biodegradability of pha

8/4/2019 Biodegradability of PHA

1/14

212

Abstract The biodegradability of microbial polythioesters(PTEs), a novel class of biopolymers which were discov-ered recently and can be produced by polyhydroxyalkano-ate (PHA)-accumulating bacteria, was studied. Using poly(3-hydroxybutyrate-co-3-mercaptopropionate) [poly(3HB-co-3MP)] as sole carbon source for screening, 22newbacterial strains were isolated and characterized. Interest-ingly, none of the PHA-degrading bacteria was able to uti-lize the homopolymer poly(3MP) as a carbon source forgrowth or to form clear zones on poly(3MP)-containingagar plates. The extracellular PHA depolymerases fromtwo strains (Schlegelella thermodepolymerans, Pseudo-monas indica K2) were purified to electrophoretic homo-geneity and biochemically characterized. The PHA de-polymerase of S. thermodepolymerans exhibited a tem-perate optimum of about 75C to 80C and was stable at70C for more than 24h. Regarding the substrate speci-

ficities of the PHA depolymerase ofS. thermodepolymer-ans, enzyme activities decreased significantly with in-

creasing 3MP content of the copolymer substrates. Inter-estingly, no activity could be detected with homoPTEsconsisting only of 3MP or of 3-mercaptobutyrate. Similarresults were obtained with the PHA depolymerases PhaZ2,PhaZ5 and PhaZ7 ofPaucimonas lemoignei which werealso investigated. The PHA depolymerase of Ps. indicaK2 did not cleave any of the investigated polymers con-taining 3MP. Gas chromatography, infrared and 1H-NMRspectrometry and matrix-assisted laser desorption/ioniza-tion time-of-flight analysis revealed that 3MPs containingoligomers were enriched in the water-insoluble fractionremaining after partial digestion of poly(3HB-co-3MP)by purified poly(3HB) depolymerase ofS. thermodepoly-merans. In contrast, 3HB was enriched in the water-soluble fraction, which also contained 3HB-co-3MP dimerobtained by partial digestion of this copolymer by theenzyme. This study clearly indicates that PHA depoly-

merases are specific for oxoester linkages of PHAs andthat the thioester bonds of PTEs cannot be cleaved by thistype of enzyme.

Keywords Polythioester PTE Poly(3-mercaptopropionate) Polyhydroxyalkanoate PHA Biodegradation PHA depolymerase

Introduction

Waste disposal is becoming an increasingly difficult prob-lem as available landfills diminish. Currently, plastic ac-counts for about 7% by weight and 18% by volume ofmunicipal solid waste, with half of this plastic waste pre-viously used for packaging (Thayer 1990). Microbial bio-degradable plastics such as polyhydroxyalkanoates (PHAs)

are synthesized intracellularly during unbalanced growthin many bacteria as a carbon and energy storage com-pound. PHAs have attracted the attention as environmen-tal friendly materials for many applications in industry,agriculture and medicine (Anderson and Dawes 1990;Steinbchel et al. 1995; Hocking and Marchessault 1994).To date, more than 140 different hydroxyalkanoates (HAs)

Khaled Elbanna Tina Ltke-Eversloh

Dieter Jendrossek Heinrich Luftmann

Alexander Steinbchel

Studies on the biodegradabilityof polythioester copolymers and homopolymers

by polyhydroxyalkanoate (PHA)-degrading bacteria and PHA depolymerases

Arch Microbiol (2004) 182 : 212225DOI 10.1007/s00203-004-0715-z

Received: 9 February 2004 / Revised: 13 July 2004 / Accepted: 16 July 2004 / Published online: 31 August 2004

ORIGINAL PAPER

This publication is dedicated to Prof. Dr. Hans G. Schlegelin honor of his 80th birthday

K. Elbanna T. Ltke-Eversloh A. Steinbchel ()Institut fr Molekulare Mikrobiologie und Biotechnologie,Westflische Wilhelms-Universitt Mnster,Corrensstrae 3, 48149 Mnster, GermanyTel.: +49-251-8339821, Fax: +49-251-8338388,e-mail: [email protected]

D. JendrossekInstitut fr Mikrobiologie, Universitt Stuttgart,Allmandring 31, 70569 Stuttgart, Germany

H. LuftmannInstitut fr Organische Chemie,Westflische Wilhelms-Universitt Mnster,Corrensstrae 40, 48149 Mnster, Germany

Present address:T. Ltke-EverslohDepartment of Chemical Engineering,Massachusetts Institute of Technology,77 Massachusetts Avenue 56422, Cambrige, MA 02139, USA

Springer-Verlag 2004


2/14

have been described as PHA constituents. These comprisedifferent carbon chain lengths and might also contain var-ious substituents at different positions (Steinbchel andValentin 1995). Since only a few PHA constituents can beobtained from simple carbon sources such as glucose,metabolic engineering is frequently applied to avoid feed-ing of expensive precursor carbon sources and to synthe-size respective substrates of PHA synthases from cheap and

abundantly available renewable resources (Steinbcheland Ltke-Eversloh 2003).The biodegradability of PHAs in different natural envi-

ronments has been investigated and more than 700 strainsof PHA-degrading microorganisms have been identified(Mergaert and Swings 1996). PHA-degrading microorgan-isms excrete PHA depolymerases, which hydrolyze thepolymer extracellularly to water-soluble products and uti-lize the hydrolysis products as carbon and energy sourcesfor growth (Delafield et al. 1965; Jendrossek et al. 1996;Jendrossek 2002). Several poly(3-hydroxybutyrate [poly(3HB)] depolymerases have been isolated from variousmicroorganisms and analyzed on the level of their struc-

tural genes (for a recent summary, see Jendrossek andHandrick 2002 and references therein). While most PHA-degrading bacteria apparently contain only one depoly-merase, Paucimonas lemoignei possesses seven extracel-lular PHA depolymerases, comprising different substratespecificities (Jendrossek and Handrick 2002).

Increasing temperature has a significant influence onthe bioavailability and solubility of organic compounds(Mller et al. 1998). At higher temperatures, poly(3HB) isconsidered to be amorphous and more sensitive to degrad-ing enzymes, allowing its rapid degradation (Takeda et al.1998). While many studies have been carried out withmesophilic polymer-degrading bacteria, only a few stud-

ies have been made with thermophilic bacteria. A ther-mophile extracellular poly(3HB) depolymerase has beenisolated and characterized from Caldimonas manganoxi-dans (Takeda et al. 1998, 2000, 2002). A new type of ther-moalkalophilic hydrolase isolated from Pa. lemoignei(PhaZ7) with high specificity for amorphous polyester ofshort chain-length HAs has been described (Handrick etal. 2001). A PHA depolymerase exhibiting an optimumreaction temperature of 70C was purified from the cul-ture broth ofComamonas testosteroni ATSU (Kasuya etal. 1994), but this soil bacterium did not retain its activityat this optimum reaction temperature. Therefore, only alittle information is available about thermophilic PHA-de-grading microorganisms and thermostable PHA depoly-merases.

A novel class of biopolymers was discovered recentlyin the PHA-accumulating bacterium Ralstonia eutropha,comprising a completely different linkage type, whichwas assigned as a polythioester (PTE). In addition to3HB, these polymers contained 3-mercaptopropionate(3MP) or 3-mercaptobutyrate (3MB) as constituents. Thepeculiarity of poly(3HB-co-3MP) and poly(3HB-co-3MB)is the occurrence of thioester linkages in the polymerbackbone (Ltke-Eversloh et al. 2001a, b). Recently, thelipase-catalyzed synthesis of PTEs, either by copolymer-

ization of lactones with mercaptoalkanoates or by trans-esterification of polyesters with mercaptoalkanoates, wasdescribed (Iwata et al. 2003). Studies on the physicalproperties of PTEs demonstrated that the characteristicsof PTEs, such as solubility, crystallinity and thermal sta-bility, differ considerably from those of the oxygen-analo-gous PHAs (Ltke-Eversloh et al. 2002b; Kawada et al.2003).

Due to the novelty of microbial PTEs, no investiga-tions on the biodegradation of PTEs have yet been pub-lished. Recently, the thermophilic poly(3HB-co-3MP)-de-grading bacterium Schlegelella thermodepolymerans wascharacterized and classified as gen. nov. sp. nov. (Elbannaet al. 2003). In the present study, the biodegradability ofPTEs, comprising both copolymers and homopolymers assubstrates, was investigated.

Materials and methods

Bacterial strains and culture conditions

All bacterial strains used in this study, including S. ther-modepolymerans (DSM 15344),Pseudomonas indica K2(DSM 16298) and other poly(3HB-co-3MP)-degradingbacteria which were isolated from activated sludge underaerobic and thermophilic conditions in this study accord-ing to Elbanna et al. (2003), are listed in Table 1.

Bacteria degrading poly(3HB-co-3MP) were routine-ly grown in MSM (Schlegel et al. 1961), with concen-trations of carbon sources as indicated in the text. Solidmedia were prepared with 1.8% (w/v) agar. Poly(3HB),poly(3HB-co-3MP) and poly(3HO-co-3HHx) overlay agarplates containing 0.2% (w/v) of the respective polymer

were prepared as described by Jendrossek et al. (1993). Allisolates were stored at 70C in glycerol (20%, v/v) ordimethylsulfoxide (7.5%, v/v).

Taxonomic studies

Morphological, physiological and biochemical characteri-zations of poly(3HB-co-3MP)-degrading bacteria werecarried out using procedures described by Elbanna et al.(2003).

Isolation of genomic DNA, amplificationand DNA sequencing of the 16S rRNA geneofPs. indica K2

Extraction of genomic DNA, PCR amplifications of the16S rRNAs and purification of PCR products ofPs. in-dica were carried out using procedures described byRainey et al. (1996). The PCR products were purified us-ing the Nucleotrap PCR extraction kit (Macherey-Nagel,Dren, Germany). The PCR product of the 16S rRNAgene from Ps. indica K2 was ligated into the vectorPGEG-T Easy (Promega) and transformed to competent

213


3/14


4/14

Escherichia coli XL-1 Blue (Stratagene). Plasmid DNAwas isolated according to Birnboim and Doly (1979).

The sequence of the 16S rRNA ofPs. indica was deter-mined by the chain-termination method, using an auto-matic LI-COR model 4000L sequencer (MWG-Biotech,Germany). The following oligonucleotides were used asprimers: 27f (5-GAGTTTGATCCTGGCTCAG-3), 343r(5-CTGCTGCCTCCCGTA-3), 357f (5-TACGGGAG-

GCAGCAG-3), 519r (5-G(T/A)ATTACCGCGGC(T/G)GCTG-3), 536f (5-CAGC(C/A)GCCGCGCGGTAAT(T/A)C-3), 803f (5-ATTAGATACCCTAGGTAG-3), 907r (5-CCGTCAATTCATTTGAGTTT-3), 1114f (5-GCAACG-AGCGCAACCC-3), 1385r (5-CGGTGTGT(A/G)CAA-GGCCC-3) and 1525r (5-AGAAAGGAGGTGATCCA-GCC-3). The 16S rRNA sequences were initially ana-lyzed using the BLAST program (National Center Bio-technology information, http://www.ncbi.nml.nih.gov). Theconsensus sequence ofPs. indica K2, strains belonging tothe same phylogenetic group and other representatives ofthe gamma-group of the proteobacteria (retrieved from theEMBL database) were aligned using the Clustal X pro-

gram (Thompson et al. 1997); and the resulting trees weredisplayed with Tree View (Page 1996). The phylogenetictree was calculated using the neighbor-joining method(Saitou and Nei 1987); and the neighbor-joining tree of

Ps. indica K2 was inferred, using TreeCon withAcineto-bacter calcoaceticus andE. coli as outgroups.

Production and preparation of polymers

Poly(3HB) and poly(3HB-co-3MP) were isolated fromR. eutropha H16 (DSM428). Cultivations ofR. eutrophaH16 in 26-l batches were carried out in a stirred (200

400rpm) and aerated (1520 l min

1

) 30-l stainless steelfermenter (Biostat UD30; B. Braun Biotech International,Melsungen, Germany). Fermentations were carried out inMSM with sodium gluconate and 3,3-thiodipropionicacid (TDP), respectively, as carbon sources and the pHwas adjusted to7.0 (Ltke-Evesloh et al. 2001a).

Poly(3MP) and poly(3MB) homoPTEs were obtainedfrom cultures ofE. coli JM109 harboring plasmid pBPP1as described by Ltke-Eversloh et al. (2002a). For poly-mer isolation, 40g lyophilized cells were resuspended in1,600 ml cold Tris-EDTA (33mM Tris-HCl, 1mM EDTA,pH 7.2) and disintegrated by sonification (MS 72; Bra-delen Electronic, Germany). The PTE granules were cen-trifuged for 60 min (35,000g) and washed several timeswith water. Subsequently, 40 ml SDS solution (25%, w/v)were added and the suspension was incubated for 30 minat 37C on a rotary shaker. After cell lysis and DNasetreatment, the proteins were digested with 120mg pro-teinase K for 23 h at 37C and the remaining materialwas collected by centrifugation for 45 min (35,000g).After washing twice with water, the white pellet was re-suspended in 2 l sodium hypochlorite solution (5%, v/v)for 48h, centrifuged for 60min at 10,000g and finallydried under vacuum. To remove contaminating glycogen,PTE granules were resuspended in 3 N HCl, incubated at

215

TDP

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

3MP

+

+

+

+

+

+

+

+

+

Poly(3HB)

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

Poly(3HO)

+

+

+

+

Poly(3HB-

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

co-3MP)

Poly(3MP)

Denitri-

nd

nd

nd

nd

+

+

+

+

+

+

+

fication


5/14

100C for 20 min, washed with water and acetone (threetimes each) and finally filtered through filter paper(Schleicher & Schuell, Dassel, Germany). Contaminatinglipids were removed from the filtration residue by extrac-tion with acetone/diethyl ether (2:1, v/v) for 48h in aSoxhlet apparatus.

Poly(3-hydroxyoctanoate-co-3-hydroxyhexanoate) [poly(3HO-co-3HHx)] was isolated from Ps. putida KT 2440

grown in MSM in a 30-l fermenter (Biostat UD30; B.Braun Biotech International). Fermentations were carriedout according to Timm and Steinbchel (1990). This poly-ester contained approximately 1012mol% 3HHx and8890mol% 3HO.

Suspensions of poly(3HO-co-3HHx), poly(3HB) andpoly(3HB-co-3MP) were prepared according to Jendros-sek et al. (1993) and Schirmer et al. (1993). A stable sus-pension of PTE granules was obtained by ultrasonicationin 5 mM phosphate buffer for 15min (TG 250; Schoeller,Frankfurt, Germany).

PHA depolymerase assays

PHA depolymerase activities were assayed by differentmethods, according to Jendrossek et al. (1993) and Schir-mer et al. (1993).

For the clear-zone method, 50l purified PHA depoly-merase were transferred onto opaque agar containing0.150.3% (w/v) poly(3HO-co-3HHx), poly(3HB), poly(3HB-co-3MP), or poly(3MB), respectively, in 100 mMTris-HCl, pH 8, 1 mM CaCl2 and incubated at 30C or45C for 24h, or at 70C for 3h, respectively. Clear zoneformation on these plates indicated the presence of activePHA depolymerases.

PHA depolymerases were assayed as p-nitrophenyl-octanoate (PNPO) and pnitrophenylbutyrate (PNPB) es-terase activity at 30C. The concentrated pnitrophenolsreleased by hydrolysis of PNPO or PNPB substrates weremeasured colorimetrically at 410nm; and an extinctioncoefficient of=14.925 mM1 cm1 was used for calcula-tions. The reaction mixture was prepared by combining30l PNPO or PNPB (10mM in ethanol) with 970lpotassium phosphate buffer (50 mM, pH7.9) to a totalvolume of 1 ml. After preincubation for 35 min at 30C, thereaction was started by the addition of 550l enzyme so-lution. One unit of PNPO or PNPB esterase activity was de-fined as the hydrolysis of 1mol PNPO or PNPB in 1minat 30C.

Spectrophotometric assays of PHA depolymeraseswere performed as follows: for PHA depolymerase activ-ities ofPs. indica K2, the reaction mixture was preparedby combining 100200l PHA or PTE suspension (1%w/v) with 800900l Tris-HCl buffer (100 mM, pH8.5)in a 1-ml cuvette. Before adding the enzyme, the suspen-sion was incubated at 30C for 35 min. The optical den-sity (OD) was measured at 650 nm over a period of 30minat 35C. For the measurement of PHA depolymerase ac-tivities of S. thermodepolymerans, the reaction mixturewas prepared by combining 100200l poly(3HB), poly

(3HB-co-3MP), poly(3MP) or poly(3MB) suspensions(1% w/v), respectively, with 1,1001,200 l Tris-HClbuffer (50 mM, pH8.5) in a 1.5-ml cuvette. This suspen-sion was heated to 90C for 35 min using a spectropho-tometer (Pharmacia Biotech, Ultraspec 2000) which wasconnected to a thermal unit to provide circulating hot wa-ter according to the specified temperature and continu-ously monitored using a thermometer; and the reaction

was initiated by the addition of the enzyme solution. Thedecrease in OD was measured at 650 nm. One unit of thePHA depolymerase activity was defined as the amount ofprotein that was required to decrease the OD by 1OD unitmin1.

Protein assay and gel electrophoresis

Protein was determined by the method of Bradford (1976).Sodium dodecyl sulfatepolyacrylamide gel electrophore-sis (SDS-PAGE) was performed according to Laemmli(1970). Proteins were heated to 95C for 10 min in the

presence of denaturing buffer. A low molecular weightcalibration kit (LMW; Amersham Pharmacia Biotech,Freiburg, Germany) was used as a molecular mass stan-dard. After electrophoresis, proteins were visualized bysilver staining (Heukeshofen and Dernick 1985).

Purification of the poly(3HB) depolymerasefrom S. thermodepolymerans

The poly(3HB) depolymerase ofS. thermodepolymeranswas purified by a heat-denaturing step and selective bind-ing to poly(3HB) granules. For this, cells ofS. thermode-

polymerans were cultivated in 4 l MSM containing 0.2%(w/v) poly(3HB) as sole carbon source and incubated at50C for 1517h at 130 rpm on a rotary shaker. After cen-trifugation, the cell-free supernatant was concentrated200-fold by passage through an ultrafiltration nitrocellu-lose membrane (YM30; Amicon, Beverly, Mass.). The con-centrated protein solutions were diafiltered with 50mMTris-HCl buffer (pH 8) at 4C, and subsequently heated to70C for 30 min. Precipitated proteins were removed bycentrifugation at 20,000g (Centricon T1080 with TFT65.13 rotor; Kontron Instruments, Neufarn, Germany).Subsequently, a suspension of poly(3HB) was added to aconcentration of 0.05% (w/v) for selective binding of thepoly(3HB) depolymerase at 50C. The resulting mixtureof enzyme and poly(3HB) were filtered through a What-man No. 5 filter paper, and the enzyme was eluted with400 mM KCl in 50 mM Tris-HCl buffer (pH8.2). The elu-ent was dialyzed against 50 mM Tris-HCl buffer (pH 8.2).

Purification of the poly(3HO) depolymerasefromPs. indica K2

Cells ofPs. indica K2 were grown in 2-l Erlenmeyer flaskscontaining 500 ml MSM. Each flask contained 0.15%

216


6/14

(w/v) poly(3HO-co-3HHx) as thin films on the bottom,cast from a solution of polyester in chloroform. Afterevaporation of chloroform, the flasks were autoclaved andsterile MSM was added. Each flask was inoculated with50 ml seed culture ofPs. indica K2 (15h old) grown on0.8% sodium gluconate and incubated on a rotary shakerat 130 rpm for 36h at 30C. After growth, cells were sep-arated by centrifugation and 5l of the cell-free super-

natant were concentrated to 25 ml by passage through aYM 10 ultrafiltration nitrocellulose membrane (Amicon).All subsequent steps were carried out at 4C and in thepresence of 50 mM potassium phosphate buffer (pH8.2).The concentrated protein solution was applied onto anoctyl-sepharose column (25 ml bed vol.), and after wash-ing with two bed vol. buffer, active poly(3HO) depoly-merase protein was eluted by 50 ml of buffer and anethanediol gradient (080%, v/v). Fractions of 2 ml werecollected and those containing poly(3HO) depolymeraseactivity were combined, concentrated 30-fold by passagethrough a YM10 membrane and applied onto a gel filtra-tion column (2.660 cm) containing 330 ml BV Superdex

200 HR (Amersham Pharmacia Biotech) and equilibratedwith 50mM potassium phosphate buffer (pH8.2). Thepoly(3HO) depolymerase was eluted with the same buffer.All fractions (0.5 ml) containing high poly(3HO) depoly-merase activity were concentrated using an YM10 ultra-filtration membrane and then stored at 20C.

Gas chromatographic and elemental analysisof polymers and degradation products

Gas chromatographic analysis was done after methanoly-sis of the polymers in the presence of sulfuric acid and

methanol, as described by Brandl et al. (1988) and Ltke-Eversloh et al. (2001a). Elemental sulfur analysis was per-formed by the Mikroanalytisches Labor Beller (Gttin-gen, Germany) according to the method of Grote andKrekeler (Deutsches Institut fr Normung DIN 51768).

Infrared spectroscopic analysis

The infrared (IR) spectra of polymer samples were takenwith a Fourier transform spectrometer (IFS28; Bruker,Bemen, Germany). For this, samples were deposited as afilm on a sodium chloride disk, as described by Ltke-Eversloh et al. (2001a).

Electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI/MS; Cole1997) was done with a Quattro LCZ mass spectrometer(WatersMicromass, Manchester, UK) with a nanosprayinlet and a double quadrupol analyzer with a collisionchamber for MS/MS experiments. The capillary voltagewas within the range 0.81.4kV and the cone voltage wasoptimized for maximum intensity.

Matrix-assisted laser desorption/ionization time-of-flightanalysis

Matrix-assisted laser desorption/ionization time-of-flight(MALDI-TOF) analysis was done in a Reflex IV spec-trometer (BrukerDaltoniks, Bremen, Germany) equippedwith a nitrogen laser (337 nm, 3 ns, reflector mode) and2,5-dihydroxybenzoic acid was used as matrix. Matrix so-

lution (1l, at 20mgml1

in ethylacetate) was applied tothe target plate and 1l saturated methanolic NaBF4 wasadded. After evaporation, a smooth layer of crystals wasformed. On this, 1l sample solution (5mgml1 in CHCl3)was applied and evaporated. The laser power was ad-justed for good ion yield and 100200spectra were accu-mulated to improve the S/N ratio (Pasch and Schrepp2003).

Results

Isolation and characterization

of poly(3HB-co-3MP)-degrading bacteria

In this study, 22aerobic bacteria able to grow withpoly(3HB-co-3MP) as sole carbon source were isolatedfrom different environmental samples, such as soil, acti-vated sludge and cow dung. These isolates were biochem-ically and physiologically characterized in comparisonwith well known PHA-degrading bacteria such as Pa.lemoignei and Ps. fluorescens GK13 (Table 1). All iso-lates were oxidase- and catalase-positive and were able toutilize a wide variety of carbon sources, such as glucose,fructose, gluconate, succinate, lactate, acetate, valerate,3HB and TDP.

Only strain K2, which was identified asPs. indica (seebelow), was able also to grow with poly(3HO-co-3HHx)as sole carbon source. In addition, it was the only isolateutilizing hexanoate and octanoate for growth. Interest-ingly, this strain was able to utilize PHAs of both shortand medium chain length, indicating the presence of atleast two different PHA depolymerases. This Gram-nega-tive strain formed motile rods and grew between 20C and40C, with an optimum temperature at 30C. (It did notgrow at 4C or >50C). The strain hydrolyzed gelatine,starch, Tween 20 and Tween 80 and could also utilize awide range of other carbon sources (Table 1). Colonies onmineral or complex medium were opaque and very vis-cous. The 16S rRNA sequence data revealed an affiliationof strain K2 to the genusPseudomonas, with 98% identityto the butane-utilizing species Ps. indica IMT37. Thislatter strain was previously classified by Pandey et al.(2002). The poly(3HB) and poly(3HO) depolymerase ac-tivities ofPs. indica K2 were measured qualitatively bythe clear-zone technique, using poly(3HO-co-3HHx),poly(3HB) and poly(3HB-co-3MP) overlay agar plates,respectively. During growth of Ps. indica K2 onpoly(3HB-co-3MP), active poly(3HB) depolymerase wassecreted into the culture medium. This strain exhibitedmaximal growth and activity of poly(3HB) depolymerase

217


7/14

218

after 1012h (Fig.1). Poly(3HO) depolymerase activitywas not observed during growth on poly(3HB).

Furthermore, strain K14, which was previously identi-fied as S. thermodepolymerans gen. nov. sp. nov. (Elbanna

et al. 2003), was chosen for a more detailed characteriza-tion. During growth of S. thermodepolymerans onpoly(3HB-co-3MP), the poly(3HB) depolymerase was se-creted into the culture medium. This strain exhibited max-imal growth and activity of its thermotolerant poly(3HB)depolymerase after 14h (Fig.2). The degradation poten-tial ofS. thermodepolymerans was also monitored usingthe clear-zone technique for both poly(3HB) and poly(3HB-co-3MP). The degradation potential was deter-mined by measuring the diameter of clear zones occurringon MSM-agar overlay plates containing poly(3HB) andpoly(3HB-co-3MP) as sole carbon source at 50C for 9

days. As shown in Fig.3, this bacterium could degradeboth polymers. However, the clear-zone diameters in-creased at a significantly higher rate with poly(3HB) thanwith poly(3HB-co-3MP). This strain was of particular in-

terest, due to its growth being optimum at a temperatureof 55C and the temperature optimum of its poly(3HB)depolymerase being between 75C and 90C (see below).

Purification and biochemical characterizationof the thermotolerant poly(3HB) depolymerasefrom S. thermodepolymerans

The extracellular poly(3HB)-depolymerase was purifiedto electrophoretic homogeneity from 4 l culture super-natant ofS. thermodepolymerans grown at 50C in MSM

Fig. 1 Growth ofPs. indicaK2 on poly(3HB-co-3MP) andsecretion of poly(3HB) depoly-merase. Bacterial growth andsecretion of the poly(3HB) de-polymerase ofPs. indica K2was monitored by countingviable cells and measuring theprotein concentration of cell-free supernatant. The culture

supernatant was concentrated100-fold by ultrafiltration; andpoly(3HB) depolymerase ac-tivity was measured at the indi-cated times. UUnits of activity

Fig. 2 Growth ofS. thermo-depolymerans on poly(3HB-co-3MP) and secretion ofpoly(3HB) depolymerase.Bacterial growth and secretionof the poly(3HB) depolymeraseofS. thermodepolymerans weremonitored by counting viablecells and measuring the proteinconcentration of cell-free su-pernatant. The culture super-natant was concentrated 100-fold by ultrafiltration; andpoly(3HB) depolymerase ac-tivity was measured at the in-dicated times


8/14

containing 0.2% (w/v) poly(3HB) as sole carbon source.The purification of the enzyme was achieved in only twosteps, exploiting the thermostability of the enzyme and itshigh affinity to poly(3HB) as shown in Table 2. Attemptsto purify this enzyme by other methods, such as gel filtra-tion, hydrophobic interaction chromatography on butylsepharose and procion green, were not successful. In ad-dition, this enzyme was only slightly bound to DEAEsepharose and Mono Q sepharose.

The molecular mass of the poly(3HB) depolymerasesubunits ofS. thermodepolymerans was 40.01.5 kDa, as

determined by SDS-PAGE (Fig. 4a). The pH optimumranged widely around pH8.2 in both Tris-HCl and potas-sium phosphate buffer; and activity was detectable overpH 6.010.5.

Interestingly, the poly(3HB) depolymerase exhibitedactivity up to 90C, with an temperature optimum be-tween 75C and 90C; and it retained 69% of its activityafter incubation for 24 h at 70C. After storage for 24h attemperatures of 50C or lower, enzyme activities of >90%were measured. After storage at 20C for more than 1 year,about 70% of the enzyme activity could be recovered.However, no activity was detected after incubation for 1 hat 80C or higher temperatures.

The activity of the thermotolerant poly(3HB) depoly-merase was completely inhibited by diisopropylfluo-rophosphate (DFP), dithiothreitol (DTT), disodium ethyl-

diamine tretraacetate (EDTA), dithiodinitrobenzoate (DTNB),phydroxymercurybenzoate, iodoacetate, sodium azide, andmercaptoethanol at concentrations between 1 mM and 10mM. Furthermore, strong inhibitory effects were observedwith 10% (v/v) ethanol, 10% (v/v) n-propanol and TritonX100 (0.21.0 mM), whereas phenylmethanesulfonylfluo-ride (PMSF, 110 mM) and Tween20 (2 mM) reduced theactivity to 40% and 66%, respectively. The activity of this

poly(3HB) depolymerase was also partially inhibited byhigh concentrations of KCl and NaCl salts.The rate of poly(3HB) depolymerization was exam-

ined under a constant concentration of 1 g ml1 purifiedenzyme and was indicated by a decrease in OD. The rateof depolymerization was almost linear up to 100 g ml1

poly(3HB). At concentrations of poly(3HB) up to 350 gml1, only a slight increase in the rate of poly(3HB) hy-drolysis was observed. A linear relationship was noticedin the double-reciprocal plot of the initial velocity of en-zyme activity with varying concentrations of poly(3HB).The apparent Km value of the purified thermotolerantpoly(3HB) depolymerase was 45 g ml1 for poly(3HB).

Purification and biochemical characterizationof poly(3HO) depolymerase fromPs. indica K2

Extracellular poly(3HO) depolymerase was purified from5 l cell-free supernatant of aPs. indica K2 culture grownat 30C in MSM containing 0.15% (w/v) poly(3HO-co-3HHx) as sole carbon source. After 200-fold concentra-tion of the cell-free supernatant, the poly(3HO) depoly-merase was purified by hydrophobic interaction chromato-graphy on an octyl sepharose column. The poly(3HO) de-polymerase was completely eluted by 10% (v/v) ethane-

diol. To remove the remaining contaminating proteins,fractions containing high enzyme activity were combined,concentrated and diafiltered. The concentrated proteinswere applied onto a gel filtration column. Fractions con-taining high enzyme activity were combined, concen-trated, washed by ultrafiltration and stored at 20C. Thepurification of poly(3HO) depolymerase from Ps. indicaK2 is summarized in Table 3.

SDS-PAGE of the purified enzyme revealed a singleband at a molecular mass of 281 kDa (Fig.4b, lane2).The partial N-terminal amino acid sequence of the puri-fied poly(3HO) depolymerase was SERSATLLLPAKVSand exhibited 71% similarity to the poly(3HO) depoly-merase ofPs. fluorescens GK13 (Schirmer et al. 1993).

The poly(3HO) depolymerase ofPs. indica K2 showedactivity at a pH range over6.810.0, with an optimum at

219

Table 2 Purification of thepoly(3HB) depolymerase fromS. thermodepolymerans

Purification step Volume Protein Volume Specific Total Recovery(ml) (mg ml1) activity activity activity (%)

(Uml1) (U mg1) (U)

Concentrated culture supernatant 5 0.45 5,700 13,000 29,000 100

Heat shock at 70C for 30 min 5 0.20 3,900 19,000 19,000 65.5

Affinity binding to poly(3HB) 1 0.12 2,900 25,000 3,000 10.3

Fig. 3 Degradation of poly(3HB) and poly(3HB-co-3MP) byS. thermodepolymerans. Cells were cultivated on MSM-agar platescontaining poly(3HB) or poly(3HB-co-3MP) at 50C for 9daysand the diameters of clear zones were determined. Pale columnssClear-zone diameter on poly(3HB) agar plates, dark columnsclear-zone diameter on poly(3HB-co-3MP) agar plates. dDays


9/14

around pH 8.5 in both Tris-HCl and potassium phosphatebuffer. Interestingly, this enzyme exhibited very high

poly(3HO) depolymerase activity (210,000 U mg

1

) and26Umg1pnitrophenyloctanoate (PNPO) esterase activity,but no activity with poly(3HB). The poly(3HO) depoly-merase exhibited activity in a temperature range of 1550C, with an optimum at 35C. The protein was stable at4C, 20C and 70C for more than 6months.

The activity of poly(3HO) depolymerase ofPs. indicaK2 was drastically inactivated by the serine esterase in-hibitor PMSF (110mM) and completely inhibited by di-isopropylfluorophosphate (DFP). In addition, the activityof poly(3HO) depolymerase was decreased by severalsulfhydryl reagents, such as phydroxymercurybenzoate

(PHMB), iodoacetate, sodium azide and EDTA at concen-trations of 110 mM. Strong inhibitory effects were alsoobserved with non-ionic detergents, such as Triton X-100and Tween 80 (110 mM). In contrast, the enzyme wasnot inhibited by dithiothreitol (DTT).

Substrate specificities of PHA depolymerases

For isolation of the new polymer-degrading bacteria,poly(3HB-co-3MP) was used as a carbon source, becausehomoPTEs were not available at the beginning of thisstudy. All of the 22 isolates and some well studied PHA-degrading bacteria were then also tested for their ability tohydrolyze poly(3MP) (Table 1). Although the copolymerpoly(3HB-co-3MP) was utilized as sole carbon source by allinvestigated bacteria, except Ps. fluorescens DSM7139,the homopolymer poly(3MP) could not be hydrolyzed byany of these strains.

Also, purified PHA depolymerases were investigatedfor their ability to cleave PTEs (Table 4). The substrate

specificity of the poly(3HB) depolymerase ofS. thermo-depolymerans was investigated by the provision of sub-strates containing varying 3MP contents: the specific ac-tivity with poly(3HB) was 25,000 unitsmg1, whereas thecopolymer poly(3HB-co-3MP) was hydrolyzed with aspecific activity of only 9,500 unitsmg1. However, no en-zyme activity was detected with poly(3MP) or poly(3MBhomopolymers. Activities with the latter polymers werealso not measurable using the enzyme fromPs. indica K2.

Other PHA-hydrolyzing enzymes such as PhaZ2,PhaZ5 or PhaZ7 fromPa. lemoignei also revealed no ac-tivity with either PTE homopolymer (Table4), regardlessof whether a drop test on polymer agar plates or an OD

photometric test was applied. However, PhaZ5 and PhaZ2showed significant hydrolysis of poly(3HB-co-3MP),whereas enzyme activity decreased proportionally withhigher 3MP contents of the copolymers.

In order to investigate a putative inhibition of the en-zyme by the sulfhydryl groups released during an initialcleavage of the PTEs, equal volumes of suspensions ofpoly(3HB) granules and poly(3MB) were mixed and theactivity of PhaZ5 was measured. Interestingly, an inter-mediate activity of PhaZ5 was found and variations in thesequential order of addition of the polymers and enzymedid not influence the depolymerase activity.

220

Table 3 Purification of poly(3HO) depolymerase fromPs. indica K2

Purification step Volume protein Esterase activity (pNPO) Poly(3HO) depolymerase activity (OD test)(ml) (mg ml1)

Volume Specific Total Recov- Volume Specific Total Recov-activity activity activity ery activity activity activity ery(Uml1) (U mg1) (U) (%) (U ml1) (U mg1) (U) (%)

Concentrated supernatant 25 0.750 3.82 127 95.5 100 2,500 83,000 62,500 100

Octyl sepharose 30 0.021 0.23 328 6.9 7.2 8,070 11,500,000 242,000 387

Gel filtration 1 0.013 0.34 26 0.34 0.4 2,710 209,000 2,700 4(Sepharose 2000)

Fig. 4 a,b SDS-PAGE of PHA depolymerases of S. thermode-polymerans andPs. indica K2 at different purification levels. SDS-denatured samples (2040l) were separated in a 12% SDS poly-acrylamide gel and silver-stained. a Poly(3HB) depolymerase ofS. thermodepolymerans. Lane 1 Molecular mass standard, lane 26g protein in concentrated culture supernatant, lane 3 3 g pro-tein in the supernatant after incubation at 70C for 30min, lane 42g protein in the supernatant after incubation with 1% (w/v)poly(3HB) suspension at 70C for 15min. b Poly(3HO) depoly-merase of Ps. indica K2. Lane1 molecular mass standard, lane 22g protein in the concentrated pool after gel filtration


10/14

Determination of hydrolysis productsof poly(3HB-co-3MP)

Poly(3HB-co-3MP) containing 23mol% 3MP was incu-bated with the purified PHA depolymerase ofS. thermo-depolymerans; and the compounds remaining from par-tial degradation of the copolymer in the water-insoluble orwater-soluble fractions, respectively, were analyzed by var-ious chemical analyses (Fig. 5). GC analysis of the origi-nal polymer before degradation gave a 3HB:3MP mole ra-tio of 3.4:1.0 (Fig.5a). After incubation of the polymerwith the enzyme, 3MP was enriched in the insoluble frac-

tion (3HB:3MP=1.0:3.7), whereas 3HB was enriched inthe soluble fraction (3HB:3MP=8.7:1.0; Fig.5b). This in-dicated a selective cleavage of oxoester bonds and prefer-ential release of low molecular weight degradation prod-ucts containing 3HB, whereas 3MP remained mostly in the

high molecular weight insoluble fraction. The IR spectra ob-

221

Enzyme Relative activity

Poly(3HO) Poly(3HB) Poly(3HB- Poly(3HB- Poly(3MP) Poly(3MB)co-12%3MP) co36%3MP)

Poly(3HB) depolymerase +++ (100%) ++ (38%) + (10%) ofS. thermodepolymeransa,b

Poly(3HO) depolymerase +++ (100%) ofPs. indica K2a,b

Poly(3HB) depolymerase (PhaZ2) +++ ++ + nd ndofPa. lemoigneib

Poly(3HB) depolymerase (PhaZ5) +++ ++ + nd ofPa. lemoigneib

Poly(3HB) depolymerase (PhaZ7) nd ofPa. lemoigneib

Table 4 Relative activities of PHA depolymerases with PHAsand PTEs. PHA depolymerase activities were determined as in-dicated by superscript letters. PhaZ7 activity was confirmed byhydrolysis of native (amorphous) poly(3HB) granules. +++ High

activity/clear zone formation, ++ medium activity/clear zone for-mation, + low activity/clear zone formation, no activity/clearzone formation detected, ndnot determined

aPHA depolymerase activities were determined by spectrophoto-metric OD assay

bPHA depolymerase activities were determined by the clear-zonemethod on indicator plates containing 100 mM Tris-HCl (pH 8)containing 1 mM CaCl2 and 0.20.3% (w/v) polymer granules

Fig. 5 ad Analysis of degradation products of poly(3HB-co-3MP) obtained with purified poly(3HB) depolymerase ofS. ther-modepolymerans. In a total volume of 90ml Tris-HCl buffer (50mM, pH8.5), 90 mg of poly(3HB-co-3MP) containing 23mol%3MP and 300 g purified S. thermodepolymerans PHA depoly-merase were incubated for 24 h at 50C. Thereafter, the reactionmixture was centrifuged for 30 min at 13,000rpm to separate itinto a water-insoluble (pellet) and a water-soluble fraction (super-natant). Both fractions were subsequently lyophilized and themasses of the lyophilized materials were determined. These sam-ples and the original copolymer were then dissolved separately in

10 ml chloroform/methanol mixture (1:1, v/v), vortexed and cen-trifuged for 30min at 13,000rpm. Aliquots of all samples weresubjected to GC and GC/MS analysis and analyzed for 3HB (opensquares) and 3MP (filled squares) contents as described in the Ma-terials and methods (a, b). Aliquots of the water-insoluble fractionwere also subjected to IR spectroscopy (c) and MALDI-TOFanalysis (d), as described in the Materials and methods


11/14

tained from the samples confirmed the GC data and showedthat the intensity of the absorption band at 1,688cm1 (rep-resenting the C=O valence vibration of the thioester bond)increased to about the same extent in comparison with theabsorption band at 1,735cm1 (representing the oxoesterbond), as shown in the IR spectrum of the water-insolublematerial (Fig.5c). In addition, elemental sulfur analysisshowed an increase in the sulfur content of this fractionin comparison with the original polymer (data not shown).1H-NMR spectroscopic analysis of the water insoluble frac-tion (data not shown) confirmed the presence of 3MP andthioester bonds in the compounds present in this fraction.When the remaining water-insoluble fraction was analyzed by

MALDI-TOF, the m/z values of the detected compounds in-dicated the presence of oligomers of 3MP (Fig.5d). The m/zvalues fit to oligo-3MP (716units) with a 3HB moiety atthe end, linked as a thioester. The ionization was accom-plished by sodium addition (Na+). This could be only ex-plained by the occurrence of a non-statistical distribution of3MP moieties in the poly(3HB-co-3MP) copolymer.

However, ESI/MS/MS analysis revealed the presenceof oligomeric compounds in the soluble fraction. Depend-ing on the ionization method, different negatively chargedmolecules were detected, indicating incomplete degrada-tion of the copolymer. The main degradation products were

dominated by a peak at m/z=103, corresponding to the3HB monomer. Also, m/z peaks corresponding to dimers,trimers or oligomers of 3HB and/or 3MP were detected,although their relative abundance was less than 4% (datanot shown). For example, the daughter ions at m/z=103,105 from the [M-H] peak detected at m/z=191 identifiedthe corresponding molecule as a 3HB-co-3MP dimer (Fig. 6).

Discussion

The copolymer poly(3HB-co-3MP), which was obtainedfrom the well known PHA-accumulating bacteriumR. eu-

tropha, was the first example of a biosynthetic PTE (Ltke-Eversloh et al. 2001a). Later, copolymers containing othermercaptoalkanoic acids as constituents and also variousPTE homopolymers became available (Ltke-Eversloh etal. 2001b, 2002a). The discovery of this novel type ofbiopolymer raised the question whether PTEs, which aresynthesized by PHA synthases, are also susceptible to thehydrolytic attack of PHA depolymerases. The main goalof this study was to evaluate whether PHA depolymerasesare equally unspecific as PHA synthases towards thepolymer linkage type. In this study, we isolated more than20 bacteria able to grow with poly(3HB-co-3MP) as solecarbon source. All bacteria isolated revealed good growth

and formed clear zones on overlay agar plates containingpoly(3HB-co-3MP). However, no PHA-degrading bac-terium was identified which could also use poly(3MP)homopolymer as sole carbon source for growth.

After taxonomic and physiological characterization, twostrains were selected for further detailed analysis: S. ther-modepolymerans and Ps. indica K2. S. thermodepoly-merans was recently classified as a new thermophilicPHA-degrading bacterium (Elbanna et al. 2003). Here, wedescribe the purification and biochemical characterizationof the extracellular poly(3HB) depolymerase from thisbacterium, which exhibited an unusual thermotolerance.This poly(3HB) depolymerase exhibited activity up to90C, with an optimum temperature between 75C and80C, which is higher than that of the other thermophilicpoly(3HB) depolymerases, such as the poly(3HB) de-polymerase (70C) of C. manganoxidans (Takeda et al.1998, 2002) and the thermoalkalophilic hydrolase of

Pa. lemoignei (Handrick et al. 2001). Furthermore, thepoly(3HB) depolymerase from S. thermodepolymeranswas highly stable up to 70C for more than 24h. Com-plete inhibition ofS. thermodepolymerans poly(3HB) de-polymerase activity by DFP or DTT and partial inhibitionby EDTA indicated this enzyme belongs to the serine hy-drolase family like other PHA depolymerases, in that the

222

Fig. 6 Occurrence of a 3HB-co-3MP dimer in the water-soluble degradation productsafter incubation of poly(3HB-co-3MP) containing 23mol%3MP with purified S. thermo-depolymerans poly(3HB) de-polymerase. Structural analy-sis of ions represented by thepeaks at m/z=191 was done by

ESI/MS/MS in the negativemode. The fragmentation pat-tern of the peak at m/z=191confirmed the proposed struc-ture for the 3HB-co-3MP dimer


12/14

enzyme requires some metal ions and disulfide bonds playan essential role in enzyme activity (Mller and Jendros-sek 1993; Jendrossek et al. 1995). The activity of thethermophilic poly(3HB) depolymerase from S. thermode-

polymerans was inhibited by PMSF and sodium azide. Incontrast, the thermophilic poly(3HB) depolymerase ofC. manganoxidans (Takeda et al. 1998, 2002) was not in-hibited. A strong sensitivity of poly(3HB) depolymerase

from S. thermodepolymerans against low and high con-centrations of nonionic detergents such as Triton X-100and Tween 20 suggested that a hydrophobic region maybe located near or at the active site (Kim et al. 2002). Sim-ilar to the poly(3HB) depolymerase from A. faecalis T1,the activity of the poly(3HB) depolymerase from S. ther-modepolymerans was completely abolished by trypsintreatment (Shiraki et al. 1995). Other properties of the en-zyme, like molecular mass and pH optimum, were similarto those of other extracellular poly(3HB) depolymerases(Jendrossek and Handrick 2002).

Ps. indica K2 was selected due to its ability to hydro-lyze PHAs of both short chain length and medium chain

length (PHASCL, PHAMCL), which is rare and has been ob-served in only a few other bacteria (Klingbeil et al. 1996;Kim et al. 2000, 2003). To our knowledge, this is the firstreport of a Pseudomonas strain which produces two dif-ferent depolymerases, one for PHASCL and one forPHAMCL. To confirm this, the extracellular poly(3HO) de-polymerase of Ps. indica K2 was purified to electro-phoretic homogeneity and biochemically characterized.The purified poly(3HO) depolymerase fromPs. indica K2is relatively similar to those from Ps. alcaligenes LB 19(Kim et al. 2002) and Streptomyces sp. KJ-72 (Kim et al.2003) in its molecular mass, optimum temperature andsensitivity to some inhibitors, such as DFP, Triton X-100 and

Tween 80. In contrast, like otherPseudomonas PHAMCLdepolymerases, the poly(3HO) depolymerase fromPs. in-dica K2 was not inhibited by DTT, suggesting that disul-fide bonds in the active site of these enzymes are not es-sential for their activities (Schirmer et al. 1993; Kim et al.2002).

Investigations on the substrate specificity of the puri-fied poly(3HB) depolymerase from S. thermodepoly-merans included PTEs with varying 3MP content. In com-parison with the poly(3HB) homopolymer, enzyme activ-ity decreased significantly according to the 3MP content;and finally no activity was detected with poly(3MP). Sim-ilar results were obtained with the three well studied PHAdepolymerases (PhaZ2, PhaZ5, PhaZ7) fromPa. lemoignei,which serves as a model organism for PHA degradation(Jendrossek and Handrick 2002), as compiled in Table 4.Further, poly(3MP), poly(3MB) as a second represen-tative of PTE homopolymers was also not hydrolyzedby any of these enzymes. According to the compoundsdetected by analysis of the chemical composition ofthe degradation products, the lower biodegradability ofpoly(3HB-co-3MP), compared with that of poly(3HB), isdue to the incorporation of 3MP units into polymer, form-ing thioester linkages in the polymer backbone that arenot susceptible to the poly(3HB) depolymerase from

S. thermodepolymerans. Similar observations were madeby Mller and Jendrossek (1993) and Oda et al. (1997),who found that the activity of PHA depolymerases de-creased as the proportion of 3-hydroxyvalerate (3HV) in-creased in poly(3HB-co-3HV) copolymers.

Analysis of the degradation products also provided evi-dence that the sequence distribution of the comonomers3HB and 3MP in poly(3HB-co-3MP) is not random and

does not obey Bernoullian statistics, because oligomersconsisting of up to about 12 3MP moieties were detectedin the water-insoluble fraction of the degradation prod-ucts. Such block copolymers were in the past also ob-tained for other PHAs, for example poly(3HB-co-4HB)and poly(3HV-co-4HB), when the producing bacteriawere cultivated on a mixture of two carbon sources (Gotoet al. 1989; Choi et al. 2003) as was also the case in thisstudy.

In conclusion, this work clearly shows that PHA de-polymerases are specific for oxoester linkages and do nothydrolyze thioester bonds. It may be assumed that, if 3MPcontaining copolymers are provided as substrates, cleav-

age of the copolymers occurs at the oxoester linkagesonly, releasing 3HB moieties which are then utilized as acarbon source. Fractions of the polymer consisting mainlyof 3MP moieties and most likely representing oligomersof 3MP are probably left and cannot be further degraded.Since bothPs. indica K2 and S. thermodepolymerans use3MP as sole carbon source for growth, the inability tocleave the thioester bonds in the polymer and probablyalso the oligomers prevents these bacteria from utilizingpoly(3MP) as a carbon source.

This study clearly shows that PHA-degrading enzymesare not able to hydrolyze PTEs; and therefore new ap-proaches must be applied to identify PTE-degrading en-

zymes. It seems likely that biologically synthesized PTEsare also biodegradable, although the biodegradation ofthese novel polymers may be catalyzed by different en-zymes. Since homoPTEs recently became available bybiotechnological production employing engineered strainsofE. coli(Ltke-Eversloh et al. 2002a), sufficient amountsof these novel biopolymers can now be used for thescreening of bacteria and/or fungi able to degrade PTEs.

Acknowledgements Provision of a fellowship to K.E. by thegovernment of the Arab Republic of Egypt is gratefully acknowl-edged. The authors are also grateful to Dr. Klaus Bergander forNMR spectroscopic analysis.

References

Anderson AJ, Dawes EA (1990) Occurrence, metabolism, meta-bolic role, and industrial use of bacterial polyhydroxyalkanoates.Microbiol Rev 54:450472

Birnboim HC, Doly J (1979) A rapid alkaline procedure forscreening recombinant plasmid DNA. Nucleic Acids Res 7:-15131523

Bradford MM (1976) A rapid and sensitive method for quantita-tion of microgram quantities of protein utilizing the principleof proteindye binding. Anal Biochem 72:248254

223


13/14

Brandl H, Gross RA, Lenz RW, Fuller RC (1988) Pseudomonasoleovorans as source of poly(-hydroxyalkanoates) for poten-tial applications as biodegradable polyesters. Appl EnvironMicrobiol 54:19771982

Choi MH, Lee HJ, Rho JK, Yoon SC, Nam JD, Lim D, Lenz RW(2003) Biosynthesis and local sequence specific degradation ofpoly(3-hydroxyvalerate-co-4-hydroxybutyrate) in Hydrogeno- phaga pseudoflava. Biomacromolecules 4:3845

Cole RB (1997) Electrospray ionization mass spectrometry. Wiley,New York

Delafield FP, Doudoroff M, Palleroni NJ, Lusty CJ, ContopoulosR (1965) Decomposition of poly--hydroxybutyrate byPseudo-monas. J Bacteriol 90:14551466

Elbanna K, Ltke-Eversloh T, Van Trappen S, Mergaert J, SwingsJ, Steinbchel A (2003) Schlegelella thermodepolymerans gen.nov., sp. nov., a novel thermophilic bacterium that degradespoly(3-hydroxybutyrate-co-3-mercaptopropionate). Int J SystEvol Microbiol 53:11651168

Goto Y, Kamiya N, Yamamoto Y, Inoue Y, Chujo R, Kunioka M,Doi Y (1989) NMR analysis of microstructure in bacterialpoly(3-hydroxybutyrate-co-4-hydroxybutyrate). Rep Prog PolymPhys Jpn 32:639642

Handrick R, Reinhardt S, Letizia-Focarete M, Scandola M,Adamus G, Kowalczuk M, Jendrossek D (2001) A new typeof thermoalkanophilic hydrolase of Paucimonas lemoigneiwith high specificity for amorphous polyesters of short chain-

length hydroxyalkanoic acids. J Biol Chem 276:3621536224Heukeshofen J, Dernick R (1985) A simplified method for silverstaining of proteins in polyacrylamide gels and the mechanismof silver staining. Electrophoresis 6:103112

Hocking PJ, Marchessault RH (1994) Biopolyesters. In: GriffinGJL (ed) Chemistry and technology of biodegradable poly-mers. Chapman and Hall, London, pp 4896

Iwata S, Toshima K, Matsumura S (2003) Enzyme-catalyzedpreparation of aliphatic polyesters containing thioester link-ages. Macromol Rapid Commun 24:467471

Jendrossek D (2002) Extracellular polyhydroxyalkanoate depoly-merases: the key enzymes of PHA degradation. In: Doi Y,Steinbchel A (eds) Biopolymers, vol 3b. WileyVCH, Wein-heim, pp 4177

Jendrossek D, Handrick R (2002) Microbial degradation of poly-hydroxyalkanoates. Annu Rev Microbiol 56:403432

Jendrossek D, Knoke I, Habibian RB, Steinbchel A, Schlegel HG(1993) Degradation of poly(3-hydroxybutyrate), PHB, by bac-teria and purification of a novel PHB depolymerase fromComamonas sp. J Environ Polymer Degrad 1:5363

Jendrossek D, Backhaus M, Andermann M (1995) Characterizationof the extracellular poly(3HB) depolymerase of Comamonassp. and its structural gene. Can J Microbiol 41[Suppl1]:160169

Jendrossek D, Schirmer A, Schlegel HG (1996) Biodegradation ofpolyhydroxyalkanoic acids. Appl Microbiol Biotechnol 46:451463

Kasuya K, Doi Y, Yao T (1994) Enzymatic degradation of poly[(R)-3-hydroxybutyrate] by Comamonas testosteroni ATSU of soilbacterium. Polym Degrad Stab 45:379386

Kawada J, Ltke-Eversloh T, Steinbchel A, Marchessault RH(2003) Physical properties of microbial polythioesters: charac-terization of poly(3-mercaptoalkanoates) synthesized by engi-neeredEscherichia coli. Biomacromolecules 4:16981702

Kim H, Ju HS, Kim J (2000) Characterization of an extracellularpoly(3-hydroxy-5-phenylvalerate) depolymerase fromXantho-monas sp. JS02. Appl Microbiol Biotechnol 53:323327

Kim DY, Yun JH, Kim HW, Bae KS, Rhee YH (2002) Purifica-tion and characterization of poly(3-hydroxybutyrate) depoly-merase from a fungal isolate,Emericellopsis minima W2. J Mi-crobiol 40:129133

Kim HJ, Kim DY, Nam JS, Bae KS, Rhee YH (2003) Characteri-zation of an extracellular medium chain-length poly(3-hydroxy-alkanate) depolymerase from Streptomyces sp. strain KJ-72.Antonie van Leeuwenhoek 83:183189

Klingbeil B, Kroppenstedt RM, Jendrossek D (1996) Taxonomicidentification ofStreptomyces exfoliatus K10 and characteriza-tion of its poly(3-hydroxybutyrate) depolymerase gene. FEMSMicrobiol Lett 142:215221

Laemmli UK (1970) Cleavage of structural proteins during the as-sembly of the head of bacteriophage T4. Nature 227:680685

Ltke-Eversloh T, Bergander K, Luftmann H, Steinbchel A(2001a) Identification of new class of biopolymer: bacterialsynthesis of a sulfur-containing polymer with thioester link-ages. Microbiology 147:1119

Ltke-Eversloh T, Bergander K, Luftmann H, Steinbchel A(2001b) Biosynthesis of poly(3-hydroxybutyrate-co-3-mer-captobutyrate) as a sulfur analogue to poly(3-hydroxybutyrate)(PHB). Biomacromolecules 2:10611065

Ltke-Eversloh T, Fischer A, Remminghorst U, Kawada J,Marchessault RH, Bgershausen A, Kalwei M, Eckert H,Reichelt R, Liu S, Steinbchel A (2002a) Biosynthesis of novelthermoplastic polythioesters by engineered Escherichia coli.Nat Mater 1:236240

Ltke-Eversloh T, Kawada J, Marchessault RH, Steinbchel A(2002b) Characterization of biological polythioesters: physi-cal properties of novel copolymers synthesized by Ralstoniaeutropha. Biomacromolecules 3:159166

Mergaert J, Swings J (1996) Biodiversity of microorganisms thatdegrade bacterial and synthetic polyesters. J Ind Microbiol 17:463469

Mller B, Jendrossek D (1993) Purification and properties ofpoly(3-hydroxyvaleric acid) depolymerase fromPseudomonaslemoignei. Appl Microbiol Biotechnol 38:487492

Mller R, Antranikian G, Maloney S, Sharp R (1998) Thermo-philic degradation of environmental pollutants. Adv BiochemEng Biotechnol 61:155169

Oda Y, Osake H, Urakami T, Tonomura K (1997) Purification andproperties of poly(3-hydroxybutyrate) depolymerase from thefungusPaecilomyces lilacimus D218. Curr Microbiol 34:230232

Page RDM (1996) Tree View: an application to display phylo-genetic trees on personal computers. Comput Appl Biosci 12:357358

Pandey KK, Mayilraj S, Chakrabarti T (2002) Pseudomonas in-dica sp. nov., a novel butane-utilizing species. Int J Syst EvolMicrobiol 52:15591567

Pasch H, Schrepp W (2003) MALDI-TOF mass spectrometry ofsynthetic polymers. Springer, Berlin Heidelberg New YorkRainey FA, Rainey NW, Kroppenstedt RM, Stackebrandt E (1996)

The genusNocardiopsis represents a phylogenetically coherenttaxon and distinct actinomycete lineage: proposal ofNocardio-paceae fam. nov. Int J Syst Bacteriol 46:10881092

Saitou N, Nei M (1987) The neighbor-joining method: a newmethod for reconstructing phylogenetic trees. Mol Biol Evol4:406425

Schirmer A, Jendrossek D, Schlegel HG (1993) Degradation ofpoly(3-hydroxyoctanoic acid) [P(3HO)], by bacteria. Purifica-tion and properties of a P(3HO) depolymerase ofPseudomonasfluorescens GK13 biovar V. Appl Environ Microbiol 59:12201227

Schlegel HG, Kaltwasser H, Gottschalk G (1961) Ein Submersver-fahren zur Kultur wasserstoffoxidierender Bakterien: wachs-tumsphysiologische Untersuchungen. Arch Mikrobiol 38:209222

Shiraki M, Shimada T, Tatsumichi M, Saito T (1995) Purificationand characterization of extracellular poly(3HB) depolymerases.J Environ Polym Degrad 3:1321

Steinbchel A, Ltke-Eversloh T (2003) Metabolic engineeringand pathway construction for biotechnological production ofrelevant polyhydroxyalkanoates in microorganisms. BiochemEng J 16:8196

Steinbchel A, Valentin HE (1995) Diversity of bacterial poly-hydroxyalkanoic acids. FEMS Microbiol Lett 128:219228

224


14/14

225

Steinbchel A, Aerts K, Babel W, Fllner C, Liebergesell M,Madkour MH, Mayer F, Pieper-Frst U, Pries A, ValentinHE, Wieczorek R (1995) Consideration on the structure andbiochemistry of bacterial polyhydroxyalkanoic acids inclu-sions. Can J Microbiol 41[Suppl 1]:94105

Takeda M, Koizumi J, Yaebe K, Adachi K (1998) Thermostablepoly(3-hydroxybutyrate) depolymerase of a thermophilic strainofLeptothrix sp. isolated from hot spring. J Ferment Bioeng85:375380

Takeda M, Kitashima K, Adachi K, Hanaoka Y, Suzuki I, Koizumi

J (2000) Cloning and expression of the gene encoding ther-mostable poly(3-hydroxybutyrate) depolymerase. J Biosci Bio-eng 90:416421

Takeda M, Kamagata Y, Ghirose WC, Hanada S, Koizumi J(2002) Caldimonas manganoxidans gen. nov., sp nov., apoly(3-hydroxybutyrate)-degrading, manganese-oxidizingthermophile. Int J Syst Evol Microbiol 52:895900

Thayer AM (1990) Degradable plastics generate controversy insolid waste issues. Chem Eng News 25:714

Thompson JD, Gibson TJ, Plewinak F, Jeanmougin F, Higgins DG(1997) The Clustal X windows interface: flexible strategies formultiple sequence alignment aided by quality analysis tools.Nucleic Acids Res 25:48674882

Timm A, Steinbchel A (1990) Formation of polyesters ofmedium-chain-length 3-hydroxyalkanoic acids from gluconateby Pseudomonas aeruginosa and other fluorescent pseudo-monads. Appl Environ Microbiol 56:33603367

biodegradability of pha

Documents