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(-)-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 purified PhaZ2 and found that the purified enzyme could degrade amorphous PHB . We describe here some of the properties of PhaZ2 and its role in PHB degradation in R . eutropha .

 
Bacterial strains, plasmids, and culture. The bacterial strains and plasmids used in the present study are listed in Table 1 . All Escherichia coli strains were grown in Luria-Bertani medium . E . coli BLR(DE3)/pLysS was used as the host cell for the recombinant plasmids carrying phaZ1 and phaZ2 . E . coli S17-1(pir) was used for mobilization of suicide vector into R . eutropha. All R . eutropha strains were grown in nutrient-rich medium as described previously (17) . For the selection and maintenance of mutants, nutrient-rich media were supplemented with antibiotics . The final concentrations were 50 µg/ml for kanamycin and 20 µg/ml for ampicillin . To produce PHB, R . eutropha cells grown on a nutrient-rich medium were transferred to a nitrogen-free mineral medium containing 2% (wt/vol) fructose and were cultured for 3 days as described previously (17) .

 
Construction of R . eutropha mutants. A part of phaZ2 (306 bp, PstI fragment) was inserted into a suicide vector, kanamycin-resistant pJP5603, which can replicate in E . coli but not in R . eutropha . Using the plasmid (pJPPOH), a phaZ2-null mutant (OH1) was constructed from H16, and a double-null mutant lacking both phaZ1and phaZ2 (DO1) was constructed from H16-SK1544 as described previously (16, 17, 26) . These mutants were confirmed based on antibiotic susceptibility, Southern blotting, and Western blotting .

Purification of PhaZ1 and PhaZ2 from E . coli. E . coli BLR(DE3)/pLysS was transformed with pET171H to obtain His-tagged PhaZ1 . The transformed cells were grown in Luria-Bertani medium with ampicillin (50 µg/ml), tetracycline (12.5 µg/ml), and chloramphenicol (34 µg/ml) at 37°C with vigorous shaking . At an A₆₀₀ of 0.6, the culture temperature was reduced to 22°C, and the protein expression was induced with IPTG (isopropyl-ß-D-thiogalactopyranoside) at a final concentration of 10 µM . The cultures were incubated overnight . All subsequent procedures were carried out at 4°C . Bacteria were harvested by centrifugation, and the cells were suspended in 20 mM Tris-HCl (pH 8.0) . The resuspended cells were sonicated on ice for 4 min (20 kHz, 30 W) and centrifuged at 10,000 x g for 30 min .

The supernatant from E . coli harboring pET171H was applied to a cochelating column (1 ml) and washed with 20 mM sodium phosphate (pH 7.4) containing 20% glycerol and 0.5 M NaCl . PhaZ1 was eluted with a linear gradient of imidazole (total volume, 15 ml; 0 to 500 mM), and the purified enzyme was dialyzed against 10 mM Tris-HCl (pH 8.0) containing 50% glycerol overnight .

To produce PhaZ2, pETOH that was constructed from phaZ2 with its own stop codon was used . Cultivation conditions of the transformed E . coli were similar to those for E . coli harboring pET171H except for when 1 mM IPTG was used . The supernatant from E . coli harboring pETOH was applied to a Toyopearl DEAE-650M (Tosoh, Tokyo, Japan) column (1.5 by 23 cm) and washed with 10 mM Tris-HCl (pH 8.0) . PhaZ2 was eluted with a linear gradient of NaCl (total volume, 220 ml; 0 to 0.2 M) . The eluted enzyme was mixed with (NH₄)₂SO₄ (final concentration, 1 M) and applied to a Toyopearl Butyl-650M (Tosoh, Tokyo, Japan) column (1.5 by 7 cm) equilibrated with 20 mM Tris-HCl (pH 8.0) containing 1 M (NH₄)₂SO₄ . The column was washed with the equilibrium buffer and 20 mM Tris-HCl (pH 8.0) containing 0.5 M (NH₄)₂SO₄, and then the enzyme was eluted with a linear gradient of (NH₄)₂SO₄ (total volume of 80 ml, 0.5 to 0.1 M) . The eluted enzyme mixed with (NH₄)₂SO₄ (final concentration, 1 M) was applied to a phenyl-Sepharose HP column (1 ml) equilibrated with 50 mM sodium phosphate (pH 7.0) containing 1 M (NH₄)₂SO₄ . The column was washed with the equilibrium buffer and 50 mM sodium phosphate (pH 7.0) containing 0.25 M (NH₄)₂SO₄, and the enzyme was eluted with a linear gradient of (NH₄)₂SO₄ (total volume of 13 ml, 0.25 to 0 M) . The eluted enzyme was dialyzed against 10 mM Tris-HCl (pH 8.0) overnight .

Enzyme assays. For PHB depolymerase activity, the released 3HB was routinely assayed by the enzymatic method by using R(-)-3HB dehydrogenase and hydrazine hydrate (30) . The reaction mixture (100 µl) was composed of 100 mM Tris-HCl (pH 8.5), artificial amorphous PHB granules (1 mg/ml as a solid), and enzyme . The reaction was started by the addition of substrate at 30°C . The reaction was stopped by the addition of 6 M HCl to pH 2, followed by boiling for 5 min . The reaction mixture was centrifuged at 15,000 x g for 10 min . The supernatant fraction was used for the quantification of 3HB . To quantify 3HB-oligomers in the supernatant fraction, they were first completely hydrolyzed by 0.05 U of extracellular 3HB-oligomer hydrolase from Ralstonia pickettii (previously known as Alcaligenes faecalis) T1 (25) . For 3HB-oligomer hydrolase activity, 3HB-oligomers (10 mM) were used as substrates instead of artificial amorphous PHB granules . The K_m and V_max values of 3HB-oligomer hydrolase were measured with automatic titration equipment (pH stat) (25) . The assay mixture comprised 3HB-oligomers and 3HB-oligomer hydrolase in double-distilled water . The reaction was started by the addition of enzyme . The released free acid was titrated with 5 mM NaOH (pH 8.5) at 30°C under a nitrogen atmosphere . The linear 3HB-oligomers were prepared as described previously (29) . The cyclic 3HB-oligomers were obtained from D . Seebach (21) . Artificial PHB granules and 3HB-oligomers used were stable at pH 8.5 for at least 30 min, and these esters were also stable upon treatment with 5 min of boiling at pH 2 . R(-)-3HB dehydrogenase activity was assayed by measuring the increase of NADH as described previously (15) . Artificial PHB granules were prepared according to a previous report (17) except that sodium deoxycholate was used instead of sodium oleate .

Sucrose density gradient. The wild-type cells of R . eutropha grown in PHB-accumulating conditions were collected and resuspended in 5 volumes of 50 mM Tris-HCl (pH 7.5) . The suspended cells were sonicated on ice for 10 min (20 kHz, 40 W) and centrifuged at 500 x g for 10 min . The supernatant (1 ml) was added on a linear sucrose gradient (10 ml), which was prepared from 1 to 2 M sucrose . After centrifugation for 4 h at 60,000 x g and 4°C, 1.1 ml of each fraction was collected .

Immunoblot analysis. Samples of sucrose density gradient fractions were subjected to immunoblot analysis according to standard procedures with a 1:5,000 dilution of rabbit antisera against PhaZ1, PhaZ2, or poly(3-hydroxyalkanoate) (PHA) synthase as a primary antiserum and alkaline phosphatase-conjugated goat anti-rabbit antisera as a secondary antibody . PHA synthase used to produce antiserum was purified from E . coli harboring phaC of R . eutropha according to the methods described by Gerngross et al . (4) . The immunocomplex was visualized by using nitroblue tetrazolium and BCIP (5-bromo-4-chloro-3-indolylphosphate) p-toluidine .

Other methods. Protein concentrations were measured by the method of Lowry et al . (11) with bovine serum albumin as the standard . The purified proteins were examined for purity and size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described by Laemmli (10) . Proteins on the gel were stained with Coomassie brilliant blue R250 . The PHB content was measured by gas chromatography as described previously (17) .

 
Enzyme purification. PhaZ2 was purified from E . coli BLR(DE3)/pLysS harboring phaZ2 of R . eutropha with a 13% yield (Table 2) . The final preparation showed apparent homogeneity upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 78 kDa (data not shown) . The chemically determined amino acid sequences at the N terminus of the purified enzyme corresponded to those deduced from the nucleotide sequence of phaZ2 (18) (data not shown) .

 
Substrate specificity. The substrate specificity of the purified enzyme was determined (Table 3) . The purified enzyme hydrolyzed a linear 3HB-dimer, trimer, tetramer, and pentamer at similar efficiency as judged from values of V_max/K_m . These data indicate that PhaZ2 acts on these 3HB-oligomers in an exo-fashion . This was confirmed by the product analyses (data not shown) . The purified enzyme, however, also degraded efficiently all cyclic 3HB-oligomers examined . Although PhaZ2 did not hydrolyze crystalline PHB, it hydrolyzed artificial amorphous PHB granules at a similar rate to PhaZ1 (Fig . 1A) . The specific activity assessed from the initial release of 3HB was ca . 0.1 µmol/min/mg, which is much smaller than the values obtained for 3HB-oligomers . PhaZ2 released only 3HB from amorphous PHB, although PhaZ1 produced mainly 3HB-oligomers from amorphous PHB as described previously (17) . Some synergistic effect of PhaZ2 on the degradation of PHB by PhaZ1 was also observed under certain conditions (Fig . 1B and C) . When PhaZ2 was added to the reaction mixture consisting of 3 µg of PhaZ1, the synergistic effect of PhaZ2 was optimal (Fig . 1B) . A combination of 3 µg of PhaZ1 and 0.1 µg of PhaZ2 enhanced the release of 3HB with incubation time (Fig . 1C) .

 
Subcellular localization of PhaZ2. The subcellular localization of PhaZ2 in R . eutropha was examined by sucrose density gradient centrifugation (Fig . 2) . PhaZ1 was found only in the fractions containing PHB, as judged from the intensity of immunostained bands, and seemed to localize solely in PHB granules in accordance with a previous report (17) . PHB depolymerase activity, however, was found both in the granule fractions and in the soluble fractions, which indicates the existence of another PHB depolymerase in addition to PhaZ1 . PhaZ2 was found both in the granules and in the soluble fractions . The activity of 3HB dehydrogenase used as a control was detected only in the supernatant fraction of the cells .

 
From the intensity of the immunostained bands, the molecules ratio PhaZ1, PhaZ2, and PHA synthase in PHB inclusion bodies was estimated to be 2.7:1:500 (data not shown) .

PHB accumulation in the phaZ-null mutants. Since PhaZ2 was located in PHB granules and hydrolyzed amorphous PHB in vitro, the effect of mutation of PhaZ2 on the accumulation of PHB in vivo was investigated (Fig . 3) . Strain D1 lacking PhaZ1, strain OH1 lacking PhaZ2, and strain DO1 lacking both PhaZ1 and PhaZ2 equally revealed similar growth in a nutrient-rich medium (Fig . 3A) . When R . eutropha (wild-type) was cultivated in the nutrient-rich medium, a transient PHB accumulation was observed (Fig . 3B) . The content of PHB was maximized in the log phase of growth (12 h, ca . 14% of dry cell weight) and then decreased to a low level (ca . 1%) in the stationary phase . In D1(phaZ1) and OH1(phaZ2), the PHB content in the stationary phase increased to ca . 5%, although the content in the log phase was little affected . The double-null mutant, DO1(phaZ1 phaZ2), increased the maximum amount of PHB in the log phase (ca . 20%) and showed a much elevated PHB content in the stationary phase (ca . 8%) compared to the wild type . Similar results were obtained when the total amounts of PHB in culture was plotted (Fig . 3C) .

 
In the present study, PhaZ2 was purified apparently homogenously from recombinant E . coli, and the kinetic properties of the purified enzyme were examined . PhaZ2 efficiently hydrolyzed all linear and cyclic 3HB-oligomers examined regardless of their sizes . The enzyme seems to hydrolyze linear oligomers such as an exo-type hydrolase, although it can degrade cyclic 3HB-oligomers . Therefore, it utilizes, like Aspergillus fumigatus extracellular PHB depolymerase, both endo and exo modes of hydrolysis (22) . The kinetic properties toward linear 3HB-oligomers were very similar to those of the extracellular 3HB-oligomer hydrolase from R . pickettii T1, which was concluded to be an exo-type hydrolase (25) . Since PhaZ2 can degrade amorphous PHB as efficiently as PhaZ1 (Fig . 1), PhaZ2 should be regarded as a novel iPHB depolymerase .

PhaZ2 localized both in the supernatant fraction and in PHB granules . It is reasonable to consider PhaZ2 a component of PHB granules . Immunostain analysis showed that the amount of PhaZ1 or PhaZ2 in PHB granules was very small compared with that of PHA synthase . Kawaguchi et al . estimated the amount of PHA synthase at 18,000 molecules in an R . eutropha cell (8) . Using this value, PhaZ1 and PhaZ2 in PHB granules were determined to be 97 and 36 molecules per cell, respectively . From its behavior on Toyopearl Butyl-650M chromatograpy, PhaZ2 is probably not very hydrophobic . Whether PhaZ2 binds directly to PHB in the granules or binds to the granules by protein-protein interaction remains to be solved . The localization of PhaZ2 in PHB granules is very important for PHB degradation in R . eutropha . Since the major products of the degradation of amorphous PHB by PhaZ1 are 3HB-oligomers, PhaZ2, which has a broad substrate specificity for 3HB oligomers of various lengths, probably has an important role in degrading the resulting 3HB-oligomers to monomers . Colocalization of PhaZ1 and PhaZ2 in PHB granules ensures a rapid degradation of PHB in vivo . The PhaZ2-null mutants increased the content of PHB in R . eutropha cells, a finding which again confirms the important role of PhaZ2 in the mobilization of PHB in vivo . Since even in a mutant lacking both PhaZ1 and PhaZ2, which showed elevated PHB accumulation both in the growth phase in nutrient-rich medium a considerable decrease of PHB was observed (Fig . 3), the existence of another depolymerase is suggested .

It is interesting that R . eutropha accumulates PHB in the log phase and then degrades it quickly in the nutrient-rich medium . The synthesis and degradation of PHB seem to occur simultaneously, since the double mutant showed an explicit increased PHB deposition in the log phase . Such a turnover of PHB was already pointed out (2, 8, 28) . In view of economy, a quick turnover of PHB seems to be a disadvantage to bacteria . The rapid metabolism of sugar through glycolysis in the presence of rich nutrients, however, probably elevates NAD(P)H/NAD(P)⁺ ratio in the cell, and the high ratio would inhibit the tricarboxylic acid cycle (27) . It is possible that the accumulation of PHB works as a electron sink to enhance the tricarboxylic acid cycle via reduction of the NAD(P)H/NAD(P)⁺ ratio, since rapid growth likely to be a primary objective for bacteria . Therefore, the accumulation of PHB in the log phase in R . eutropha seems to be a physiological phenomenon for bacterial growth as described in Azobobacter beijerinkii (23, 24) . Futile cycling of glycogen in Fibrobacter succinogenes was also reported (3) .

The regulation of phaZ is important in the mobilization of PHB in R . eutropha . PhaZs in R . eutropha seemed to work in the growth phase but not the stationary phase in the N-rich medium (Fig . 3) . The synthesis of PhaZ1 and PhaZ2 stringently correlates with the production of PHB (17, 18) . Certain factors are probably involved at the transcriptional, translational, or posttranslational level (9) and remain to be eclucidated .

The steps for in vivo degradation of PHB appear to be as follows . (i) PhaZ1 or PhaZ2 makes several nicks in the chains of amorphous PHB molecules . (ii) As a result, medium sized 3HB-oligomers that still bind to the granules due to their hydrophobicity, some loosened 3HB ends of PHB chains protruding from the granules, and a small amount of 3HB monomer/short-chain 3HB-oligomers that diffuse from the granules are produced . (iii) PhaZ2 degrades 3HB-oligomers on PHB granules and the loosened ends of amorphous PHB chains to 3HB in an exo-fashion . (iv) Finally, PhaZ2 localized in the cytosol hydrolyzes the diffused 3HB-oligomers .

The participation of some unknown hydrolase(s) in PHB degradation was suggested by the findings of the present study . Certainly, this point should be clarified further .

 
This study was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (Sustainable Biodegradation Plastics, no . 11217214 [1999]) and a Grant-in-Aid for the High-Tech Research Center Project from the Ministry of Education, Culture, Sports, Science, and Technology of Japan .

We thank D . Seebach, ETH-Zentrum, Zurich, Switzerland, and D . Jendrossek of the Institüte für Mikrobiologie der Universität Stuttgart in Germany for the linear and cyclic 3HB-oligomers and for mutant H16-SK, respectively .

 
* Corresponding author . Mailing address: Laboratory of Molecular Microbiology, Department of Biological Sciences, Faculty of Science, Kanagawa University, 2946 Tsuchiya, Hiratsuka, Kanagawa 259-1293, Japan . Phone: 81-463-59-8673 . Fax: 81-463-59-8673 . E-mail: 43saito@bio.kanagawa-u.ac.jp.

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