Purification and Properties of an Intracellular 3-Hydroxybutyrate-Oligomer Hydrolase (PhaZ2) in Ralstonia eutropha H16 and Its Identification as a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase

Abstract

An intracellular 3-hydroxybutyrate (3HB)-oligomer hydrolase (PhaZ2 Reu ) of Ralstonia eutropha was purified from Escherichia coli harboring a plasmid containing phaZ2 Reu . The purified enzyme hydrolyzed linear and cyclic 3HB-oligomers. Although it did not degrade crystalline poly(3-hydroxybutyrate) (PHB), the purified enzyme degraded artificial amorphous PHB at a rate similar to that of the previously identified intracellular PHB (iPHB) depolymerase (PhaZ1 Reu ). The enzyme appeared to be an endo-type hydrolase, since it actively hydrolyzed cyclic 3HB-oligomers. However, it degraded various linear 3HB-oligomers and amorphous PHB in the fashion of an exo-type hydrolase, releasing one monomer unit at a time. PhaZ2 was found to bind to PHB inclusion bodies and as a soluble enzyme to cell-free supernatant fractions in R. eutropha; in contrast, PhaZ1 bound exclusively to the inclusion bodies. When R. eutropha H16 was cultivated in a nutrient-rich medium, the transient deposition of PHB was observed: the content of PHB was maximized in the log growth phase (12 h, ca. 14% PHB of dry cell weight) and decreased to a very low level in the stationary phase (ca. 1% of dry cell weight). In each phaZ1-null mutant and phaZ2-null mutant, the PHB content in the cell increased to ca. 5% in the stationary phase. A double mutant lacking both phaZ1 and phaZ2 showed increased PHB content in the log phase (ca. 20%) and also an elevated PHB level (ca. 8%) in the stationary phase. These results indicate that PhaZ2 is a novel iPHB depolymerase, which participates in the mobilization of PHB in R. eutropha along with PhaZ1.Poly(3-hydroxybutyrate) (PHB), a homopolymer of R(Ϫ)-3-hydroxybutyrate (3HB), is a storage material produced by some bacteria under certain conditions (1). In the past few decades, the application of this biopolymer to biodegradable polymers or plastics has been studied extensively (12). In these studies, the extracellular metabolism of PHB has been clarified in many bacteria and some fungi (6, 7). However, only a few studies on the intracellular degradation of PHB have been published (13,14,17,19,20). An intracellular PHB (iPHB) depolymerase system in Rhodospirillum rubrum was first reported in 1964 and consisted of a thermostable activator and a thermolabile esterase (13). This system is still not well understood in spite of a recent reinvestigation (14). The molecular cloning of an iPHB depolymerase from Ralstonia eutropha H16 has been also reported (17). This enzyme (PhaZ1 Reu ) degraded artificial amorphous PHB granules but not crystalline PHB. A mutant lacking PhaZ1 Reu showed a higher PHB content compared to the wild-type in a nutrient-rich medium, but in this mutant the mobilization of PHB was not inhibited completely, suggesting that the cloned depolymerase gene is not the only gene responsible for the biodegradation of PHB in this bacterium (5, 17). In regard to this point, recently we found another esterase (PhaZ2 Reu ) that hydrolyzes 3HB-oligomers and cloned its gene (18). We examined the properties of the ...

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

“…PhaZa1 Weu (formerly PhaZ1 Reu ) exists in PHB inclusion bodies and degrades artificial amorphous PHB (20,30). The existence of PhaZa1 Weu homologs has also been reported in W. eutropha H16 (29,44).…”

“…In Wautersia eutropha (formerly Ralstonia eutropha) H16, three types of intracellular PHB depolymerase or 3HB oligomer hydrolase genes have been cloned and some features of the gene products have been reported (19,20,21,31,32). PhaZa1 Weu (formerly PhaZ1 Reu ) exists in PHB inclusion bodies and degrades artificial amorphous PHB (20,30).…”

“…Additionally, enzymes with properties similar to PhaZa1 Weu have been studied in other bacteria (6,18). PhaZb Weu (formerly PhaZ2 Reu ) (20,32) and PhaZc Weu (19) mainly degrade 3HB oligomers and the degradation product is 3HB monomer. Another PHB depolymerase or related hydrolase in W. eutropha H16 with different properties from these enzymes has been reported (35), but it has not been fully characterized.…”

“…The reaction mixture (50 l) was composed of 100 mM Tris-HCl (pH 8.5), PHB granules (0.5 mg/ml as a solid), and enzyme. The reaction was started by the addition of substrate at 30°C (20). To quantify the amount of 3HB oligomer released from PHB by the enzyme, the supernatant of the reaction mixture was treated with 1.1 U (3HB dimer-hydrolyzing activity) of 3HB oligomer hydrolase from W. eutropha H16 (PhaZb Weu ) (20,32).…”

Production, optimization and characterization of polyhydroxybutyrate, a biodegradable plastic by Bacillus spp.

“…8) However, PHB possesses many deficiencies to be used as a practical polymer material such as high crystallinity and thermal instability near the melting point. 9) Thus, physical blending and chemical modifications have been carried out to improve its properties such as strength, film formation, mechanical strength, amphilicity, biodegradability, and biocompatibility. 10) Though much advancement has taken place regarding the microbial biosynthesis of PHB, industrial-scale production and application are still at far away from reality.…”