Partial purification and characterization of ?-hydroxybutyric acid dehydrogenase of a methylotrophic bacterium, Pseudomonas 135

Abstract

An intracellular enzyme, D(-)-fl-hydroxybutyric acid dehydrogenase involved in an intracellular poly-D(-)-fl-hydroxybutyric acid degradation was isolated from a facultative methylotrophic bacterium, P s e u d o m o n a s 135, grown on methanol as a sole carbon and energy source. This enzyme was partially purified to l l.6-fold by ammonium sulphate fractionation and a dye-affinity chromatography. The enzyme catalysed simultaneously the oxidation of D(-)-fl-hydroxybutyric acid (Dfl-HB) and the reduction of acetoacetate. The optimum pH was 8.5 for the oxidation reaction and 5.5-6.0 for the reduction reaction, and the enzyme was stable for 2 weeks at -20 ° C. The Km values for oxidation and reduction reactions were determined as 1.84 mM for Dfl-HB, 0.244 mM for NAD +, 0.319 mM for acetoacetate and 0.032 mM for NADH, respectively. It was also found that D-lactate and NADH significantly inhibited the oxidation reaction by competitive inhibition, and acetoacetate by non-competitive inhibition, respectively. The inhibition constants were determined as 1.49 mM for D-lactate, 0.196mM for NADH and 1.82mM for acetoacetate, respectively. According to an experiment with resting cells, it seemed that the enzyme was constitutive.

Molecular Basis of Substrate Recognition in D‐3‐Hydroxybutyrate Dehydrogenase from Pseudomonas putida

“…It is well known that D ‐lactate is a competitive inhibitor of D ‐3‐HBDH, so it was felt it might be useful to examine this compound for comparison. For the wild‐type enzyme from P. putida we determined an inhibition constant of 0.095 m M , a value about two to seven times lower than those reported for other HBDHs 13–15. The competitive inhibition mode was confirmed in our studies.…”

Heterologous expression, characterization and evolution prediction of a diaphorase from Geobacillus sp. Y4.1MC1

“…The quantitative analysis of acetone by gas chromatography can only determine the concentration of total ketones in serum that lack of speci city (Deng et al 2004). Nowadays, enzymatic method by diaphorase and β-hydroxybutyrate dehydrogenase has been developed (Daniel et al 1992). Under the speci c oxidation of β-hydroxybutyric acid dehydrogenase and NAD + , β-hydroxybutyric acid produces acetoacetic acid and NADH.…”

Accumulation of PHA and its copolyesters by Methylobacterium sp. KCTC 0048

“…It has been argued that methanol would appear to be an attractive substrate for P(3HB) production due to several advantages of low price, complete water miscibility, moderate requirement of oxygen, and so on (Byrom, 1987). Although a few methanol-utilizing bacteria were recently studied as PHA producers (Suzuki et al, 1986, Daniel et al, 1992, Ueda et al, 1992, little is known about methylotrophs having ability of producing the copolyesters other than P(3HB) or P(3HB-co-3HV). Recently, we observed the possibility of production of PHA and its copolyesters, P(3HB-co-3HV), P(3HB-co-4HB) and P(3HB-co-3HP) from various types of carbon substrates by a methylotroph isolated in our labaratory (Kang et al, 1992).…”