Degradation of poly (3-hydroxybutyrate) and its copolymer poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by a marine Streptomyces sp. SNG9.

A marine Streptomyces sp. SNG9 was characterized by its ability to utilize poly(3-hydroxybutyrate) (PHB) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate P (3HB-co-HV). The bacterium grew efficiently in a simple mineral liquid medium enriched with 0.1% poly(3-hydroxybutyrate) powder as the sole carbon source. Cells excreted PHB depolymerase and degraded the polymer particles to complete clarity in 4 days. The degradation activity was detectable by the formation of a clear zone around the colony (petri plates) or a clear depth under the colony (test tubes). The expression of PHB depolymerase was repressed by the presence of simple soluble carbon sources. Bacterial degradation of the naturally occurring sheets of poly(3-hydroxybutyrate) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was observed by scanning electron microscopy (SEM). Morphological alterations of the polymers sheets were evidence for bacterial hydrolysis.     

Degradation of poly (3-hydroxybutyrate) and its copolymer poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by a marine Streptomyces sp. SNG9

A marine Streptomyces sp. SNG9 was characterized by its ability to utilize poly(3-hydroxybutyrate) (PHB) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate P (3HB-co-HV). The bacterium grew efficiently in a simple mineral liquid medium enriched with 0.1% poly(3-hydroxybutyrate) powder as the sole carbon source. Cells excreted PHB depolymerase and degraded the polymer particles to complete clarity in 4 days. The degradation activity was detectable by the formation of a clear zone around the colony (petri plates) or a clear depth under the colony (test tubes). The expression of PHB depolymerase was repressed by the presence of simple soluble carbon sources. Bacterial degradation of the naturally occurring sheets of poly(3-hydroxybutyrate) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was observed by scanning electron microscopy (SEM). Morphological alterations of the polymers sheets were evidence for bacterial hydrolysis.     

Structure and enzymatic degradation of poly(3-hydroxybutyrate) copolymer single crystals with an extracellular PHB depolymerase from Alcaligenes faecalis T1.

Lamellar single crystals of four random copolymers of (R)-3-hydroxybutyrate with different hydroxyalkanoates: poly(3-hydroxybutyrate-co-8 mol%-3-hydroxyvalerate) (P(3HB-co-8%-3HV)), poly(3-hydroxybutyrate-co-10 mol%-4-hydroxybutyrate) (P(3HB-co-10%-4HB)), poly(3-hydroxybutyrate-co-8 mol%-3-hydroxyhexanoate) (P(3HB-co-8%-3HH)) and poly(3-hydroxybutyrate-co-10 mol%-6-hydroxyhexanoate) (P(3HB-co-10%-6HH)), were grown from dilute solutions of chloroform and ethanol. All single crystals have lath-shaped morphology and the second monomer units seem to be excluded from the P(3HB) crystal, on the basis of the electron diffraction diagrams. The enzymatic degradation of P(3HB-co-8%-3HH) and P(3HB-co-10%-6HH) single crystals was investigated with an extracellular PHB depolymerase from Alcaligenes faecalis T1. Adsorption of an extracellular PHB depolymerase, examined using an immuno-gold labelling technique, demonstrated a homogeneous distribution of enzyme molecules with a low concentration on the crystal surfaces. Enzymatic degradation of single crystals progressed from the edges and ends of crystals to yield narrow cracks along their long axes and the small crystal fragments. Lamellar thicknesses of single crystals and molecular weights of copolymer chains remained unchanged during the enzymatic hydrolysis. The above results support the hypothesis that the hydrophobic adsorption of the enzyme contributes to increase the mobility of molecular chains of single crystals and generate the disordered chain-packing regions. The active-site of PHB depolymerase takes place preferentially at the disordered chain-packing regions of crystal edges and ends with endo-exo enzymatic hydrolysis behaviour, termed processive degradation.     

Intracellular degradation of poly(3-hydroxybutyrate) granules of Zoogloea ramigera I-16-M.

Intracellular degradation of poly(3-hydroxybutyrate) (PHB) in bacteria is not yet clear. The properties of the autodigestion of native PHB granules from Zoogloea ramigera I-16-M were examined. The release of D(-)-3-hydroxybutyrate was observed only at pH values higher than about 8.5 and at relatively high ionic strength (optimal concentration 200 mM NaCl). Triton X-100 and diisopropylfluorophosphate inhibited this reaction. Addition of the supernatant fraction of Z. ramigera did not increase the release of D(-)-3-hydroxybutyrate from the native PHB granules. On the other hand, using the protease-treated PHB granules from Alcaligenes eutrophus as a substrate, PHB depolymerase activity was detected in the supernatant fraction of Z. ramigera cells. The soluble PHB depolymerase showed similar properties to the enzyme in the PHB granules. Since PHB depolymerase activity was found in fractions containing D(-)-3-hydroxybutyrate oligomer hydrolase activity, which were separated by DEAE-Toyopearl or by Sephacryl S-100, it is possible that the intracellular PHB depolymerase is identical to the oligomer hydrolase which has been purified already.     

Isolation of a Gram-positive poly(3-hydroxybutyrate) (PHB)-degrading bacterium from compost, and cloning and characterization of a gene encoding PHB depolymerase of Bacillus megaterium N-18-25-9

A Gram-positive poly(3-hydroxybutyrate) (PHB)-degrading bacterial strain was isolated from compost. This organism, identified as Bacillus megaterium N-18-25-9, produced a clearing zone on opaque NB-PHB agar, indicating the presence of extracellular PHB depolymerase. A PHB depolymerase gene, PhaZ(Bm), of B. megaterium N-18-25-9 was cloned and sequenced, and the recombinant gene product was purified from Escherichia coli. The N-terminal half region of PhaZ(Bm) shared significant homologies with a catalytic domain of other PHB depolymerases. Although the C-terminal half region of PhaZ(Bm) showed no significant similarity with those of other PHB depolymerases, that region was necessary for the PHB depolymerase activity. Therefore, this enzyme's domain structure is unique among extracellular PHB depolymerase domain structures. The addition of PHB to the medium led to a sixfold increase in PhaZ(Bm) mRNA, while the presence of glucose repressed PhaZ(Bm) expression. The maximum activity was observed at pH 9.0 at 65 degrees C.     

Intracellular degradation of poly(3-hydroxybutyrate) granules of Zoogloea ramigera I-16-M

Abstract Intracellular degradation of poly(3-hydroxybutyrate) (PHB) in bacteria is not yet clear. The properties of the autodigestion of native PHB granules from Zooglea ramigera I-16-M were examined. The release of d (−)-3-hydroxybutyrate was observed only at pH values higher than about 8.5 and at relatively high ionic strength (optimal concentration 200 mM NaCl). Triton X-100 and diisopropylfluorophosphate inhibited this reaction. Addition of the supernatant fraction of Z. ramigera did not increase the release of d (−)-3-hydroxybutyrate from the native PHB granules. On the other hand, using the protease-treated PHB granules from Alcaligenes eutrophus as a substrate, PHB depolymerase activity was detected in the supernatant fraction of Z. ramigera cells. The soluble PHB depolymerase showed similar properties to the enzyme in the PHB granules. Since PHB depolymerase activity was found in fractions containing d (−)-3-hydroxybutyrate oligomer hydrolase activity, which were separated by DEAE-Toyopearl or by Sephacryl S-100, it is possible that the intracellular PHB depolymerase is identical to the oligomer hydrolase which has been purified already.     

Purification and characterization of an extracellular poly(3-hydroxybutyrate-co-3-hydroxyvalerate) depolymerase from Acidovorax sp. HB01

The poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)-degrading strain Acidovorax sp. HB01 was isolated from an activated sludge sample. A novel PHBV depolymerase with a molecular weight of 43.4 kDa was purified to homogeneity from the culture supernatant of the HB01 strain. The optimum pH and temperature of the PHBV depolymerase were 7.0 and 50 °C, respectively. The PHBV depolymerase can also degrade polyhydroxybutyrate, poly (3-hydroxybutyrate-co-4-hydroxybutyrate), and poly(caprolactone); however, the PHBV degradation activity of the depolymerase is higher than its activity against the other polymers. Effect of metal ions and various inhibitors on the PHBV depolymerase activity was examined. The addition of Na(+), K(+), and Ca(2+) markedly increased the hydrolysis rate, whereas the enzyme activity was inhibited by Zn(2+), Mg(2+), Mn(2+), and particularly by Cu(2+) and Fe(2+). Ethylenediaminetetraacetic acid was found to have a significant inhibitory effect. The main degradation product of depolymerase was identified as the 3-hydroxybutyric acid monomer and 3-hydroxyvaleric acid monomers via mass spectrometry.     

Polyhydroxybutyrate: An intriguing biopolymer

The microbial polymer poly-3-hydroxybutyrate (PHB) and related poly-hydroxyalkanoates, such as poly-3-hydroxyvalerate and poly-3-hydroxyoctanoate, are unique biodegradable thermoplastics of considerable commercial importance. The structure, properties and regulation of synthesis and degradation of PHB are reviewed and the microbial production of copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate, with properties varying according to copolymer composition, is discussed.     

Mobilization of poly(3-hydroxybutyrate) in Ralstonia eutropha

Ralstonia eutropha H16 degraded (mobilized) previously accumulated poly(3-hydroxybutyrate) (PHB) in the absence of an exogenous carbon source and used the degradation products for growth and survival. Isolated native PHB granules of mobilized R. eutropha cells released 3-hydroxybutyrate (3HB) at a threefold higher rate than did control granules of nonmobilized bacteria. No 3HB was released by native PHB granules of recombinant Escherichia coli expressing the PHB biosynthetic genes. Native PHB granules isolated from chromosomal knockout mutants of an intracellular PHB (i-PHB) depolymerase gene of R. eutropha H16 and HF210 showed a reduced but not completely eliminated activity of 3HB release and indicated the presence of i-PHB depolymerase isoenzymes.     

Extracellular poly(hydroxyalkanoate) depolymerases and their inhibitor from Pseudomonas lemoignei.

Enzymatic degradation processes of microbial copolyesters, poly(3-hydroxybutyrate-co-3-hydroxyvalerate): P(3HB-co-3HV) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate): P(3HB-co-4HB), were studied by the weight loss (erosion) of copolyester films. These studies employed three extracellular depolymerases which degrade poly(3-hydroxybutyrate): P(3HB). Two enzymes were purified from the culture supernatant of Pseudomonas lemoignei and one from Alcaligenes faecalis T1. The rate of enzymatic degradation of microbial copolyester films with various compositions showed an almost similar tendency to three different P(3HB) depolymerases, and decreased in the following order: P(3HB-co-4HB) greater than P(3HB) greater than P(3HB-co-3HV). An inhibitory protein of P(3HB) depolymerases in the succinate culture medium of P. lemoignei was isolated and characterized. The molecular weight of P(3HB) depolymerase inhibitor was 35,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. This inhibitor of a single polypeptide chain may reversibly bind the serine residues at the active site of P(3HB) depolymerase. This inhibitory protein was not induced in the culture medium when P. lemoignei was grown on P(3HB) as the sole carbon source.     

A new type of thermoalkalophilic hydrolase of Paucimonas lemoignei with high specificity for amorphous polyesters of short chain-length hydroxyalkanoic acids.

A novel type of hydrolase was purified from culture fluid of Paucimonas (formerly Pseudomonas) lemoignei. Biochemical characterization revealed an unusual substrate specificity of the purified enzyme for amorphous poly((R)-3-hydroxyalkanoates) (PHA) such as native granules of natural poly((R)-3-hydroxybutyrate) (PHB) or poly((R)-3-hydroxyvalerate) (PHV), artificial cholate-coated granules of natural PHB or PHV, atactic poly((R,S)-3-hydroxybutyrate), and oligomers of (R)-3-hydroxybutyrate (3HB) with six or more 3HB units. The enzyme has the unique property to recognize the physical state of the polymeric substrate by discrimination between amorphous PHA (good substrate) and denatured, partially crystalline PHA (no substrate). The pentamers of 3HB or 3HV were identified as the main products of enzymatic hydrolysis of native PHB or PHV, respectively. No activity was found with any denatured PHA, oligomers of (R)-3HB with five or less 3HB units, poly(6-hydroxyhexanoate), substrates of lipases such as tributyrin or triolein, substrates for amidases/nitrilases, DNA, RNA, casein, N-alpha-benzoyl-l-arginine-4-nitranilide, or starch. The purified enzyme (M(r) 36,209) was remarkably stable and active at high temperature (60 degrees C), high pH (up to 12.0), low ionic strength (distilled water), and in solvents (e.g. n-propyl alcohol). The depolymerase contained no essential SH groups or essential disulfide bridges and was insensitive to high concentrations of ionic (SDS) and nonionic (Triton and Tween) detergents. Characterization of the cloned structural gene (phaZ7) and the DNA-deduced amino acid sequence revealed no homologies to any PHB depolymerase or any other sequence of data banks except for a short sequence related to the active site serine of serine hydrolases. A classification of the enzyme into a new family (family 9) of carboxyesterases (Arpigny, J. L., and Jaeger, K.-E. (1999) Biochem. J. 343, 177-183) is suggested.     

Poly(3-hydroxyvalerate) depolymerase of Pseudomonas lemoignei

Pseudomonas lemoignei is equipped with at least five polyhydroxyalkanoate (PHA) depolymerase structural genes (phaZ1 to phaZ5) which enable the bacterium to utilize extracellular poly(3-hydroxybutyrate) (PHB), poly(3-hydroxyvalerate) (PHV), and related polyesters consisting of short-chain-length hxdroxyalkanoates (PHA(SCL)) as the sole sources of carbon and energy. Four genes (phaZ1, phaZ2, phaZ3, and phaZ5) encode PHB depolymerases C, B, D, and A, respectively. It was speculated that the remaining gene, phaZ4, encodes the PHV depolymerase (D. Jendrossek, A. Frisse, A. Behrends, M. Andermann, H. D. Kratzin, T. Stanislawski, and H. G. Schlegel, J. Bacteriol. 177:596-607, 1995). However, in this study, we show that phaZ4 codes for another PHB depolymeraes (i) by disagreement of 5 out of 41 amino acids that had been determined by Edman degradation of the PHV depolymerase and of four endoproteinase GluC-generated internal peptides with the DNA-deduced sequence of phaZ4, (ii) by the lack of immunological reaction of purified recombinant PhaZ4 with PHV depolymerase-specific antibodies, and (iii) by the low activity of the PhaZ4 depolymerase with PHV as a substrate. The true PHV depolymerase-encoding structural gene, phaZ6, was identified by screening a genomic library of P. lemoignei in Escherichia coli for clearing zone formation on PHV agar. The DNA sequence of phaZ6 contained all 41 amino acids of the GluC-generated peptide fragments of the PHV depolymerase. PhaZ6 was expressed and purified from recombinant E. coli and showed immunological identity to the wild-type PHV depolymerase and had high specific activities with PHB and PHV as substrates. To our knowledge, this is the first report on a PHA(SCL) depolymerase gene that is expressed during growth on PHV or odd-numbered carbon sources and that encodes a protein with high PHV depolymerase activity. Amino acid analysis revealed that PhaZ6 (relative molecular mass [M(r)], 43,610 Da) resembles precursors of other extracellular PHA(SCL) depolymerases (28 to 50% identical amino acids). The mature protein (M(r), 41,048) is composed of (i) a large catalytic domain including a catalytic triad of S(136), D(211), and H(269) similar to serine hydrolases; (ii) a linker region highly enriched in threonine residues and other amino acids with hydroxylated or small side chains (Thr-rich region); and (iii) a C-terminal domain similar in sequence to the substrate-binding domain of PHA(SCL) depolymerases. Differences in the codon usage of phaZ6 for some codons from the average codon usage of P. lemoignei indicated that phaZ6 might be derived from other organisms by gene transfer. Multialignment of separate domains of bacterial PHA(SCL) depolymerases suggested that not only complete depolymerase genes but also individual domains might have been exchanged between bacteria during evolution of PHA(SCL) depolymerases.     

Studies on intracellular degradation of polyhydroxyalkanoic acid-polyethylene glycol copolymer accumulated by Azotobacter chroococcum MAL-201

Azotobacter chroococcum MAL-201 accumulates poly(3-hydroxybutyric acid) [PHB] when grown in glucose containing nitrogen-free Stockdale medium. The same medium supplemented with valerate alone and valerate plus polyethylene glycol (PEG) leads to the accumulation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [PHBV] and PEG containing PHBV-PEG polymers, respectively. The intracellular degradation of these polymers as studied in carbon-free Stockdale medium showed a rapid degradation of PHB followed by PHBV, while it was least in case of PHBV-PEG. The rate of such degradation was 44.16, 26.4 and 17.0 mg h(-1)l(-1) for PHB, PHBV and PHBV-PEG, respectively. During the course of such of PHBV and PHBV-PEG degradation the 3HB mol% of polymers decreased significantly with increase of 3HV mol fraction, the EG mol% in PHBV-PEG, however, remained constant. After 50h of degradation the decrease in intrinsic viscosity and molecular mass of PHBV-PEG were 37.5 and 43.6%, respectively. These values appeared low compared to PHB and PHBV. Moreover, the increasing EG content of polymer retarded their extent of degradation. Presence of PEG, particularly of low molecular weight PEG was inhibitory to intracellular PHA depolymerise (i-PHA depolymerase) activity and the relative substrate specificity of the i-PHA depolymerase of MAL-201 appeared to be PHB > PHBV > PHBV-PEG.     

Accumulation of a poly(hydroxyalkanoate) copolymer containing primarily 3-hydroxyvalerate from simple carbohydrate substrates by Rhodococcus sp. NCIMB 40126

A number of taxonomically-related bacteria have been identified which accumulate poly(hydroxyalkanoate) (PHA) copolymers containing primarily 3-hydroxyvalerate (3HV) monomer units from a range of unrelated single carbon sources. One of these, Rhodococcus sp. NCIMB 40126, was further investigated and shown to produce a copolymer containing 75 mol% 3HV and 25 mol% 3-hydroxybutyrate (3HB) from glucose as sole carbon source. Polyesters containing both 3HV and 3HB monomer units, together with 4-hydroxybutyrate (4HB), 5-hydroxyvalerate (5HV) or 3-hydroxyhexanoate (3HHx), were also produced by this organism from certain accumulation substrates. With valeric acid as substrate, almost pure (99 mol% 3HV) poly(3-hydroxyvalerate) was produced. N.m.r. analysis confirmed the composition of these polyesters. The thermal properties and molecular weight of the copolymer produced from glucose were comparable to those of PHB produced by Alcaligenes eutrophus.     

Identification of the intracellular polyhydroxyalkanoate depolymerase gene of Paracoccus denitrificans and some properties of the gene product

Paracoccus denitrificans degraded poly(3-hydroxybutyrate) (PHB) in the cells under carbon source starvation. Intracellular poly(3-hydroxyalkanoate) (PHA) depolymerase gene (phaZ) was identified near the PHA synthase gene (phaC) of P. denitrificans. Cell extract of Escherichia coli carrying lacZ--phaZ fusion gene degraded protease-treated PHB granules. Reaction products were thought to be mainly D(--)-3-hydroxybutyrate (3HB) dimer and 3HB oligomer. Diisopropylfluorophosphonate and Triton X-100 exhibited an inhibitory effect on the degradation of PHB granules. When cell extract of the recombinant E. coli was used, Mg(2+) ion inhibited PHB degradation. However, the inhibitory effect by Mg(2+) ion was not observed using the cell extract of P. denitrificans.     

The "intracellular" poly(3-hydroxybutyrate) (PHB) depolymerase of Rhodospirillum rubrum is a periplasm-located protein with specificity for native PHB and with structural similarity to extracellular PHB depolymerases

Rhodospirillum rubrum possesses a putative intracellular poly(3-hydroxybutyrate) (PHB) depolymerase system consisting of a soluble PHB depolymerase, a heat-stable activator, and a 3-hydroxybutyrate dimer hydrolase (J. M. Merrick and M. Doudoroff, J. Bacteriol. 88:60-71, 1964). In this study we reinvestigated the soluble R. rubrum PHB depolymerase (PhaZ1). It turned out that PhaZ1 is a novel type of PHB depolymerase with unique properties. Purified PhaZ1 was specific for amorphous short-chain-length polyhydroxyalkanoates (PHA) such as native PHB, artificial PHB, and oligomer esters of (R)-3-hydroxybutyrate with 3 or more 3-hydroxybutyrate units. Atactic PHB, (S)-3-hydroxybutyrate oligomers, medium-chain-length PHA, and lipase substrates (triolein, tributyrin) were not hydrolyzed. The PHB depolymerase structural gene (phaZ1) was cloned. Its deduced amino acid sequence (37,704 Da) had no significant similarity to those of intracellular PHB depolymerases of Wautersia eutropha or of other PHB-accumulating bacteria. PhaZ1 was found to have strong amino acid homology with type-II catalytic domains of extracellular PHB depolymerases, and Ser(42), Asp(138), and His(178) were identified as catalytic-triad amino acids, with Ser(42) as the putative active site. Surprisingly, the first 23 amino acids of the PHB depolymerase previously assumed to be intracellular revealed features of classical signal peptides, and Edman sequencing of purified PhaZ1 confirmed the functionality of the predicted cleavage site. Extracellular PHB depolymerase activity was absent, and analysis of cell fractions unequivocally showed that PhaZ1 is a periplasm-located enzyme. The previously assumed intracellular activator/depolymerase system is unlikely to have a physiological function in PHB mobilization in vivo. A second gene, encoding the putative true intracellular PHB depolymerase (PhaZ2), was identified in the genome sequence of R. rubrum.     

An extracellular poly (3-hydroxybutyrate) depolymerase from Penicillium sp. DS9713a-01

Summary Penicillium sp. DS9713a-01 was obtained by ultraviolet (u.v.) light mutagenesis from the Penicillium sp. DS9713a which can degrade poly (3-hydroxybutyrate) (PHB). The enzymatic activity of DS9713a-01 was 97% higher than that of the wild-type strain. The DS9713a-01 mutant could completely degrade PHB films in 5 days; however, the wild-type strain achieved only 61% at the same time. The extracellular PHB depolymerase was purified from the culture medium containing PHB as the sole carbon source by filtration, ammonium sulfate precipitation and chromatography on Sepharose CL-6B. The molecular weight of the PHB depolymerase was about 15.1kDa determined by SDS-polyacrylamide gel electrophoresis. The optimum activity of the PHB depolymerase was observed at pH 8.6 and 50 °C. The enzyme was stable at temperatures below 37 °C and in the pH range from 8.0 to 9.2. The activity of PHB depolymerase could be activated or inhibited by some metal ions. The apparent K _m value was 0.164 mg ml⁻¹. Mass spectrometric analysis of the water-soluble products after enzymatic degradation revealed that the primary product was the monomer, 3-hydroxybutyric acid.     

Ralstonia eutropha H16 encodes two and possibly three intracellular Poly[D-(-)-3-hydroxybutyrate] depolymerase genes

Intracellular poly[D-(-)-3-hydroxybutyrate] (PHB) depolymerases degrade PHB granules to oligomers and monomers of 3-hydroxybutyric acid. Recently an intracellular PHB depolymerase gene (phaZ1) from Ralstonia eutropha was identified. We now report identification of candidate PHB depolymerase genes from R. eutropha, namely, phaZ2 and phaZ3, and their characterization in vivo. phaZ1 was used to identify two candidate depolymerase genes in the genome of Ralstonia metallidurans. phaZ1 and these genes were then used to design degenerate primers. These primers and PCR methods on the R. eutropha genome were used to identify two new candidate depolymerase genes in R. eutropha: phaZ2 and phaZ3. Inverse PCR methods were used to obtain the complete sequence of phaZ3, and library screening was used to obtain the complete sequence of phaZ2. PhaZ1, PhaZ2, and PhaZ3 share approximately 30% sequence identity. The function of PhaZ2 and PhaZ3 was examined by generating R. eutropha H16 deletion strains (Delta phaZ1, Delta phaZ2, Delta phaZ3, Delta phaZ1 Delta phaZ2, Delta phaZ1 Delta phaZ3, Delta phaZ2 Delta phaZ3, and Delta phaZ1 Delta phaZ2 Delta phaZ3). These strains were analyzed for PHB production and utilization under two sets of conditions. When cells were grown in rich medium, PhaZ1 was sufficient to account for intracellular PHB degradation. When cells that had accumulated approximately 80% (cell dry weight) PHB were subjected to PHB utilization conditions, PhaZ1 and PhaZ2 were sufficient to account for PHB degradation. PhaZ2 is thus suggested to be an intracellular depolymerase. The role of PhaZ3 remains to be established.     

Properties of a novel intracellular poly(3-hydroxybutyrate) depolymerase with high specific activity (PhaZd) in Wautersia eutropha H16

A novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase (PhaZd) of Wautersia eutropha (formerly Ralstonia eutropha) H16 which shows similarity with the catalytic domain of the extracellular PHB depolymerase in Ralstonia pickettii T1 was identified. The positions of the catalytic triad (Ser190-Asp266-His330) and oxyanion hole (His108) in the amino acid sequence of PhaZd deduced from the nucleotide sequence roughly accorded with those of the extracellular PHB depolymerase of R. pickettii T1, but a signal peptide, a linker domain, and a substrate binding domain were missing. The PhaZd gene was cloned and the gene product was purified from Escherichia coli. The specific activity of PhaZd toward artificial amorphous PHB granules was significantly greater than that of other known intracellular PHB depolymerase or 3-hydroxybutyrate (3HB) oligomer hydrolases of W. eutropha H16. The enzyme degraded artificial amorphous PHB granules and mainly released various 3-hydroxybutyrate oligomers. PhaZd distributed nearly equally between PHB inclusion bodies and the cytosolic fraction. The amount of PHB was greater in phaZd deletion mutant cells than the wild-type cells under various culture conditions. These results indicate that PhaZd is a novel intracellular PHB depolymerase which participates in the mobilization of PHB in W. eutropha H16 along with other PHB depolymerases.     

Cloning of an intracellular Poly[D(-)-3-Hydroxybutyrate] depolymerase gene from Ralstonia eutropha H16 and characterization of the gene product

An intracellular poly[D(-)-3-hydroxybutyrate] (PHB) depolymerase gene (phaZ) has been cloned from Ralstonia eutropha H16 by the shotgun method, sequenced, and characterized. Nucleotide sequence analysis of a 2.3-kbp DNA fragment revealed an open reading frame of 1,260 bp, encoding a protein of 419 amino acids with a predicted molecular mass of 47,316 Da. The crude extract of Escherichia coli containing the PHB depolymerase gene digested artificial amorphous PHB granules and released mainly oligomeric D(-)-3-hydroxybutyrate, with some monomer. The gene product did not hydrolyze crystalline PHB or freeze-dried artificial amorphous PHB granules. The deduced amino acid sequence lacked sequence corresponding to a classical lipase box, Gly-X-Ser-X-Gly. The gene product was expressed in R. eutropha cells concomitant with the synthesis of PHB and localized in PHB granules. Although a mutant of R. eutropha whose phaZ gene was disrupted showed a higher PHB content compared to the wild type in a nutrient-rich medium, it accumulated PHB as much as the wild type did in a nitrogen-free, carbon-rich medium. These results indicate that the cloned phaZ gene encodes an intracellular PHB depolymerase in R. eutropha.