Purification of an extracellular D-(-)-3-hydroxybutyrate oligomer hydrolase from Pseudomonas sp. strain A1 and cloning and sequencing of its gene

Abstract

An extracellular D-(؊)-3-hydroxybutyrate oligomer hydrolase was purified from a poly(3-hydroxybutyrate)-degrading bacterium, Pseudomonas sp. strain A1. The purified enzyme hydrolyzed the D-(؊)-3-hydroxybutyrate dimer and trimer at similar rates. The enzyme activity was inhibited by a low concentration of diisopropylfluorophosphate. The molecular weight of the hydrolase was estimated to be about 70,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A 10-kbp DNA fragment of A1 was detected by hybridization with the gene (2 kbp) of an extracellular poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis. Subsequent subcloning showed that a SmaI-KpnI fragment (2.8 kbp) was responsible for expression of the hydrolase in Escherichia coli and an in vitro transcription-translation system. The expressed protein detected by immunostaining had the same molecular weight as the purified enzyme from A1. The protein band detected in the in vitro transcription-translation system had a molecular size of 72 kDa. The nucleotide sequence of the SmaI-KpnI fragment was determined, and one open reading frame (2,112 nucleotides) was found. It specifies a protein with a deduced molecular weight of 72,876 (704 amino acids). In this sequence, the consensus sequence of serine-dependent hydrolysis, G-X-S-X-G, did not exist.Poly-3-hydroxybutyrate (PHB) is a unique intracellular reserve of organic carbon and/or chemical energy found in a wide variety of bacteria (6, 9) and is regarded as a potential biodegradable thermoplastic not derived from petroleum (12). Recently similar bacterial polyesters have been found, and these polyesters including PHB were called "polyhydroxyalkanoates" (PHAs) (7). Extracellular PHB metabolism is important in the application of these bacterial polyesters as biodegradable plastics. Many microorganisms that are capable of degrading PHB exist in the environment, and they secrete PHB depolymerases to degrade this polymer. There have been many studies on the extracellular PHB depolymerases from several bacteria and fungi (2,3,4,11,15,22,26,29). Genes for some enzymes were also cloned and sequenced (10,19,21). Alcaligenes faecalis T1, a PHB-degrading bacterium, secretes a D-(Ϫ)-3-hydroxybutyric acid oligomer hydrolase (3HB-oligomer hydrolase) (EC 3. 1.1.22) (23) besides a PHB depolymerase. Although the physiological role of the 3HB-oligomer hydrolase is not clear, it may be important to study this hydrolase in relation to extracellular degradation of PHB. Similar enzymes were found within the bacterial cells (8,16,25). Pseudomonas sp. strain A1 was isolated from activated sludge as a PHB-degrading bacterium (22) and was found to have an extracellular 3HB-oligomer hydrolase besides a PHB depolymerase. In this communication, we describe the purification and characterization of the 3HB-oligomer hydrolase and cloning and sequencing of its gene. MATERIALS AND METHODSBacterial strains, plasmids, and culture. Pseudomonas sp. strain A1 was isolated from activated sludge of the sewage treatment facility of...

Cloning of an Intracellular Poly[d(−)-3-Hydroxybutyrate] Depolymerase Gene fromRalstonia eutrophaH16 and Characterization of the Gene Product

“…To hydrolyze the resulting 3HB oligomers, 0.05 U of 3HB oligomer hydrolase (31) was added, and the mixture was incubated at 30°C for 15 min and centrifuged. The 3HB in the supernatant fraction was measured enzymatically with 3HB dehydrogenase as described previously (31). One unit of the enzyme catalyzes the formation of 1 mol of 3HB per min under the assay conditions used.…”

“…After metal chelating purification, the protein was finally purified by a continuous elution sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (Prep cell model 491; Bio-Rad). The antibody against purified His-PHB depolymerase was prepared in rabbits as described previously (31).…”

Properties of a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase with High Specific Activity (PhaZd) in Wautersia eutropha H16

“…The amount of 3HB was measured by an enzymatic method using 3HB dehydrogenase and hydrazine hydrate as described previously (20). Hydrolytic activity toward p-nitrophenyl esters was determined spectrophotometrically and lipase activity was assayed by titration of the acid released from olive oil as described previously (45). To investigate the effect of reagents, they were added to the reaction mixture containing the enzyme, and then the mixture was incubated for 10 min at room temperature.…”

Novel Intracellular 3-Hydroxybutyrate-Oligomer Hydrolase in Wautersia eutropha H16

“…For 3HB-oligomer hydrolase activity, all 3HB oligomers were dissolved in distilled water at a concentration of 10 mM and used as substrates at a concentration of 2 mM instead of PHB granules. The rate of hydrolysis of p-nitrophenyl esters was determined spectrophotometrically, and lipase activity was assayed by titration of the acid released from olive oil, as described previously (33).…”