Cloning of poly(3-hydroxybutyrate) depolymerase from a marine bacterium, Alcaligenes faecalis AE122, and characterization of its gene product.
A DNA fragment that carries the gene coding for poly(3-hydroxybutyrate) (PHB) depolymerase was cloned from the chromosomal DNA of Alcaligenes faecalis AE122 isolated from seawater. The open reading frame encoding the precursor of the PHB depolymerase was 1905 base pairs (bp) long, corresponding to a protein of 635 amino acid residues (M(r) = 65,208). The promoter site, which could be recognized by Escherichia coli RNA polymerase, was upstream from the gene, and the sequence adhering to the ribosome-binding sequence was found in front of the gene. The deduced amino acid sequence agreed with the N-terminal amino acid sequence of the purified PHB depolymerase from amino acid 28 onwards. Analysis of the deduced amino acid sequence revealed the domain structure of the protein; a signal peptide of 27 amino acids long was followed by a catalytic domain of about 400 amino acids, a fibronectin type III module sequence, and a putative substrate binding domain. The molecular mass (62,526) of the mature protein deduced from the nucleotide sequence was significantly lower than the value (95 kDa) estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but coincided well with the value (62,426) estimated from matrix-assisted laser desorption ionization mass spectra. By comparison of the primary structure with those of other PHB depolymerases, the substrate binding domain was found to consist of two domains, PHB-specific and poly(3-hydroxyvalerate)-specific ones, connected by a linker region. The PHB depolymerase gene was expressed in Escherichia coli under the control of the tac promoter. The enzyme expressed in E. coli was purified from culture broth and showed the same catalytic properties as the enzyme from A. faecalis.
Kita
K
K
Department of Biotechnology, Tottori University, Koyama, Japan. kita@bio.tottori-u.ac.jp
Mashiba
S
S
Nagita
M
M
Ishimaru
K
K
Okamoto
K
K
Yanase
H
H
Kato
N
N
D000421:Alcaligenes / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016415:Sequence Alignment
D004247:DNA; D002265:Carboxylic Ester Hydrolases; C022417:poly-beta-hydroxybutyrate depolymerase