Substrate specificities of poly(hydroxyalkanoate)-degrading bacteria and active site studies on the extracellular poly(3-hydroxyoctanoic acid) depolymerase of<i>Pseudomonas fluorescens</i>GK13

Schirmer, Andreas; Matz, Claudia; Jendrossek, Dieter

doi:10.1139/m95-184

Cited by 61 publications

(36 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…This method is practicable when the degradation of macroscopically visible specimens of polymer samples (e.g., PHB films) is determined over a time scale of many hours. For short assays of PHB depolymerase activity, most laboratories determine the rate of clearing of a turbid suspension of nPHB or dPHB granules for (1,13,22,24,28,32,45,46). However, this method is limited principally to very diluted suspensions of PHB granules.…”

Section: Discussionmentioning

confidence: 99%

Assay of Poly(3-Hydroxybutyrate) Depolymerase Activity and Product Determination

Gebauer

Jendrossek

2006

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

Two methods for accurate poly(3-hydroxybutyrate) (PHB) depolymerase activity determination and quantitative and qualitative hydrolysis product determination are described. The first method is based on online determination of NaOH consumption rates necessary to neutralize 3-hydroxybutyric acid (3HB) and/or 3HB oligomers produced during the hydrolysis reaction and requires a pH-stat apparatus equipped with a softwarecontrolled microliter pump for rapid and accurate titration. The method is universally suitable for hydrolysis of any type of polyhydroxyalkanoate or other molecules with hydrolyzable ester bonds, allows the determination of hydrolysis rates of as low as 1 nmol/min, and has a dynamic capacity of at least 6 orders of magnitude. By applying this method, specific hydrolysis rates of native PHB granules isolated from Ralstonia eutropha H16 were determined for the first time. The second method was developed for hydrolysis product identification and is based on the derivatization of 3HB oligomers into bromophenacyl derivates and separation by highperformance liquid chromatography. The method allows the separation and quantification of 3HB and 3HB oligomers up to the octamer. The two methods were applied to investigate the hydrolysis of different types of PHB by selected PHB depolymerases.Polyhydroxyalkanoates (PHAs) are typical storage compounds of carbon and energy and are widely found in prokaryotes. The most common PHA is poly(3-hydroxybutyrate) (PHB), and this polymer can be accumulated at up to 90% of the cellular dry weight during unbalanced growth in some bacteria (2a, 37, 49). PHAs are thermoplasts, and despite the relatively high production costs, PHB and copolymers of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate are industrially produced on a scale of a few hundred tons per year.One reason for the interest in biologically produced PHAs lies in the environmentally friendly production from renewable resources and in the biodegradability of PHAs. The ester bonds of the PHAs are the Achilles' heel of the polymer that can be hydrolyzed by a large variety of hydrolytic enzymes (PHA depolymerases). In vivo, PHAs can be hydrolyzed by the accumulating strain itself during periods of starvation (intracellular PHA hydrolysis by intracellular PHA depolymerases) (41). PHAs that were produced by humans or that were released from PHA-accumulating microorganisms (e.g., after death) can be cleaved by extracellular PHB depolymerases, and many examples of extracellular PHA depolymerases were described during the last two decades (14, 21). A differentiation of extracellular degradation from intracellular degradation is necessary because PHA exists in two biophysical conformations: in vivo, PHB is completely amorphous (native) and is covered by a surface layer that is about half the size of a cytoplasmic membrane (4, 5, 31) but apparently consists mainly of proteins, so-called phasins (48). After the release of the polymer from the cell (e.g., after cell lysis or by solvent extraction) or after damage of the surface l...

show abstract

Section: Discussionmentioning

confidence: 99%

Assay of Poly(3-Hydroxybutyrate) Depolymerase Activity and Product Determination

Gebauer

Jendrossek

2006

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

show abstract

“…The catalytic domain (residues 1 to 306) contains a pentapeptide sequence, G-X-S 136 -X-G (lipase box), which is a characteristic of serine esterases such as lipases, esterases, and all PHA depolymerases analyzed so far. The central serine of the lipase box has been shown to be essential for activity in many lipases and several PHA depolymerases (8,23,24) and H 269 ) and one aspartate residue (D 211 ) are strictly conserved in PhaZ6 and all PHA depolymerases (Fig. 3).…”

Section: Resultsmentioning

confidence: 99%

Poly(3-Hydroxyvalerate) Depolymerase of Pseudomonas lemoignei

Schöber

Thiel

Jendrossek

2000

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

. 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 The ability to degrade extracellular poly(3-hydroxybutyrate) (PHB) and related polyesters is widely distributed among bacteria and depends on the secretion of specific polyester depolymerases which hydrolyze the water-insoluble polyester to water-soluble monomers or oligomers. One of the best-studied polyester-degrading bacteria is Pseudomonas lemoignei (5). It belongs to the beta subclass of Proteobacteria and is related to the Burkholderia-Ralstonia rRNA sublineage. The catabolic abilities of P. lemoignei are restricted to the utilization of a few organic acids (acetate, butyrate, valerate, pyruvate, succinate, and 3-hydroxybutyrate), and polyesters such as PHB, poly(3-hydroxyvalerate) (PHV), and related short-chain-length polyhydroxyalkanoates (PHA SCL ). Sugars, alcohols, and amino acids do not support growth of P. lemoignei (5, 17). Recently, P. lemoignei (strain A62) was reisolated by application of a specific enrichment procedure with PHV as the sole source of carbon and energy (16).P. lemoignei is unique among PHA-degrading bacteria because it is able to synthesize at least five different extracellular PHA depolymerases. Three PHA depolymerases are specific for PHB and copolymers of 3-hydroxybutyrate (3-HB) and 3-hydroxyvalerate (3-HV) with low 3-HV content (PHB depolymerases A, B and D). The activity of these enzymes with the homopolyester PHV is below 5% of the activity obtained with PHB as substrate. None of the three PHB depolymerases is able to produce clearing zones on opaque PHV granulecontaining agar. The two remaining PHA depolymerases (PHB depolymerases C and PHV depolymerase) also degrade PHB, but are additionally able to hydrolyze PHV, with about 15 and 30% activity compared to PHB hydrolysis. PHB depolymerase C and PHV depolymerase produce clearing zones on opaque PH...

show abstract

“…Growth of the strains was investigated in liquid MSM and on MSM agar plates and utilization of carbon sources was determined according to Schirmer et al (1995) and Gerhardt et al (1994). The dehydroabietic acid-degrading bacterium DhA-71 was cultivated as described by Mohn (1995).…”

Section: Supplemented Withmentioning

confidence: 99%

“…The susceptibility of the strains towards antibiotics was determined according to Schirmer et al (1995) on MSM agar plates for the following antibiotics (ml 21 ): 15 mg tetracycline, 100 mg streptomycin, 50 mg kanamycin, 50 mg ampicillin and 34 mg chloramphenicol.…”

Section: Supplemented Withmentioning

confidence: 99%

Schlegelella thermodepolymerans gen. nov., sp. nov., a novel thermophilic bacterium that degrades poly(3-hydroxybutyrate-co-3-mercaptopropionate)

Elbanna

Lütke‐Eversloh

Trappen

et al. 2003

International Journal of Systematic and Evolutionary Microbiology

View full text Add to dashboard Cite

Schlegelella thermodepolymerans gen. nov., sp. nov., a novel thermophilic bacterium that degrades poly(3-hydroxybutyrate-co-3-mercaptopropionate) A novel thermophilic bacterium, strain K14 T , capable of degrading poly(3-hydroxybutyrate) as well as copolymers containing 3-hydroxybutyrate and 3-mercaptopropionate linked by thioester bonds, was isolated. 16S rDNA sequence analysis showed that strain DhA-71, a dehydroabietic acid-degrading bacterium, was the nearest phylogenetic neighbour and that both strains should be placed as members of a newly created genus, Schlegelella gen. nov., in the Rubrivivax subgroup of the b-Proteobacteria. Strain K14 T (=LMG 21644 T =DSM 15344 T ) is proposed as the type strain of Schlegelella thermodepolymerans gen. nov., sp. nov. Its phylogenetic, morphological, biochemical and chemotaxonomic characteristics are described in detail.

show abstract

Substrate specificities of poly(hydroxyalkanoate)-degrading bacteria and active site studies on the extracellular poly(3-hydroxyoctanoic acid) depolymerase ofPseudomonas fluorescensGK13

Cited by 61 publications

References 31 publications

Assay of Poly(3-Hydroxybutyrate) Depolymerase Activity and Product Determination

Assay of Poly(3-Hydroxybutyrate) Depolymerase Activity and Product Determination

Poly(3-Hydroxyvalerate) Depolymerase of Pseudomonas lemoignei

Schlegelella thermodepolymerans gen. nov., sp. nov., a novel thermophilic bacterium that degrades poly(3-hydroxybutyrate-co-3-mercaptopropionate)

Contact Info

Product

Resources

About