological Sciences, Faculty of Science, Kanagawa University, 2946 Tsuchiya, Hiratsuka, Kanagawa 259-1293, Japan.
Shiraki
Mari
M
Abe
Tomoko
T
Sugiyama
Akinori
A
Saito
Terumi
T
D015153:Blotting, Western; D002265:Carboxylic Ester Hydrolases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D020118:Cupriavidus necator / Q000201:enzymology*; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D006885:Hydroxybutyrates / Q000378:metabolism; D002479:Inclusion Bodies / Q000378:metabolism; D009154:Mutation; D011091:Polyesters / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D013379:Substrate Specificity; D000094122:Polyhydroxybutyrates
D002265:Carboxylic Ester Hydrolases; D006885:Hydroxybutyrates; D011091:Polyesters; D011994:Recombinant Proteins; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C022417:poly-beta-hydroxybutyrate depolymerase; C032152:hydroxybutyrate-dimer hydrolase