Cloning and sequencing of an intracellular D(-)-3-hydroxybutyrate oligomer hydrolase from Acidovorax sp. strain SA1 and purification of the enzyme.
The gene of an intracellular D(-)-3-hydroxybutyrate oligomer hydrolase (i3HBOH) was cloned and sequenced from a poly(3-hydroxybutyrate) (PHB)-degrading bacterium, Acidovorax sp. strain SA1. The i3HBOH gene has 876 nucleotides corresponding to the deduced sequence of 292 amino acids. In this amino acid sequence, the general lipase box sequence (G-X(1)-S-X(2)-G) was found, whose serine residue was determined to the active sites serine by site-directed mutagenesis. An i3HBOH was purified to electrophoretical homogeneity from SA1. The molecular mass of the purified enzyme was estimated to be 32 kDa by SDS-PAGE. The N-terminal amino acid sequence of the purified enzyme corresponded to the deduced N-terminal amino acid sequence in the cloned i3HBOH gene. This is the first cloning and sequencing of an intracellular D(-)-3-hydroxybutyrate oligomer hydrolase gene to date.
Sugiyama
Akinori
A
Laboratory of Molecular Microbiology, Department of Biological Sciences, Faculty of Science, Kanagawa University, 2946 Tsuchiya, Hiratsuka, Kanagawa 259-1293, Japan.
Shiraki
Mari
M
Kobayashi
Teruyuki
T
Morikawa
Gakushi
G
Yamamoto
Mizuho
M
Yamaoka
Michiyo
M
Saito
Terumi
T
D020562:Betaproteobacteria / Q000201:enzymology* / Q000235:genetics; D015153:Blotting, Western; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D011091:Polyesters / Q000378:metabolism; D014176:Protein Biosynthesis; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
D011091:Polyesters; D002265:Carboxylic Ester Hydrolases; C032152:hydroxybutyrate-dimer hydrolase