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Journal of Bacteriology, June 2003, p. 3485-3490, Vol. 185, No. 12
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.12.3485-3490.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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

Teruyuki Kobayashi,1,2 Mari Shiraki,1 Tomoko Abe,1 Akinori Sugiyama,1 and Terumi Saito1,2*

Laboratory of Molecular Microbiology, Department of Biological Sciences, Faculty of Science,1 Research Institute for Integrated Science, Kanagawa University, 2946 Tsuchiya, Hiratsuka, Kanagawa 259-1293, Japan2

Received 22 January 2003/ Accepted 25 March 2003

An intracellular 3-hydroxybutyrate (3HB)-oligomer hydrolase (PhaZ2Reu) of Ralstonia eutropha was purified from Escherichia coli harboring a plasmid containing phaZ2Reu. 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 (PhaZ1Reu). 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.

* Corresponding author. Mailing address: Laboratory of Molecular Microbiology, Department of Biological Sciences, Faculty of Science, Kanagawa University, 2946 Tsuchiya, Hiratsuka, Kanagawa 259-1293, Japan. Phone: 81-463-59-8673. Fax: 81-463-59-8673. E-mail: 43saito{at}bio.kanagawa-u.ac.jp.

Journal of Bacteriology, June 2003, p. 3485-3490, Vol. 185, No. 12
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.12.3485-3490.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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