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.

FIG. 1.

Degradation of artificial amorphous PHB granules. (A) PHB degradation by PhaZ1 (•, 3HB monomer; ○, 3HB monomer plus oligomers) or PhaZ2 (▵, 3HB monomer) over 30 min. (B) PHB degradation by PhaZ2 over 30 min. Each curve contained PhaZ2 and various amounts of PhaZ1 (○, 0 μg; □, 1.5 μg; ▵, 3 μg; ⋄, 6 μg). (C) Time course of PHB degradation. Symbols: ⋄, PhaZ2 (0.1 μg); □, PhaZ1 (3 μg); ▵, PhaZ1 (3 μg) and PhaZ2 (0.1 μg). The released 3HB was measured enzymatically. Error bars (standard deviations of the mean) were derived from three independent measurements.

FIG. 2.

Subcellular localization of PhaZ2 by sucrose density gradient (total 11 ml). Each fraction (1.1 ml) was collected, and several amounts were measured. Symbols: ○, PHB depolymerase activity; •, oligomer hydrolase activity; □, 3-hydroxubutyrate dehydrogenase activity; ▵, PHB; ⋄, protein. Each fraction was analyzed by Western blotting and immunostaining with antiserum against PhaZ1 or PhaZ2. For PHB depolymerase and 3HB-oligomer hydrolase activity, the released 3HB was measured enzymatically.

FIG. 3.

Growth and accumulation of PHB in various R. eutropha phaZ-null mutants in the nutrient-rich medium. (A) A₆₀₀; (B) accumulation of PHB (percentage of dry cell weight); (C) accumulation of PHB (mg/ml of culture). Symbols: ○, wild-type; ▵, D1(ΔphaZ1); □, OH1(ΔphaZ2); ⋄, DO1(ΔphaZ1 ΔphaZ2). The results are the means for three independent measurements. The range of error was within ±5% of each value.