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Applied and Environmental Microbiology, January 1999, p. 189-197, Vol. 65, No. 1
Graduate School of Science and Engineering,
Received 11 August 1998/Accepted 27 October 1998
The extracellular polyhydroxybutyrate (PHB) depolymerase gene
(phaZPst) of Pseudomonas stutzeri
was cloned and sequenced. phaZPst was composed
of 1,728 bp encoding a protein of 576 amino acids. Analyses of the
N-terminal amino acid sequence and the matrix-assisted laser
desorption/ionization-time-of-flight (MALDI-TOF) mass spectrum of the
purified enzyme showed that the mature enzyme consisted of 538 amino
acids with a deduced molecular mass of 57,506 Da. Analysis of the
deduced amino acid sequence of the protein revealed a domain structure
containing a catalytic domain, putative linker region, and two putative
substrate-binding domains (SBDI and SBDII). The putative linker region
was similar to the repeating units of the cadherin-like domain of
chitinase A from Vibrio harveyi and chitinase B from
Clostridium paraputrificum. The binding characteristics of
SBDs to poly([R]-3-hydroxybutyrate) [P(3HB)] and chitin
granules were characterized by using fusion proteins of SBDs with
glutathione S-transferase (GST). These GST fusion proteins
with SBDII and SBDI showed binding activity toward P(3HB) granules but
did not bind on chitin granules. It has been suggested that the SBDs of
the depolymerase interact specifically with the surface of P(3HB). In
addition, a kinetic analysis for the enzymatic hydrolysis of
3-hydroxybutyrate oligomers of various sizes has suggested that the
catalytic domain of the enzyme recognizes at least two monomeric units
as substrates.
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Cloning and Characterization of the
Polyhydroxybutyrate Depolymerase Gene of Pseudomonas
stutzeri and Analysis of the Function of
Substrate-Binding Domains
*
Corresponding author. Mailing address: Polymer
Chemistry Laboratory, The Institute of Physical and Chemical Research
(RIKEN), Hirosawa, Wako-shi, Saitama 351-0198, Japan. Phone:
81-48(467)9402. Fax: 81-48(462)4667. E-mail:
ydoi{at}postman.riken.go.jp.
Applied and Environmental Microbiology, January 1999, p. 189-197, Vol. 65, No. 1
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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