BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS

An extracellular polyhydroxybutyrate depolymerasein Thermus thermophilus HB8

Christos P. Papaneophytou & Anastasia A. Pantazaki &Dimitrios A. Kyriakidis

Received: 19 February 2008 /Revised: 22 December 2008 /Accepted: 23 December 2008 / Published online: 13 February 2009# Springer-Verlag 2009

Abstract The thermophilic bacterium Thermus thermo-philus HB8 has been characterized as a polyhydroxybuty-rate (PHB)-degrading microorganism since it growsefficiently and forms clear zones on agar plates containingPHB as sole carbon source. T. thermophilus extracellularPHB depolymerase was purified to homogeneity using anaffinity chromatography protocol. The purified enzyme wasestimated to have an apparent molecular mass of 42 kDa.The extracellular PHB depolymerase gene was identified asthe TTHA0199 gene product of T. thermophilus HB8. Theamino acid sequence of the TTHA0199 gene productshared significant homologies to other carboxylesterases.A catalytic triad was identified consisting of S183, E310, andH405. A pentapeptide sequence (GX1SX2G) exists withinthe molecule, characteristic for PHB depolymerases (lipasebox) and for other serine hydrolases. Purified extracellularPHB depolymerase was stable at high temperatures with anoptimum activity at pH 8.0. The apparent Km value of thepurified enzyme for PHB was 53 μg/ml. As the mainproduct of the enzymic hydrolysis of PHB, the monomer 3-hydroxybutyrate was identified, suggesting that the enzymeacts principally as an exo-type hydrolase.

Keywords Poly-hydroxybutyrate (PHB) .

PHB-depolymerase . Thermus thermophilus

Introduction

Polyhydroxyalkanoates (PHAs) are intracellular storage com-pounds of carbon and energy that are produced by manybacteria in the form of inclusion bodies (Anderson and Dawes1990). PHAs have attracted commercial biotechnologicalinterest because of their biodegradability and biocompatibil-ity (Reddy et al. 2003). Degradation of PHAs can take placeeither extracellularly or intracellularly by specific PHAdepolymerases which are carboxyesterases (EC 3.1.1.75,EC 3.1.1.76; Jendrossek and Handrick 2002). The distinctionbetween extra- and intracellular degradation is essential sincePHA can be present in two biophysical conformations: theintracellular physiological amorphous (native) polymer(nPHA), which is covered by a surface layer consisting ofproteins and phospholipids, and the extracellular denaturedPHA (dPHA) which is deprived of the surface layer uponextraction from the cell (Merrik et al. 1965).

Extracellular PHA degradation takes place by specificPHA depolymerases that hydrolyze the water-insolublepolymer to water-soluble oligomers or monomers (Jendros-sek et al. 1996; Jendrossek and Handrick 2002). On theother hand, under carbon starvation, the intracellular PHAreservoir, accumulated in the form of polymers, is degradedby intracellular PHA depolymerases (Handrick et al. 2001).

Polyhydroxybutyrate (PHB) is the best-characterizedPHA and accounts for up to 90% of the cellular dry weightduring unbalanced growth (Doi and Steinbüchel 2001).Most enzymes that hydrolyze PHB are specific for eitherthe nPHB or the dPHB form (Handrick et al. 2004). Morethan 20 different extracellular dPHB depolymerases have

Appl Microbiol Biotechnol (2009) 83:659–668DOI 10.1007/s00253-008-1842-2

C. P. Papaneophytou :A. A. Pantazaki :D. A. Kyriakidis (*)Laboratory of Biochemistry, Department of Chemistry,Aristotle University of Thessaloniki,54124 Thessaloniki, Greecee-mail: kyr@chem.auth.gr

D. A. KyriakidisNational Hellenic Research Foundation,48 Vas. Constantinou,11636 Athens, Greece

been characterized during the last decade (Jendrossek andHandrick 2002). It is important to note that elevatedtemperatures improve dramatically the bioavailability andsolubility of organic polymeric compounds and biodegrad-able environmental pollutants allowing efficient bioremedi-ation (Müller et al. 1998). At higher temperatures, PHB isamorphous and more sensitive to degrading enzymes,allowing its rapid degradation (Takeda et al. 1998).However, while numerous reports exist on mesophilicpolymer-degrading bacteria, reports on thermophilic PHA-degrading bacteria and thermostable PHA depolymerasesappeared only recently (Elbanna et al. 2004). For example,a new type of thermoalkalophilic hydrolase of Paucimo-nas lemoigni, with high specificity for amorphous poly-esters of short-chain-length hydroxyalkanoic acids, hasbeen described (Handrick et al. 2001), while another typeof a thermoalkalophilic extracellular esterase-degradingPHB has been isolated from the thermophilic Anoxyba-cillus gonensis G2 (Çolak et al. 2005). Moreover, athermophile extracellular PHB depolymerase isolatedfrom Caldimonas manganoxidans has been characterized(Takeda et al. 1998, 2000, 2002), and a PHA depolymer-ase exhibiting optimal temperature at 70°C was purifiedfrom the culture broth of Comamonas testosteroni ATSU(Kasuya et al. 1994), although this soil bacterium did notretain its depolymerase activity at this optimal reactiontemperature. Moreover, a thermophilic PHB depolymerasehas been purified from gram-positive Streptomyces sp.,which hydrolyzed not only bacterial polyesters, like PHBand poly(3-hydroxybutyrate-co-3-hydroxyvalerate), butalso synthetic, aliphatic polyesters such as polypropiolac-tone, poly(ethylene adipate), and poly(ethylene succinate)(Calabia and Tokiwa 2006).

In this report, we describe the purification of anextracellular PHB depolymerase from Thermus thermo-philus grown in liquid minimal salt medium using PHB assole carbon source by a novel chromatographic affinitymethod. The physicochemical and catalytic properties ofthe purified enzyme were studied, and its gene wasidentified.

Materials and methods

Materials

PHB was obtained from Fluka Chemica GmbH(Steinheim, Germany). Agar (gellam gum) was obtainedfrom Roeper GmbH. Trypsin was purchased from BDH(Dorset, UK). All other chemicals were purchased fromSigma Chem. Co. (St. Louis, MO, USA). Stock solutionsof 10 mM p-nitrophenylbutyrate (pNPB) were prepared inethanol

Bacterial strain and cultivation conditions

T. thermophilus HB8 (DSM 579) was grown in a richmedium (DSMZ-74) containing per liter: 8 g tryptone, 4 gyeast extract, and 2 g NaCl. For PHB depolymeraseproduction, cultivation was carried out in mineral saltmedium (MSM) containing per liter: 10 g NaCl, 1 g NH4Cl,1.6 g K2HPO4, 0.2 mg KH2PO4, 15 mg CaCl2, 123 mgMgSO4·7H2O, 0.1 g yeast extract, 1 g PHB powder, and6 ml of a trace element solution (Pantazaki et al. 2003).PHB supplementation was in the form of thin filmsgenerated at the bottom of the flasks by dissolving theappropriate amount of PHB in chloroform, followed byevaporation. The medium was inoculated with 5% (v/v) ofan 18-h T. thermophilus culture in DSMZ-74 medium andsubsequently incubated on a rotary shaker at 70°C for 72 h.Aliquots of 10 ml were centrifuged at 6,000×g for 15 min.The cell-free supernatant was used for assaying extracellu-lar PHB depolymerase activity.

Biological determination of extracellular PHBdepolymerase activity

Extracellular PHB depolymerase activity was assessed viathe ability of T. thermophilus to degrade 0.25% (w/v) PHBthat was added in 1.1% MSM agar plates. Aliquots of 10 μlfrom of an overnight liquid culture of T. thermophilus inMSM containing 0.1% (w/v) PHB as sole carbon sourcewere gently inoculated into agar plates and incubated at 70°C. The agar plates were prepared as described previously(Augusta et al. 1993; Nishida and Tokiwa 1993).

PHB depolymerase assays

For the determination of PHB depolymerase activity of thecrude or purified enzyme, a stable suspension of PHB in a50 mM Tris–HCl buffer solution, pH 7.0, was prepared bysonication at 20 kHz for 10 min (Ghanem et al. 2005). Thestandard reaction mixture contained 50 mM Tris–HClbuffer (pH 8.0), 150 μg/ml PHB, and 2 mM CaCl2. Thereaction was started by the addition of the enzyme andfollowed by the decrease in PHB turbidity measuredspectrophotometrically at 650 nm. One unit of PHBdepolymerase activity was defined as the amount of enzymethat was required to decrease the OD650 by 0.1 min−1

(Jendrossek et al. 1993).A second approach to determine PHB depolymerase

activity was used, taking advantage of the esterase activityof PHB depolymerase when pNPB is used as a substrate(Schirmer et al. 1993). The concentrated p-nitrophenolreleased by the hydrolysis of pNPB was measuredcolorimetrically at 410 nm; 1 U of pNPB esterase activitywas defined as the hydrolysis of 1 μmole/min of pNPB at

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65°C. The reaction mixture consisted of 0.5 mM pNPB,2 mM CaCl2, and 50 mM Tris–HCl pH 8.0. Afterincubation at 65°C for 5 min, the reaction was started bythe addition of 10–100 μl of enzyme solution.

Protein determination, gel electrophoresis, and zymogramanalysis

The concentration of protein was determined by theBradford method (Bradford 1976), and bovine albuminwas used as standard. Proteins were separated by electro-phoresis in 10% (w/v) sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE) as describedpreviously (Laemmli 1970). The hydrolytic activity (car-boxylesterase activity) was detected by separating theproteins by electrophoresis in gels and by a re-naturaliza-tion process consisting of washing the gels in a solution of20 mM Tris–HCl buffer, pH 8.0, containing 0.5% (w/v)Triton X-100, for 20 min at 65°C (Fuciños et al. 2005). Theesterase activity was detected with α-naphthyl acetate and4-benzamido-2,5-dimethoxy-benzenediazonium (Fast BlueRR), as described previously (Fuciños et al. 2005)

Purification of the PHB depolymerase from T. thermophilus

The extracellular PHB depolymerase was isolated from thecell-free supernatant of T. thermophilus culture, grown at70°C in MSM, containing 0.1% (w/v) PHB as sole carbonsource as described previously (Elbanna et al. 2004) withsome modifications. Briefly, cell-free supernatant wasconcentrated 100-fold by passage through an ultrafiltrationnitrocellulose membrane (YM 30; Amicon, Beverly, MA).The concentrated protein was diluted with 50 mM Tris–HCl

buffer, pH 8 at 4°C, and subsequently heated to 80°C for30 min. The precipitated proteins were removed bycentrifugation at 20,000×g. Subsequently, a 0.1% (w/v)suspension of PHB was added for selective binding of thePHB depolymerase at 70°C under continuous stirring. Theresulting mixture of enzyme and PHB were filtered througha Whatman No. 5 filter paper, washed twice with 50 mMTris–HCl buffer pH 8.0, and the PHB depolymerase elutedwith 0.5 M KCl in the same buffer. The eluent was dialyzedagainst 50 mM Tris–HCl buffer pH 8.

Matrix-assisted laser desorption mass spectrometry

Protein samples were subjected to SDS-PAGE. Proteinbands were excised from the gel and subjected to matrix-assisted laser desorption mass spectrometry (MALDI-MS;Bruker Reflex III). Following digestion with 400 ngtrypsin, the resulting peptides were desalted using ZIP-TIPS (Millipore) and spotted onto Bruker gold anchor(AnchorChip 400) targets with 2,5-dihydroxybenzoic acidmatrix. Analysis was carried out in positive mode with amass range of 500–3,500 Da. Data analysis was performedusing Biotools software and Mascot search algorithms.

Determination of hydrolysis products of PHB

After enzymic hydrolysis of PHB by the purified PHBdepolymerase for various time intervals at 70°C, thereaction mixture was centrifuged at 12,000×g for (45 min,at 4°C). The supernatant containing the reaction productswas concentrated 10-fold in a rotary evaporator and usedfor product determination. The derivatization of PHB

Fig. 1 Growth and secretion of PHB depolymerase of T. thermophilusHB8 in liquid culture containing 0.1% (w/v) PHB as sole carbonsource. Secretion of active PHB depolymerase activity was measuredusing PHB as substrate at the indicated times. Optical density (closedsquares); extracellular protein (closed triangles); extracellular PHBdepolymerase activity (open circles)

Fig. 2 Detection of enzymes exhibiting carboxylesterase activity inSDS-PAGE. Renaturation of the proteins was performed by washingthe gel for 20 min at 65°C in 20 mM Tris–HCl buffer (pH 8.0)containing 0.5% (w/v) Triton X-100. The gel was stained with α-naphthyl acetate and Fast Blue RR. Lane 1 cell-free supernatant, lane2 prestained molecular weight markers

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hydrolysis products was performed using bromophenylacylbromide (BPB) according to Durst et al. (1975) andGebauer and Jendrossek (2006).

Detection and quantification of PHB hydrolysis products

Twenty microliters of the derivatization mixture was loadedonto a reverse-phase C18 high-performance liquid chroma-tography (HPLC) column (Separon SGX C18, 7 μm, 200×4.6 mm). The analysis was performed with a Q-Gradquaternary gradient pump of LabAlliance and a photodiodearray detector (Finnigan Spectral System UV6000LP).Samples were eluted at a flow rate of 0.6 ml/min. Elutionwas performed as reported previously (Gebauer andJendrossek 2006). The absorbance of BPB derivatives wasdetected at 254 nm.

Results

Secretion of the extracellular PHB depolymerase

Colonies of T. thermophilus grown on agar containing PHBas sole carbon source were surrounded by translucent halosindicating the hydrolysis of the insoluble PHB to water-solubleproducts due to the secretion of an extracellular PHB de-polymerase. Clear zones emerged at 24 h, and their formationwas recorded after 3 days of incubation (data not shown).

Figure 1 illustrates the growth and secretion of PHBdepolymerase of T. thermophilus HB8 in MSM containing

0.1% (w/v) PHB as sole carbon source. Despite the lowgrowth rate of T. thermophilus in this medium, extracellularPHB depolymerase activity reached the maximal value of0.16 U/ml after 24 h of incubation.

Identification of the extracellular proteins possessingesterase activity

In the cell-free supernatant, which was derived from aculture of T. thermophilus grown in 0.1% (w/v) PHB as solecarbon source, two enzymes of 42 and 23 kDa weredetected, as shown in Fig. 2.

Purification of the extracellular T. thermophilus PHBdepolymerase

Extracellular T. thermophilus PHB depolymerase waspurified by thermal treatment and affinity binding to PHB(Table 1). The enzyme was purified 28.2-fold with anoverall recovery of 8.2%. SDS-PAGE analysis of thepurified enzyme preparation revealed a single band ofmolecular mass of approximately 42 kDa (Fig. 3, lane 2).

Identification of the PHB depolymerase gene

MALDI-time-of-flight (TOF)-MS analysis revealed that thepurified enzyme matched with the protein encoded by thegene TTHA0199 of T. thermophilus (Fig. 4). Blast analysisof TTHA0199 gene product showed significant homologiesto many carboxylesterases of a number of species. In

Table 1 Purification of T. thermophilus PHB depolymerase

Purification step Total protein (mg) Total activity (U) Specific activity (U/mg) Purification fold Recovery (%)

Concentrated supernatant 110 220 2 1 100Heat treatment at 80°C 63 145 2.3 1.2 66Affinity binding to PHB 0.32 18 56.3 28.2 8.2

Fig. 3 SDS-electrophoresis ofpurified PHB depolymerase.Purified enzyme (5 μg) wassubjected to SDS-polyacryl-amide gel electrophoresis in10% (w/v) polyacrylamide. Thegel was stained with silver ni-trate. Lane 1 cell-free superna-tant, lane 2 purified PHBdepolymerase, lane 3 molecularweight markers of the indicatemasses

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addition, the characteristic region for PHB depolymerases,LpqC, was detected. Blast analysis of the TTHA0199 geneproduct with the genomes of several species revealedsimilarity to other extracellular PHB depolymerases. Thehighest degree of similarity was found with the PHB

depolymerases of Mesorhizobium loti MAFF303099 (gi:1226451; 26% identity, 40% similarity) and Ralstoniapickettii 12 J (gi: 6287209; 30% identity, 38% similarity).Interestingly, T. thermophilus extracellular PHB depolymer-ase exhibited low similarity with the PhaZ7 of Ralstonia

Fig. 4 Identification of T. ther-mophilus extracellular PHBdepolymerase as the product ofthe TTH0199 gene. Boldfaceletters indicate peptides identi-fied by MALDI-TOF-MS. A 20-amino acid-long signal peptidewas identified and marked in abox. The underlined aminoacids indicate the characteristicarea for PHB depolymerases(LpqC), containing the lipasebox. The conserved motifs ofHGGG, GX1SX2G, and the pu-tative catalytic residues com-posed of Ser183, Glu310, andHis310 of extracellular PHBdepolymerases are indicated inboldface with a box

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eutropha H16 (gi: 4456176; 27% identity, 40% similarity).The deduced amino acid sequencing included a 20-aminoacid-long signal peptide as predicted by SignalP 3.0 HMM(signal peptide possibility 0.863).

Effect of temperature on the activity and stabilityof the extracellular purified PHB depolymerase

The optimum temperature of the PHB depolymeraseenzymic reaction, using PHB as substrate, was 70°C(Fig. 5a). The effect of temperature on the stability of the

purified PHB depolymerase was investigated. One micro-gram per milliliter of the enzyme solution was incubated in50 mM Tris–HCl, pH 8 for various time periods andtemperatures (Fig. 5b), following the addition of PHBsuspension at a final concentration of 150 μg/ml for60 min. The enzyme was able to retain about 75% and50% of its initial activity at 60°C and 70°C, respectively at4 h of incubation. Finally, even at the extreme incubationconditions of 80°C for 4 h, extracellular PHB depolymeraseactivity retained 45% of its activity (Fig. 4b).

Fig. 5 Effect of temperature on purified PHB depolymerase activity(a) and stability (b). For the stability assay, the enzyme (1 μg/ml) waspre-incubated in 50 mM Tris–HCl buffer, pH 8.0 at the indicatedtemperatures, and for time intervals, using as 100% for the calculationof relative activity a reference sample which was not preheated

Table 2 Effect of different ions, inhibitors, or detergents on thepurified T. thermophilus extracellular PHB depolymerase

Addition Concentration Activity (%)

None 100CaCl2 1 mM 150

2 mM 2065 mM 14510 mM 90

MgCl2 1 mM 1202 mM 905 mM 5510 mM 30

KCl 1 mM 1402 mM 1505 mM 10010 mM 80

NaCl 1 mM 1002 mM 905 mM 6010 mM 45

ΕDTA 0.1 mM 511 mM 2410 mM 17

NaN3 1 mM 8610 mM 84

PMSF 1 mM 2410 mM 10

Triton X-100 0.1% (v/v) 1151% (v/v) 7010% (v/v) 56

Tween 20 0.1% (v/v) 1001% (v/v) 6410% (v/v) 25

Tween 80 0.1% (v/v) 801% (v/v) 6510% (v/v) 42

Methanol 10% (v/v) 137Ethanol 10% (v/v) 100Acetone 10% (v/v) 102Mercaptoethanol 1% (v/v) 20

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Optimum pH of the extracellular PHB depolymeraseactivity

The purified extracellular PHB depolymerase was assayedat pH 4–12 using 150 μg/ml PHB as substrate. PHBdepolymerase was active at the pH range of 6–9, exhibitinghighest activity at pH 8.0.

Effect of metal ions and commercial inhibitorson the extracellular PHB depolymerase

The presence of K+ at concentrations up to 2 mM slightlyincreased the activity of extracellular PHB depolymerase,while higher K+ concentrations inhibited it to some extent(Table 2). The purified PHB depolymerase activity wasincreased at concentrations of Mg2+ up to 1 mM, while itwas inhibited by higher Mg2+ concentrations (Table 2). Na+

inhibited PHB depolymerase activity, while the addition ofCa2+ at concentrations up to 2 mM increased significantlyenzyme activity (Table 2).

Purified PHB depolymerase was highly inhibited by theaddition of ethylenediamine tetraacetic acid or the serineesterase inhibitor phenylmethylsulfonyl fluoride (PMSF).The presence of NaN3 inhibited slightly the PHB depoly-merase activity. The addition of the non-ionic detergentsTriton X-100, Tween 20, and Tween 80 resulted ininhibition of the activity at high concentrations.

The presence of methanol had a stimulatory effect, butthe addition of ethanol or acetone did not influence thereaction. Finally, the reducing agent 2-mercaptoethanolgreatly reduced PHB depolymerase activity.

Kinetics of extracellular PHB depolymerase

The rate of PHB depolymerization was linear (R=0.98) upto 40 μg/ml, while at higher PHB concentrations of up to50 μg/ml, only a slight increase in the rate of PHB hydrolysiswas observed. From the double-reciprocal plots of PHBhydrolysis by the purified enzyme, the Km was found to be53 μg/ml (9.8×10−5 μM, based on an estimated PHB averagemolecular mass of 540,000 g/mol) and Vmax 1.42 U/ml (datanot shown). The hydrolysis rate of pNPB at the 5–300 μMconcentration range was also monitored, and Km and Vmaxvalues were estimated from a Lineweaver–Burk plot at33.33 μM and 0.35 U/ml, respectively (data not shown).

Substrate specificity

Regarding enzymic hydrolysis of dPHB by extracellularPHB depolymerise (1 μg/ml), a linear decrease wasobserved (Fig. 6). Purified enzyme was also able tohydrolyze pNPB but not other artificial p-nitrophenyl fattyacid esters, such as p-nitrophenyl octanoate, p-nitrophenyllaurate, indicating preference of this enzyme for C4

substrates.

Products of extracellular PHB depolymerase reaction

Following the enzymic degradation of PHB, the com-pounds remaining in the water-soluble fraction wereanalyzed by HPLC. The spectra of the original polymerbefore degradation revealed a sharp peak at 17.92 min dueto unreacted BPB (Fig. 7a). In contrast, when 5 mM of 3-hydroxybutyre (3HB) was used as standard, a single peak at19.24 min appeared (Fig. 7b). It is notable that the peak at17.92 min was very low since almost all BPB wasconsumed for the monomer derivatization. After incubationof PHB with PHB depolymerase from 0.25 to 0.5 h, the3HB monomer was detected as the main product (Fig. 7c,d, respectively), providing evidence of degradation. Theconcentration of the 3HB monomer increased upon longerincubation periods (Fig. 7e, f). When PHB was incubated at70°C for 8 h, in the absence of the enzyme, degradation didnot occur (data not shown). These results indicated that T.thermophilus extracellular PHB depolymerase degradedPHB in the fashion of an exo-type hydrolase releasingone monomer unit at a time.

Discussion

The efficient growth of T. thermophilus and the forma-tion of clear zones on agar containing PHB as sole carbonsource could be attributed to the secretion of an extracel-lular PHB depolymerase. The purification of the extracel-

Fig. 6 Enzymic and hydrolytic degradation of dPHB. Enzymicdegradation (closed squares) was carried out in 50 mM Tris–HClbuffer, pH 8.0, containing 175 μg/ml PHB, 2 mM CaCl2, and 1 μg/mlof purified PHB depolymerase at 70°C. Hydrolytic degradation (in theabsence of enzyme) was carried out as a control (closed circles) underthe same conditions

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lular PHB depolymerase was achieved by a one-step PHBsubstrate-binding affinity chromatography method, result-ing in enzyme recovery of approximately 8% and 28-foldpurification. The overall recovery and purification ofextracellular PHB depolymerase were comparable to thoseobtained by other single-step procedures (Iyer et al. 2000;Elbanna et al. 2004) or other multistep purificationprocesses that have been previously employed (Schereret al. 1999; Romen et al. 2004; Calabia and Tokiwa 2006).

PHB depolymerase of T. thermophilus revealed compa-rable structure to previously characterized extracellularPHB depolymerases of other bacteria. Almost all extracel-lular PHB depolymerases have a common structure,consisting of a catalytic domain, a linker domain, and asubstrate-binding domain (Jendrossek et al. 1995). Theindividual domains can be classified into type 1 and type 2

subgroups of catalytic domains, depending on the sequen-tial order of the catalytic triad amino acids; three subgroupsof linker domains [threonine-rich region (Thr), fibronectintype 3 (Fn3), cadherin-like domain (Cad)] and twosubgroups of substrate-binding domains (SBD1 andSBD2). The structure domain of T. thermophilus PHBdepolymerase is similar to the type-1 subgroup. Theoxyanion (H95), the putative lipase box (GX1SX2G), andthe catalytic triad amino acids (S183, E309, H405) wereidentified by comparison of the strictly conserved aminoacids (Fig. 4). These conserved amino acids have beenpreviously described to be involved in the hydrolyticactivity of several PHA depolymerase (Jendrossek andHandrick 2002). In contrast to all known PHB depoly-merases, a linker domain was not detected in theTTHA0199 gene product. Moreover, the C-terminal of the

Fig. 7 Separation of PHB prod-ucts by HPLC, derived from T.thermophilus extracellular PHBdepolymerase. Detection wasperformed at 254 nm. The pickat 17.92 min corresponded tountreated bromophenacyl bro-mide. PHB (a); 3-hydroxybuty-rate (b); digestion after 0.25 h(c); 0.5 h (d); 1 h (e) and 2 h (f)of reaction. mAU milliabsorp-tion units

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TTHA0199 gene product, containing the substrate bindingdomain, completely differs from that of other PHBdepolymerases. Similarly, in the amino acid sequence ofthe PHB depolymerase PhaZ7 of Paucimonas leimoigneiand the soluble depolymerase of Rhodospririllum rubrum,only the catalytic domain was identified (Handrick et al.2001, 2004). Moreover, the different structure of T.thermophilus PHB depolymerase is probably due to itshigh content in charged amino acids (116 amino acids outof 495, corresponding to 23.4% for E, D, R, K, and H),whereas the charged amino acids of other depolymerasesshowed an average of 9.8–13% (Romen et al. 2004; Takedaet al. 1998, 2000, 2002; Handrick et al. 2001).

All known PHB depolymerases contain six to ninecysteine residues, and a cysteine bond may be necessary forstructural maintenance of the active enzyme form. The PHBdepolymerase of T. thermophilus contains eight cysteineresidues, two (C50, and C371) of which are conserved inother extracellular PHB depolymerases. Moreover, thesurvey for specific domain hits in TTHA0199 revealedthe existence of the LpqC domain, which is characteristic ofPHB depolymerases (Fig. 4). Interestingly, the extracellularPHB depolymerase of M. loti MAFF303099 was similar tothat of T. thermophilus HB8. However, cloning of theTTHA0199 gene, which is already in progress in ourlaboratory, will shed light on the difference of the molecularmass between the TTHA0199 gene product (56 kDa) andthe purified enzyme (42 kDa).

T. thermophilus extracellular PHB depolymeraseexhibited an optimum reaction temperature of 70°C andhigh thermostability (Fig. 5). The PHA depolymeraseisolated from Schlegelella thermodepolymerans exhibitedoptimal reaction temperature at 75–80°C and was stable at70°C for more than 24 h (Elbanna et al. 2004). The highestactivity of the enzyme was obtained at pH 8.0. Similarobservations with optimum neutral or alkaline pH have alsobeen reported for many scl-PHA depolymerases (Jendrossek2001).

Purified extracellular PHB depolymerase probablydegrades PHB initially as an endo-type and subsequentlyas an exo-type hydrolase, as it was revealed by monitoringthe degradation of PHB and by the detection of thehydrolysis products. Although both hydrolysis types areutilized to degrade PHB, the exo-type seems to be the mostpreferable, since the 3HB monomer was identified as themain product. PHB depolymerase from Aspergillus fumi-gatus also degrades PHB in both endo- and exo-modes ofhydrolysis (Scherer et al. 2000), while the depolymerasefrom Comamonas sp. releases mainly monomers from thepolyester (Jendrossek et al. 1993). Other PHB depoly-merases such as PhaZ5 and PhaZ2 of Pseudomonaslemoignei produce a mixture of monomers, dimmers, ortrimers (Handrick et al. 2004).

T. thermophilus extracellular PHB depolymerase wasinhibited by PMSF (Table 2) similarly to the PHAdepolymerases of Pseudomonas alcaligenes and Pseudo-monas luteola (Yamada et al. 1993; Rhee et al. 2006). Theapparent Km value of this thermophilic PHB depolymerasewas estimated at 53 μg/ml which is as high as that reportedfor the enzyme derived from S. thermodepolymerans(45 μg/ml) (Elbanna et al. 2004).

In conclusion, extracellular PHB depolymerase is anexcellent thermostable candidate biocatalyst for environ-mental, industrial, and medical applications. Taking theadvantage that the extracellular PHB depolymerasedegrades PHB as an exo-type hydrolase, the resulting3HB monomer can be used in medical applications. Thetransformation of raw material in PHA or in PHAderivatives with added value can be considered animportant input in terms of eco-effective applicability andenvironmental biotechnology.

Acknowledgment The authors are grateful to Prof. M. Liakopoulou-Kyriakides and her group for the help on the determination of the PHBenzymic hydrolysis products by HPLC.

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