Cloning of the gene for poly(3-hydroxybutyric acid) depolymerase of Comamonas testosteroni and functional analysis of its substrate-binding domain.
A poly(3-hydroxybutyric acid) (PHB) depolymerase gene of Comamonas testosteroni YM1004 was cloned on Sau3AI fragment from genomic DNA into Escherichia coli DH5. Nucleotide sequence analysis dedicated a 1539 bp open reading frame encoding a protein 513 amino acid with a putative 25 residue signal peptide for secretion. The deduced amino acid sequence was very similar to that of PHB depolymerase of Comamonas sp. In order to understand the characteristics of substrate-binding domain of the depolymerase, we constructed its glutathione S-transferase (GST) fusion protein and investigated the ability of adsorption on PHB single crystals by using gold-conjugated antibody and transmission electron microscopy. The fusion protein adsorbed on PHB single crystals tightly and homogeneously, suggesting that binding domain contributes to the adsorption of enzyme on solid PHB without site specificity.
Shinomiya
M
M
Polymer Chemistry Laboratory, Institute of Physical and Chemical Research (RIKEN), Saitama, Japan.
Iwata
T
T
Kasuya
K
K
Doi
Y
Y
D000595:Amino Acid Sequence; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis; D005982:Glutathione Transferase / Q000235:genetics; D016946:Gram-Negative Aerobic Rods and Cocci / Q000201:enzymology* / Q000235:genetics*; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D002265:Carboxylic Ester Hydrolases; C022417:poly-beta-hydroxybutyrate depolymerase