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Journal of Bacteriology, January 2001, p. 94-100, Vol. 183, No. 1
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.1.94-100.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Cloning of an Intracellular Poly[D(-)-3-Hydroxybutyrate] Depolymerase Gene from Ralstonia eutropha H16 and Characterization of the Gene Product

Haruhisa Saegusa,1 Mari Shiraki,2 Chie Kanai,2 and Terumi Saito2,*

Research Institute of Innovative Technology for the Earth Branch in Kanagawa University1 and Laboratory of Molecular Microbiology, Department of Biological Sciences, Faculty of Science, Kanagawa University,2 2946 Tsuchiya, Hiratsuka, Kanagawa 259-1293, Japan

Received 14 August 2000/Accepted 10 October 2000

An intracellular poly[D(-)-3-hydroxybutyrate] (PHB) depolymerase gene (phaZ) has been cloned from Ralstonia eutropha H16 by the shotgun method, sequenced, and characterized. Nucleotide sequence analysis of a 2.3-kbp DNA fragment revealed an open reading frame of 1,260 bp, encoding a protein of 419 amino acids with a predicted molecular mass of 47,316 Da. The crude extract of Escherichia coli containing the PHB depolymerase gene digested artificial amorphous PHB granules and released mainly oligomeric D(-)-3-hydroxybutyrate, with some monomer. The gene product did not hydrolyze crystalline PHB or freeze-dried artificial amorphous PHB granules. The deduced amino acid sequence lacked sequence corresponding to a classical lipase box, Gly-X-Ser-X-Gly. The gene product was expressed in R. eutropha cells concomitant with the synthesis of PHB and localized in PHB granules. Although a mutant of R. eutropha whose phaZ gene was disrupted showed a higher PHB content compared to the wild type in a nutrient-rich medium, it accumulated PHB as much as the wild type did in a nitrogen-free, carbon-rich medium. These results indicate that the cloned phaZ gene encodes an intracellular PHB depolymerase in R. eutropha.

* 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: 81-463-59-4111. Fax: 81-463-58-9684. E-mail: 43saito{at}info.kanagawa-u.ac.jp.

Journal of Bacteriology, January 2001, p. 94-100, Vol. 183, No. 1
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.1.94-100.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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