title: Biochemical and genetic characterization of a D(-)-3-hydroxybutyrate dehydrogenase from Acidovorax sp. strain SA1.
abstract: D(-)-3-hydroxybutyrate dehydrogenase (BDH; EC 1.1.1.30) from a poly(D(-)-3-hydroxybutyrate) (PHB) degrading bacterium, Acidovorax sp. SA1, was purified using Toyopearl DEAE-650M, red-Sepharose CL-4B, and Q Sepharose FF. The molecular mass of the enzyme was estimated as 27 kDa by SDS-PAGE and 110 kDa by gel filtration. The gene encoding BDH was cloned and sequenced, and expressed in Escherichia coli. The gene product was purified in two steps with a high yield. The N-terminal amino acid sequence of the enzyme purified from E. coli agreed with that of the purified enzyme from strain SA1. The BDH of strain SA1 had high amino acid sequence homology to that of Ralstonia eutropha H16. The Km values for D(-)-3-hydroxybutyrate and NAD+ in the oxidation reaction were 4.5 x 10(-4) M and 8.9 x 10(-5) M, respectively. The Km values for acetoacetate and NADH in the reduction reaction were 2.4 x 10(-4) M and 2.9 x 10(-5) M, respectively.
authors.affiliation: Department of Biological Sciences, Faculty of Science, Kanagawa University, 2946 Tsuchiya, Hiratsuka, Kanagawa 259-1293, Japan.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Takanashi
authors.forename: Tadashi
authors.initials: T
authors.lastname: Shibahara
authors.forename: Mari
authors.initials: M
authors.lastname: Shiraki
authors.forename: Terumi
authors.initials: T
authors.lastname: Saito