Characterization of a Novel Subgroup of Extracellular Medium-Chain-Length Polyhydroxyalkanoate Depolymerases from Actinobacteria

Joana Gangoiti,a Marta Santos,a María Auxiliadora Prieto,b Isabel de la Mata,c Juan L. Serra,a and María J. Llamaa

Enzyme and Cell Technology Group, Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country(UPV/EHU), Bilbao, Spaina; Department of Environmental Biology, Biological Research Center, CSIC, Madrid, Spainb; and Department of Biochemistry and MolecularBiology I, Faculty of Biology, Complutense University of Madrid, Madrid, Spainc

Nineteen medium-chain-length (mcl) poly(3-hydroxyalkanoate) (PHA)-degrading microorganisms were isolated from naturalsources. From them, seven Gram-positive and three Gram-negative bacteria were identified. The ability of these microorganismsto hydrolyze other biodegradable plastics, such as short-chain-length (scl) PHA, poly(�-caprolactone) (PCL), poly(ethylene suc-cinate) (PES), and poly(L-lactide) (PLA), has been studied. On the basis of the great ability to degrade different polyesters, Strep-tomyces roseolus SL3 was selected, and its extracellular depolymerase was biochemically characterized. The enzyme consisted ofone polypeptide chain of 28 kDa with a pI value of 5.2. Its maximum activity was observed at pH 9.5 with chromogenic sub-strates. The purified enzyme hydrolyzed mcl PHA and PCL but not scl PHA, PES, and PLA. Moreover, the mcl PHA depoly-merase can hydrolyze various substrates for esterases, such as tributyrin and p-nitrophenyl (pNP)-alkanoates, with its maximumactivity being measured with pNP-octanoate. Interestingly, when poly(3-hydroxyoctanoate-co-3-hydroxyhexanoate [11%]) wasused as the substrate, the main hydrolysis product was the monomer (R)-3-hydroxyoctanoate. In addition, the genes of severalActinobacteria strains, including S. roseolus SL3, were identified on the basis of the peptide de novo sequencing of the Streptomy-ces venezuelae SO1 mcl PHA depolymerase by tandem mass spectrometry. These enzymes did not show significant similarity tomcl PHA depolymerases characterized previously. Our results suggest that these distinct enzymes might represent a new sub-group of mcl PHA depolymerases.

Biodegradability of polymers has drawn much attention as asolution to problems concerning the global environment and

biomedical technologies. Several aliphatic polyesters showingproperties comparable to those of conventional plastics have beendeveloped and used as biodegradable plastics, such as poly(3-hy-droxyalkanoate) (PHA), poly(ε-caprolactone) (PCL), poly(L-lac-tide) (PLA), and poly(ethylene succinate) (PES). They can be syn-thesized from petrochemicals (PES and PCL) or from renewableresources (PLA and PHA) (58). Among these biodegradable plas-tics, PHA is the only one that is completely synthesized by micro-organisms and accumulates intracellularly during unbalancedgrowth conditions (30). Additionally, PHA is suitable for a broadrange of applications in medicine, the pharmaceutical industry,and industry due to its biocompatibility and biodegradability (2).Moreover, all of the PHA monomers are enantiomerically pureand in the R configuration (3, 40, 44). More than 150 hydroxyal-kanoic acids (HAs) have been identified as constituents of thesemicrobial polyesters (6, 57). Interestingly, these monomers arevaluable intermediates that can be used as starting materials forthe synthesis of antibiotics, vitamins, flavors, and pheromones(1). Since chiral (R)-HAs are normally difficult to synthesize bychemical means (2), the study of enzymatic PHA hydrolysis hasattracted much attention.

The ability to degrade extracellular PHA in the environmentdepends on the release of extracellular PHA depolymerases (17)that could be specific for either short-chain-length (scl) PHA (3 to5 carbon atoms) (EC 3.1.1.75) or medium-chain-length (mcl)PHA (6 to 14 carbon atoms) (EC 3.1.1.76) (17). Depending on thedepolymerase activity, the end products are only monomers, bothmonomers and dimers, or a mixture of oligomers as a result of theenzymatic PHA degradation (17).

Extracellular PHA depolymerase-producing microorganisms

are widely distributed and have been isolated from various envi-ronments (32, 51). Currently, very few mcl PHA depolymeraseshave been characterized in comparison to the number of scl PHAdepolymerases studied (26). To date, most of the mcl PHA de-polymerases reported belong to Gram-negative bacteria, predom-inantly Pseudomonas species (22, 31). The poly(3-hydroxyoctano-ate) depolymerase from Pseudomonas fluorescens GK13(PhaZPflGK13) was the first mcl PHA depolymerase studied in de-tail at the molecular level (49–51). Additionally, several biotech-nological applications of this enzyme have been reported, includ-ing the construction of fusion proteins with affinity to mcl PHAs(13), the production of (R)-3HAs (8), and the synthesis of poly-esters (48). Thus, this enzyme is considered the prototype enzymeof extracellular mcl PHA depolymerases. In general, these en-zymes consist of a signal peptide, an N-terminal substrate bindingdomain, and a C-terminal catalytic domain (15, 22). In a recentstudy, the identification of a significantly different mcl PHA de-polymerase gene from the thermophilic bacterium Thermus ther-mophilus HB8 has been reported (36). Recently, the isolation andidentification of Streptomyces venezuelae SO1 as a novel mcl PHAdepolymerase (PhaZSveSO1) producer have been reported by ourgroup (47). However, the molecular characteristics of the genes

Received 1 June 2012 Accepted 28 July 2012

Published ahead of print 3 August 2012

Address correspondence to Juan L. Serra, juanl.serra@ehu.es.

J.G. and M.S. share the position of first author.

Supplemental material for this article may be found at http://aem.asm.org/.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.01707-12

October 2012 Volume 78 Number 20 Applied and Environmental Microbiology p. 7229–7237 aem.asm.org 7229

on April 11, 2018 by guest

http://aem.asm

.org/D

ownloaded from

encoding mcl PHA depolymerases from Streptomyces origins havenot yet been cleared. In this paper, we report the isolation of sev-eral novel extracellular mcl PHA-degrading microorganisms, pre-dominantly Streptomyces species. Two of the isolates, SL3 andSO2, have been identified to be Streptomyces roseolus and Strepto-myces omiyaensis, respectively. Furthermore, the mcl PHA depoly-merase from S. roseolus SL3 (PhaZSroSL3) has been biochemicallycharacterized. In addition, we provide for the first time informa-tion about the primary structure of the mcl PHA depolymerasesfrom Streptomyces bacteria.

MATERIALS AND METHODSChemicals. Poly(3-hydroxyoctanoate-co-3-hydroxyhexanoate [11%])[P(3HO) or mcl PHA] was supplied by Biopolis, S.A. (Valencia, Spain), andCPI (Newcastle, United Kingdom). Accurel MP-1000 was purchased fromMembrana GmbH (Obenburg, Germany). Poly(3-hydroxypropionic acid)[P(3HP)] was donated by CIBA (Manchester, United Kingdom). Chroma-tography media were obtained from GE Healthcare (Uppsala, Sweden). Mo-lecular weight standards, p-nitrophenyl (pNP)-alkanoates, poly(3-hydroxy-butyric acid) [P(3HB)], poly(3-hydroxybutyric acid-co-3-hydroxyvalericacid) {P(3HB-HV[12%])}, PCL, PES, and PLA were obtained from Sigma-Aldrich (St. Louis, MO). All other chemicals were supplied by Merck (Darm-stadt, Germany).

Preparation of biopolymer suspensions. Latex suspensions of PCLand P(3HO) were prepared as described by Schirmer and Jendrossek (50).In the case of PES and PLA, 4 volumes of water were poured into 1 volumeof polymer suspension in methylene chloride with stirring. The suspen-sions were emulsified by an ultrasonic homogenizer, and the solvent wasthen evaporated. P(3HB), P(3HP), and P(3HB-HV[12%]) suspensions ofsimilar concentration (10 mg/ml) were prepared by dispersing each poly-mer powder in water by ultrasonic treatment.

Isolation and identification of mcl PHA-degrading microorgan-isms. Several mcl PHA-degrading bacterial strains were isolated in ourlaboratory from natural environmental samples (soil, sludge, and water)taken at different places on the Leioa campus of the University of theBasque Country, Vizcaya, Spain. Serial dilutions of the homogenized sam-ples were spread on P(3HO)-mineral agar plates consisting of P(3HO)latex-covered petri plates with mineral medium such as M9 medium (45)and E medium (24). The plates were incubated for 2 to 3 days at 30°C.Those strains which showed clearing of the P(3HO) latex were selectedand isolated.

The bacteria were identified by sequence analysis of the 16S rRNAgene.

For further identification, the cultural, morphological, and physiolog-ical characteristics of the SL3 and SO2 strains were obtained by followingthe methods given in the International Streptomyces Project (ISP) (54).Aerial spore mass color and substrate mycelium color were recorded usingInter-Society Color Council, National Bureau of Standards (NBS), colorname charts (18), after incubation for 20 days at 30°C in oatmeal agarmedium (ISP medium 3). Morphological observations of spores and my-celia were made by light microscopy (Nikon Eclipse 50i A light micro-scope) and scanning electron microscopy (model JEOL 6100 scanningelectron microscope). The carbon utilization test was performed in ISP9medium with the addition of D-glucose (positive control), L-arabinose,sucrose, D-xylose, myo-inositol, D-mannitol, D-fructose, rhamnose, orraffinose and in the absence of a carbon source (negative control), asdescribed by Shirling and Gottlieb (54).

Strains SL3 and SO2 were identified from ISP (55, 56). The identifiedmcl PHA-degrading strains have been deposited in the Spanish Type Cul-ture Collection (CECT, Valencia, Spain; www.cect.org) as Streptomycesroseolus SL3 CECT 7919 and Streptomyces omiyaensis SO2 CECT 7923.

Microorganisms and growth conditions. The following microorgan-isms were used in this study: S. venezuelae SO1 CECT 7920, S. omiyaensisSO2 CECT 7923, and S. roseolus SL3 CECT 7919. All other strains arelisted in Table 1. Polymer-degrading bacteria were routinely grown insolid M9 mineral medium (45) containing 1.5% (wt/vol) agar with thecarbon sources indicated in the text. For enzyme production, S. roseolusSL3 and S. venezuelae SO1 cells were grown at 30°C in 250-ml Erlenmeyerflasks containing 100 ml of mineral medium supplemented with a film(0.15 g) of P(3HO) as the sole carbon and energy source, as described bySantos et al. (47). The strains were maintained as frozen spore suspensionsin 15% (vol/vol) glycerol at �20°C, as described by Kieser et al. (19).

For the isolation of genomic DNA, the bacteria were grown for 3 daysat 30°C in 250-ml Erlenmeyer flasks containing 100 ml of S-YEME me-dium (19) in an orbital incubator shaker at 250 rpm. Cultures were har-vested at 4°C by centrifugation (10,000 � g for 20 min). The resultingpellet was used for DNA extraction. Genomic DNAs of Streptomycesstrains were isolated as described by Kieser et al. (19).

Purification of mcl PHA depolymerases. S. roseolus SL3 and S. ven-ezuelae SO1 cells were grown in 2-liter Erlenmeyer flasks containing 800ml of mineral medium (23) supplemented with a film (1.2 g) of P(3HO).Flasks were inoculated with 100 ml of a culture of mineral medium sup-plemented with glucose (0.4%, wt/vol) that had been grown for 72 h, andthe cultures were grown for 3 days in an orbital incubator shaker at 250rpm and 30°C. Cells were harvested by filtration, and the enzyme presentin the supernatant was purified by adsorption onto porous polypropylene(Accurel MP-1000) as reported by Gangoiti et al. (8).

TABLE 1 Microbial strains isolated, their closest relative bacteria based on 16S rRNA analysis, and their polyester-degrading abilities

Strain Closest relative (GenBank accession no.) Category%similarity

Ability to degrade polyestera

P(3HO) P(3HP) P(3HB) P(HB-HV[12%]) PCL PLA PES

SL1 Pseudomonas alcaligenes (Z76653) Gammaproteobacteria 99.6 ��� ND � � � � �SL2 Streptomyces atratus (DQ026638) Actinobacteria 99.6 � � � � � � �SL3 Streptomyces roseolus (AB184168) Actinobacteria 99.8 � �� ��� ��� � � �SL6 Stenotrophomonas maltophilia (HQ406762.1) Gammaproteobacteria 99.2 � ND � ND � � �SL11 Streptomyces anulatus (AB184875) Actinobacteria 99.5 � � � � �� � �SL15 Streptomyces beijiangensis (AB249973) Actinobacteria 99.4 ��� ND �� �� � � �SO2 Streptomyces omiyaensis (AB184411) Actinobacteria 99.5 �� � �� �� ��� � �W1 Pseudomonas beteli (DQ299947.1) Gammaproteobacteria 99.0 � ND � ND � � �W2 Rhodococcus equi (X80614) Actinobacteria 99.6 � ND � � � � �W3 Streptomyces pulveraceus (AB184808) Actinobacteria 99.8 � ND � � � � �GK13 Pseudomonas fluorescens Gammaproteobacteria ��� � � � ND � �a The ability to degrade different polyesters was determined by clear-zone formation around the colony on the opaque plates after 2 to 3 days of growth at 30°C. Symbols andabbreviations: �, no clearing zone; �, small clearing zone; ��, medium clearing zone; ���, large clearing zone; P(3HP), poly(3-hydroxypropionate); P(3HB),poly(3-hydroxybutyrate); P(HB-HV[12%]), poly(3-hydroxybutyrate-co-3-hydroxyvalerate); PCL, poly-ε-caprolactone; PLA, poly(L-lactide); PES, poly(ethylene succinate); ND,not determined.

Gangoiti et al.

7230 aem.asm.org Applied and Environmental Microbiology

on April 11, 2018 by guest

http://aem.asm

.org/D

ownloaded from

Enzyme assays. Esterase activity was assayed using several pNP-al-kanoates as the substrate (8). Blanks without enzyme were performed todetermine spontaneous hydrolysis not due to enzymatic activity. One unitof esterase activity was the amount of enzyme that released 1 �mol ofp-nitrophenol per min under standard conditions. The extinction coeffi-cient (ε) for pNP at pH 9.5 was determined to be 16.635 mM�1 · cm�1.

Qualitative estimation of the hydrolytic activity of mcl PHA depoly-merase from S. roseolus SL3 toward different polymers was performed bya drop test on indicator plates (14). Briefly, 5 ml of a 1% (wt/vol) polymeremulsion was mixed with 5 ml of 1% (wt/vol) agarose in 200 mM Tris-HCl buffer, pH 8.5, and poured on a glass plate. Samples (20 �l) wereloaded in 5-mm-diameter holes made in the gel and incubated at 30°C for24 h. Similarly, qualitative determination of esterase activity was per-formed on agarose plates using tributyrin as the substrate, as described byGandolfi et al. (7). The diameters of the resulting clearing zones weresemiquantitatively correlated with the enzyme activity.

Identification of hydrolysis products of P(3HO). The hydrolysisproducts from the P(3HO) substrate catalyzed by PhaZSroSL3 were iden-tified. For this purpose, reaction mixtures containing 250 �g of P(3HO)latex in 20 mM Tris-HCl buffer, pH 8.0, and 50 �g of the purified enzymewere incubated (in 2-ml tubes) at 30°C and in an orbital shaker at 160 rpmfor various time intervals (3 h, 24 h, 48 h, and 72 h). The enzymaticreaction was stopped by incubating the tubes for 5 min at 100°C, and thenthe tubes were centrifuged at 4°C for 60 min at 14,000 � g. The degrada-tion products were isolated from supernatants and derivatized using bro-mophenacyl bromide (BPB), as described by Gebauer and Jendrossek(11). The detection and quantification of the hydrolysis products wereperformed by high-pressure liquid chromatography (HPLC)-photodiode array (PDA), and the identity of the 3-hydroxyoctanoic acid (3-HO)oligomer peaks detected at 254 nm were determined by HPLC-mass spec-trometry (MS) (8). The peak of 26.1 min corresponds to unreacted BPB.

Determination of the N-terminal protein sequences. The purePhaZSroSL3 and PhaZSveSO1 were electroblotted from an SDS-polyacryl-amide gel to a polyvinylidene difluoride (PVDF) membrane (Biotrace;Pall Corporation). Edman degradation analysis was carried out in theProteomics and Bioinformatics facility from UAB, a member of theProteoRed network.

Identification of mcl PHA depolymerases genes. In order to deter-mine the mcl PHA depolymerase sequences from Streptomyces,PhaZSveSO1 was subjected to de novo peptide sequence analysis by massspectrometry. For this purpose, a Coomassie blue-stained gel spot corre-sponding to the enzyme was excised, washed, reduced with dithiothreitol(DTT), and alkylated with iodoacetamide. The in-gel digest with trypsinwas carried out at 37°C. The resultant peptides were analyzed by matrix-assisted laser desorption ionization (MALDI)–tandem time of flight(TOF/TOF) mass spectrometry (4700 proteomics analyzer; Applied Bio-systems) in MS and MS/MS modes. To enhance the quality of the tandemMS/MS spectra for the de novo-sequencing, N-terminal chemical modifi-cation using 4-sulfophenyl isothiocyanate (SPITC) was carried out at55°C for 30 min (10). The N-terminal derivatized peptides were desaltedand concentrated using �Zip-Tips C18 (Millipore), as described by themanufacturer. The sample was spotted onto the MALDI target plate pre-spotted with alpha-cyano-4-hydroxycinnamic acid matrix.

Peptide de novo sequencing was carried out manually using the pro-gram mMass (http://www.mmass.org/). De novo-derived peptide se-quences were combined in one search query and analyzed by MS-BLAST(53). Searches were performed against nonredundant proteins withPAM30MS as the search matrix. The sequences obtained were subjectedto multiple alignments employing CLUSTALW (28).

The phaZSroSL3, phaZSveSO1, and S. omiyaensis SO2 phaZ (phaZSomSO2)genes were partially amplified by PCR using chromosomal DNA as thetemplate. The degenerated PCR primers were designed in accordancewith the N-terminal protein sequences, considering the codon usage inStreptomyces (19), as well as on the basis of the sequence of the hypothet-ical protein SVEN_7345 from S. venezuelae ATCC 10712 (GenBank ac-

cession no. CCA60631.1). PCR amplifications were performed in a Px2thermal cycler (Thermo Hybaid, United Kingdom) using the TDPfu pro-gram, adjusted for the high G�C content of Streptomyces genomes, andemploying Pfu DNA polymerase (Promega) (9). The phaZSveSO1 andphaZSomSO2 genes were partially amplified using primers VN1 (5=-CGAGGTGGACGTCGACATCGAGG-3=) and A4R (5=-GCGCAGCCACGCCGTGGTCGG-3=), whereas in the case of the phaZSroSL3 gene, primers NSL3(5=-GTSGGSACSGACTGGGACCG-3=) and A4R were used.

DNA fragments (�600 bp) amplified in each PCR were purified fromthe agarose bands. DNA sequences were determined by the dideoxy chaintermination method (46) with an automated sequencer, DNA analyzer3730 (Applied Biosystems).

Enzyme analysis. SDS-PAGE was performed as described by Laemmli(27). Two-dimensional electrophoresis was performed by isoelectric fo-cusing using immobilized pH gradient strips (pH 3 to 10) (first dimen-sion) and SDS-polyacrylamide gel electrophoresis (second dimension).The protein concentration was determined by the method of Peterson(39), using bovine serum albumin as the standard.

Nucleotide sequence accession numbers. The 16S rRNA gene se-quences from isolates obtained in this study were deposited in GenBankunder the accession numbers JX305978 to JX305987. The partial se-quences of phaZSroSL3, phaZSveSO1, and phaZSomSO2 have been depositedin GenBank under the accession numbers JX305988, JX305989, andJX305990, respectively.

RESULTSScreening of mcl PHA-degrading bacteria and their ability tohydrolyze other aliphatic polyesters. Nineteen bacteria able togrow on P(3HO) as the sole source of carbon and energy wereisolated from samples of soil, sludge, and water [the first letter(s)in the isolate designations, SO, SL, and W, respectively]. All theisolates produced a clearing zone surrounding the colony within 2to 3 days of incubation on opaque P(3HO) agar at 30°C. Tendifferent bacteria were identified from their 16S rRNA gene se-quences (Table 1), including three Gram-negative and sevenGram-positive bacteria. The closest relative strain to each isolatehas also been included in Table 1. Interestingly, six of these bacte-ria belonged to the genus Streptomyces.

The isolated bacteria were screened for polymer-degrading ca-pacity using the clear-zone method. None of the Gram-negativebacteria were able to hydrolyze scl PHA. In contrast, all Strepto-myces strains showed rapid growth and degradation of scl PHA, aswell as PCL (Table 1). However, none of the isolated bacteria wereable to hydrolyze PES and PLA.

Characterization of strains SO2 and SL3. On the basis of theirgreat ability to degrade different polyesters, the mcl PHA-degrad-ing SL3 and SO2 strains, isolated from sludge and soil, respec-tively, were selected to study the degradation of P(3HO) in detail.SL3 and SO2 are Gram-positive, aerobic, and nonmotile filamen-tous bacteria with branching vegetative hyphae embedded in thesubstrate and aerial hyphae bearing spores. The spores of bothbacteria show a smooth surface and occur in rectiflexible chainscontaining more than 10 spores per chain (see Fig. S1 in the sup-plemental material). Strain SL3 developed an aerial mycelium inthe red color series and a yellow-brownish substrate mycelium. Incontrast, the color of the aerial mycelium of strain SO2 on ISP3was gray, while that of the substrate mycelium was yellow-brown-ish. These bacteria did not produce diffusible pigments in any ofthe media tested. SL3 and SO2 strains utilized D-glucose, D-xylose,and rhamnose but were unable to use myo-inositol, D-mannitol,sucrose, and raffinose. SL3 utilized L-arabinose and D-fructose,whereas only a trace of growth was observed in the case of SO2 in

mcl PHA Hydrolysis by Novel PHA Depolymerase Producers

October 2012 Volume 78 Number 20 aem.asm.org 7231

on April 11, 2018 by guest

http://aem.asm

.org/D

ownloaded from

the presence of these sugars. On the basis of phylogenetic analysesof the sequence of the 16S rRNA gene and morphological andphysiological characteristics, strains SL3 and SO2 were identifiedas Streptomyces roseolus and Streptomyces omiyaensis, respectively(see Materials and Methods for details). The results shown in Ta-ble 1 suggest that S. roseolus SL3 and S. omiyaensis SO2 may syn-thesize at least two different PHA depolymerases specific for scl ormcl PHAs, as it has been suggested for S. exfoliatus (25).

Biochemical properties of mcl PHA depolymerase from S.roseolus SL3. The molecular mass of the purified enzyme from S.roseolus SL3, determined by SDS-PAGE analysis, was approxi-mately 28 kDa (Fig. 1). Nondenaturing (ND)-PAGE analysesshowed only one enzyme form with an estimated native molecularmass of 28 kDa, indicating that this native enzyme consists of asingle polypeptide chain. Besides, the isoelectric point ofPhaZSroSL3 was about 5.2. The effect of pH on PhaZSroSL3 activitywas examined at pH values ranging from 6.0 to 12.0, using pNP-octanoate (pNPO) as the substrate. This enzyme exhibited itsmaximum activity at pH 9.5 and retained more than 60% of thisactivity over a pH range from 8.0 to 10.5. The N-terminal aminoacid sequence of the mature PhaZSroSL3 was determined by Edmandegradation to be AIPPVGTDWDRP (Fig. 1). This sequenceshowed at least 50% identity only to that of PhaZSspKJ-72 (23).However, it showed low identity to the sequences correspondingto Pseudomonas species (22), indicating that the mcl PHA depoly-merases produced by Streptomyces strains may be encoded by adifferent type of gene.

Substrate specificity of PhaZSroSL3 depolymerase. PhaZSroSL3

hydrolyzes mcl PHA and PCL (see Fig. S2 in the supplementalmaterial), forming large clearing zones after 24 h of incubation at30°C. These results suggest that the depolymerase is able to hydro-lyze ester bonds of �- and �-polyhydroxyalkanoates with a rela-tively long side chain. However, as expected, no hydrolytic activitywas detected with scl PHA, such as P(3HB), P(3HP), and P(3HB-HV[12%]). Moreover, the enzyme was unable to hydrolyze PESand PLA, a poly(alkenedicarboxylate) and a polyester consistingof �-hydroxyalkanoate repeating units, respectively. Besides,PhaZSroSL3 showed slight activity toward tributyrin, which is a typ-ical substrate for esterases. However, after 3 days of reaction at30°C, the enzyme was unable to hydrolyze olive oil, which is asuitable substrate for lipases, indicating that this depolymerasedoes not show lipase activity (data not shown). Similar substrate

specificity was observed with PhaZSveSO1 (47) and PhaZSomSO2

(unpublished data). As described before (14), the prototypePhaZPflGK13 did not hydrolyze scl PHA or PLA. In addition, similarto Streptomyces enzymes, in this study no hydrolytic activity wasobserved in the presence of PES or olive oil using PhaZPflGK13 asthe catalyst. However, PhaZPflGK13 was not able to hydrolyze tribu-tyrin, and only a small clearing zone was observed in PCL-agaroseplates after 24 h at 30°C (data not shown).

Moreover, the esterase activity of PhaZSroSL3 was assayed usingseveral pNP-alkanoates as the substrates (Table 2). The enzymeshowed the highest esterase activity with pNPO (4.1 U/mg pro-tein), whereas it was unable to hydrolyze pNP-hexadecanoate. Onthe other hand, its activity with scl pNP-alkanoates was signifi-cantly lower. Similar substrate specificities for pNP-alkanoateswere described for PhaZSspKJ-72 (23) and PhaZSveSO1 (47). In con-trast, PhaZPflGK13 showed maximum esterase activity when pNP-tetradecanoate was used as the substrate (8).

Products of extracellular mcl PHA depolymerase from S.roseolus SL3 reaction. Enzymatic degradation of P(3HO) latexcatalyzed by PhaZSroSL3 was followed by HPLC-PDA, and theidentity of the resulting peaks was determined by HPLC-MS. Thecomposition and relative amounts of the hydrolysis productsidentified were significantly dependent on the time of hydrolysisused (Fig. 2). Thus, during the early enzymatic period (3 h), trimer3-HO-HO-HO (�41%) was the main hydrolysis product de-tected. However, longer periods of incubation yielded higher con-

FIG 1 SDS-PAGE analysis of the purified mcl PHA depolymerase from S.roseolus SL3. Proteins were separated in a homogeneous 12% (wt/vol) acryl-amide gel and revealed by Coomassie brilliant blue R-250. Lane M, molecularmass markers; lane 1, purified enzyme. The N-terminal amino acid sequenceof the enzyme, in one-letter code, was determined by Edman degradation.

TABLE 2 Relative activity of mcl PHA depolymerase of S. roseolus SL3a

Substrate Relative activity (%)

pNP-acetate 0.5pNP-butyrate 3pNP-valerate 30pNP-octanoate 100pNP-decanoate 93pNP-dodecanoate 87pNP-hexadecanoate 13pNP-octadecanoate 4a The pure enzyme was assayed with the indicated chromogenic substrates at a finalconcentration of 0.3 mM, in all cases. One hundred percent activity corresponded to4.1 U/mg protein.

FIG 2 Evolution with the hydrolysis time of the abundance of P(3HO) prod-ucts catalyzed by the S. roseolus SL3 mcl PHA depolymerase. The products areindicated as follows: HO, HO-HO, and HO-HO-HO, monomer, dimer, andtrimer of 3-hydroxyoctanoic acid, respectively; HO-HX and HO-HO-HX,dimer and trimer of 3-hydroxyoctanoic acid and 3-hydroxyhexanoic acid,respectively. The P(3HO) used as a substrate was a copolymer composed of89% 3-HO and 11% 3-HX.

Gangoiti et al.

7232 aem.asm.org Applied and Environmental Microbiology

on April 11, 2018 by guest

http://aem.asm

.org/D

ownloaded from

centrations of 3-HO monomers, whereas those of trimers mark-edly decreased. In fact, after 72 h of enzymatic hydrolysis, 3-HOmonomers were the main degradation products (�57%) ob-tained, while the trimers were almost absent (Fig. 2). The trimer3-HO-HO-HX and the dimer 3-HO-HX could also be detected.However, it was difficult to determine the relative amount of themonomer 3-hydroxyhexanoic acid (3-HX) since it showed thesame retention time as the unreacted BPB (26.1 min). WhenP(3HO) was incubated at 30°C for 72 h in the absence of theenzyme, no degradation occurred (data not shown).

Identification of mcl PHA depolymerases from Actinobacte-ria. To identify the depolymerase-encoding genes from Actino-bacteria, the amino acid sequences of four peptides of the pu-rified PhaZSveSO1 (47) were determined by de novo sequencinganalysis as VDLEHIGSAGHSQGGAAAVNAAIDAR, DSSHLPAVYGEVR, APTTAWIR, and RNWHNGDENAR. MS-BLASTanalysis of these peptide sequences revealed a best match with ahypothetical protein from Streptosporangium roseum DSM43021(34) (GenBank accession no. YP_003340976). The mcl PHA-de-grading ability of this bacterium was confirmed by the clear-zoneformation method (data not shown). Furthermore, this proteinexhibited high amino acid similarity (more than 69%) with thehypothetical proteins of other Actinobacteria species, includingtwo sequences from Rhodococcus erythropolis strains (Table 3).

In parallel with this work, the complete genome of S. venezu-elae ATCC 10712 was elucidated (41). Although a putativeP(3HB) depolymerase (GenBank accession no. CCA60573.1) wasannotated, no open reading frame (ORF) encoding a mcl PHAdepolymerase was identified. Interestingly, among the aminoacid sequences obtained by BLAST analysis, the hypotheticalprotein SVEN_7345 (GenBank accession no. CCA60631.1)from this bacterium was found (Table 3). On the basis of theDNA sequence of this protein, as well as on the N-terminalsequences determined by Edman degradation (see Materialsand Methods for details), DNA fragments of �600 bp of thephaZSroSL3, phaZSveSO1, and phaZSomSO2 genes were amplifiedusing their corresponding isolated chromosomal DNAs as thetemplate (Fig. 3). The deduced amino acid sequences sharedsignificantly high similarity (71 to 94%) with all the hypothet-ical mcl PHA depolymerase proteins identified by de novo se-quencing and homology search.

The identified mcl PHA depolymerase gene sequences (overallG�C content range, 65 to 74% mol) encoded proteins consistingof �279 to 293 amino acids (29.4 to 30.5 kDa). All these sequencesincluded a classical N-terminal signal peptide of 25 to 35 aminoacids, as predicted by the SignalP (version 4.0) program (38) (Fig.3). The calculated molecular masses of the mature proteins rangedfrom 26.1 to 27.8 kDa. In addition, the high content of aromatic

(7.2 to 9.4%) and uncharged aliphatic (44.3 to 50.8%) side chainresidues in these amino acid sequences suggested that these en-zymes were strongly hydrophobic. In general, these proteinsshowed a larger number of charged amino acids (17.8 to 21.8% forE, D, R, K, and H) than the mature enzyme of P. fluorescens GK13(15%). On the other hand, these sequences did not show signifi-cant similarity to any of the already known extracellular mcl PHAdepolymerases. In fact, no more than 32.5% and 22.1% similari-ties were observed between these proteins and PhaZPflGK13 andPhaZTthHB8, respectively (see Table S3 in the supplemental mate-rial). However, similar to all extracellular PHA depolymerases, theprimary structure corresponding to Actinobacteria strains con-tained strictly conserved amino acids (Ser-Asp-His) that comprisea catalytic triad in the active center (Table 4). Moreover, the cat-alytic domain of these proteins contained the consensus lipase boxpentapeptide of serine hydrolases G-X1-S-X2-G, in which X1 is aHis residue and X2 is a Gln residue. Additionally, Table 4 showsthose residues identified to be possible oxyanion hole amino acids,on the basis of the homology modeling of the mcl PHA depoly-merase from S. venezuelae ATCC 10712 (see Fig. S4 in the supple-mental material).

DISCUSSION

In this work, 10 mcl PHA-degrading depolymerase producer bac-teria were isolated from natural samples and their abilities to de-grade different aliphatic biodegradable polyesters were evaluated.Among our identified bacteria, only three of them, Pseudomonasalcaligenes, Stenotrophomonas maltophilia, and Rhodococcus equi,have already been described to be extracellular mcl PHA-degrad-ing bacteria (20, 29, 42). Previous work (22, 33) demonstratedthat Gram-negative bacteria belonging to Pseudomonas andStenotrophomonas species are the predominant mcl PHA degrad-ers in soil and marine environments. However, six Streptomycesstrains were identified among our isolates. Additionally, onlythose strains belonging to the Streptomyces genus showed the abil-ity to degrade not only mcl PHA but also scl PHA and PCL. Theseresults indicated that streptomycetes may play an important rolein the degradation of polyesters. However, none of the isolates candegrade PLA and PES.

The majority of the PHA-degrading microorganisms areknown to express only one type of PHA depolymerase that actsupon either scl PHA or mcl PHA (17). However, the ability todegrade scl PHA and mcl PHA by producing two types of depoly-merases is rare and has been reported in only a few bacteria (5, 23,25, 36, 47). In this work, the mcl PHA degraders S. roseolus SL3and S. omiyaensis SO2 were also found to express scl PHA depoly-merase in the presence of P(3HB). Additionally, when S. roseolusSL3 was grown in the presence of P(3HO) as the sole carbon

TABLE 3 Similarity between amino acid sequences of Sros_5476 and hypothetical proteins identified from sequenced microbial genomes

Protein Source/microorganismGenBankaccession no.

% identity/% similarity Reference

Hypothetical protein Sros_5476 Streptosporangium roseum DSM 43021 YP_003340976 Nolan et al. (34)Hypothetical acetyl xylan esterase SPW_6174 Streptomyces sp. W007 ZP_09405870 78/85 UnpublishedHypothetical acetyl xylan esterase SACT1_2252 Streptomyces griseus Xyleb KG-1 ZP_08235685 81/85 Grubbs et al. (12)Hypothetical protein SGR_2003 Streptomyces griseus subsp. griseus NCBR 13350 YP_001823515 80/84 Ohnishi et al. (35)Hypothetical protein SVEN_7345 Streptomyces venezuelae ATCC 10712 CCA60631 59/70 Pullan et al. (41)Hypothetical protein RHOER0001_1689 Rhodococcus erythropolis SK121 ZP_04385744 57/69 UnpublishedPutative hydrolase RER_58150 Rhodococcus erythropolis PR4 YP_002769262 56/69 Sekine et al. (52)

mcl PHA Hydrolysis by Novel PHA Depolymerase Producers

October 2012 Volume 78 Number 20 aem.asm.org 7233

on April 11, 2018 by guest

http://aem.asm

.org/D

ownloaded from

source, it produced one polypeptide chain of mcl PHA depoly-merase with a mass of �28 kDa and a pI of �5.2. These results aresimilar to those for several mcl PHA depolymerases characterizedfrom other sources (47) but significantly different from those forthe P(3HO) depolymerase from Pseudomonas fluorescens GK13(dimer; 48 kDa; pI �7).

As previously reported by Santos et al. (47), it is likely that themcl PHA depolymerases produced from Streptomyces strains have

a wider range of substrate specificity. In this work, the substratespecificity of PhaZSroSL3 confirms this hypothesis. In fact, in con-trast to the mcl PHA depolymerases from Pseudomonas, the en-zyme degrades PCL and tributyrin but not olive oil. However,none of the mcl PHA depolymerases reported so far exhibiteddetectable activities against PLA (14, 20, 23, 47) and PES (47).

The pure PhaZSroSL3 mainly hydrolyzed P(3HO) to the mono-meric unit of 3-hydroxyoctanoate (3-HO) after 72 h of reaction.

FIG 3 Alignment of amino acid sequences of mcl PHA depolymerases. Identical amino acids are shaded in gray. The lipase consensus sequence is marked in bold.Amino acids that might constitute a catalytic triad and the possible oxyanion are indicated in bold and by asterisks. The signal peptides predicted by the SignalP(version 4.0) program are boxed. The N-terminal amino acid sequences determined by Edman degradation are indicated in italics. 1, S. venezuelae SO1; 2, S.omiyaensis SO2; 3, S. roseolus SL3; 4, S. venezuelae ATCC 10712 (GenBank accession no. CCA60631); 5, R. erythropolis SK121 (GenBank accession no.ZP_04385744); 6, R. erythropolis PR4 (GenBank accession no. YP_002769262); 7, S. griseus Xyleb KG-1 (GenBank accession no. ZP_08235685); 8, S. griseussubsp. griseus NCBR 13350 (GenBank accession no. YP_001823515); 9, Streptomyces sp. strain W007 (GenBank accession no. ZP_09405870); 10, S. roseum DSM43021 (GenBank accession no. YP_003340976).

Gangoiti et al.

7234 aem.asm.org Applied and Environmental Microbiology

on April 11, 2018 by guest

http://aem.asm

.org/D

ownloaded from

In this sense, PhaZSroSL3 behaves like the extracellular mcl PHAdepolymerases of P. alcaligenes LB19 (20) and S. venezuelae SO1(47). On the other hand, PhaZPflGK13 (49) and PhaZSspKJ-72 (23)mainly hydrolyzed P(3HO) to the dimeric form of 3-hy-droxyoctanoate. Thus, PhaZSroSL3 appears to have a promisingpotential for biotechnological application in the production ofenantiomerically pure (R)-3-HO monomers. Several mcl PHAdepolymerases have been biochemically characterized. However,only the PhaZPflGK13-coding gene and a few other homologousgenes have been cloned and sequenced (21, 29, 37, 50), includingan mcl PHA depolymerase from the predator Bdellovibrio bacte-riovorus (31). Additionally, in a recent work, a significant differentgene from a thermophilic bacterium, T. thermophilus HB8, hasbeen identified (36). However, no gene sequence of the genusStreptomyces has been reported so far.

In this work, de novo sequencing of PhaZSveSO1 allowed theidentification of a novel subgroup of mcl PHA depolymerasesfrom Actinobacteria. These new types of mcl PHA depolymerasesshowed high sequence similarity (more than 60%) to each other(see Table S3 in the supplemental material), as well as to thededuced amino acid sequences of PhaZSroSL3, PhaZSveSO1, andPhaZSomSO2. Inspection of the amino acid sequences revealed nosignificant similarity to previously characterized mcl PHA depoly-merases (less than 33%). The primary structure of these enzymesshowed the signal peptide domain typical of mcl PHA depoly-merases. Besides, like most serine hydrolases, these enzymesshowed the catalytic triad amino acids (Ser, Asp, His) and thelipase consensus pentapeptide Gly-X1-Ser-X2-Gly. In all the en-zymes identified in this work, X1 was a His residue and X2 was aGln residue. Similarly, in true lipases, the X1 residue is generallyoccupied by His or Tyr, whereas X2 is variable (50). However, in allmcl PHA depolymerases of Pseudomonas strains analyzed so far,X1 was an Ile residue and X2 was a Ser residue. Interestingly, con-trary to Pseudomonas enzymes, PhaZSroSL3, PhaZSomSO2, andPhaZSveSO1 can degrade PCL and tributyrin as bacterial lipases.The presence of a His residue instead of a hydrophobic one in theX1 position would be a possible explanation for the differences insubstrate specificities observed between mcl PHA depolymerasesfrom Pseudomonas and Streptomyces origins. Moreover, the mclPHA depolymerases described in this study contained a largernumber of charged amino acids (�18 to 22%) than the matureenzyme from P. fluorescens GK13 (15%). An increased number ofintramolecular ion bonds by charged amino acids is known tocontribute to the thermal stability of enzymes by conferring rigid-ity on them (36, 43). This fact is in accordance with previousresults that demonstrated that PhaZSveSO1 showed higher thermo-stability than its PhaZPflGK13 counterpart (8, 47).

The three-dimensional (3D) model structure of the mcl PHAdepolymerase of S. venezuelae ATCC 10712 was deduced by ho-mology modeling using Pseudomonas mendocina lipase as thetemplate (see Fig. S4 in the supplemental material). This modelrevealed that the enzyme consisted of an �/� hydrolase core withthe catalytic triad (Ser125-Asp169-His199) at its surface, being veryexposed to the solvent, and Gln147 as the oxyanion hole aminoacid that stabilized the tetrahedral transition estate. Therefore, it isassumed that this enzyme does not undergo the typical phenom-enon known as interfacial activation described for several lipasesand for the intracellular mcl PHA depolymerase from Pseudomo-nas putida KT2442. Similar conclusions were deduced by de Eu-genio et al. (4) on the basis of the 3D model of PhaZPflGK13. Similar

TA

BLE

4A

lignm

ent

ofam

ino

acidsequ

ences

ofm

clPH

Adepolym

erasesin

neigh

borhood

ofpu

tativeactive

sitesa

Strain

Sequen

ceat

the

followin

gactive-site

positionb

Referen

ceor

source

Ser(S)

Asp

(D)

His

(H)

Oxyan

ionh

ole

S.venezuelaeA

TC

C10712

154VDLDHIASAGHSQGGAAA

198YLAGQRDLTVW

228RGAGHLSSIGDG

176DTAVPIQPGPLTDPD

Pu

llanet

al.(41)R

.erythropolisP

R4

152VDLEHIGASGHSQGGAAA

196YLAGQADAIVW

226RGATHFGTAING

174DTAVAIQPGPLNDVD

Sekine

etal.(52)

R.erythropolis

SK112

152VDLEHIGASGHSQGGAAA

196YLAGQADAIVW

226RGATHFGTAING

174DTAVAIQPGPLNDVD

Un

publish

edStreptosporangium

roseumD

SM43021

144VDLDRIGASGHSQGGAAA

188ILAGQRDSIVW

218RGADHFTVVGAP

166DTVVPIQPGPLADAD

Nolan

etal.(34)

S.griseussu

bsp.griseusN

CB

R13350

99VDLEHIGAVGHSQGGSAA

149LLAGQRDSIVL

173RGADHFTVVGDP

121DTVLPIQPGPLADID

Oh

nish

ietal.(35)

S.griseusX

ylebK

G-1

156VDLEHIGAVGHSQGGSAA

200LLAGQRDSIVL

230RGADHFTVVGDP

105DTVLPIQPGPLADID

Gru

bbset

al.(12)Streptom

ycessp.W

007147

VDLEHIGASGHSQGGAAA

191LLAGQRDSIVF

221RGADHFTVVGDP

169DTILPIQPGPLANID

Un

publish

edS.roseolus

SL3VDLARIGSAGHSQGGAAA

YLAGERDLTVW

RGAGHLSSIGDG

DTAVPIQPGPLTDPD

Th

isstu

dyS.venezuelae

SO1

VDLEHIGSAGHSQGGAAA

YLAGQRDLTVW

RGAGHLSSIGDG

DTAVPIQPGPLTDPD

Th

isstu

dyS.om

iyaensisSO

2VDLEHIGSAGHSQGGAAA

YLAGQRDLTVW

RGAGHLSSIGDG

DTAVPIQPGPLTDPD

Th

isstu

dyC

onsen

sus

VDL-:I

�--GHSQGG-AA

-LAG--D--V*

RGA-H*--*--

DT***IQPGPL--*D

Th

isstu

dyP

.fluorescensG

K13

172LNAQRQYATGISSGGYNT

228FLHGFVDAVVP

260PLGGHEWFAASP

111VQNLLDHGYAVIAP

Schirm

eran

dJen

drossek(50)

Con

sensu

soflipases

-V-**GhS-G

�---

-----D-*v

---H*------

--***HG*-----

Jendrossek

(16)a

Th

eposition

sofSer,A

sp,His,an

dth

epu

tativeoxyan

ionh

olein

the

prematu

redepolym

eraseprotein

sare

indicated.C

onsen

sus

sequen

cesare

presented,in

wh

icham

ino

acidscon

servedin

allthe

analyzed

sequen

cesare

indicated

bycapitalletters

and

inw

hich

symbols

areas

follows:*,am

ino

acidsw

ithh

ydrophobic

sidech

ains;�

,amin

oacids

with

asm

allsidech

ain;:,am

ino

acidsw

ithch

argedside

chain

s.Th

ecorrespon

ding

sequen

cesofth

eP

(3HO

)depolym

eraseofP

.fluorescensG

K13

aregiven

.Th

econ

sensu

ssequ

ences

oflipasesare

alsosh

own

,inw

hich

amin

oacids

conserved

inallth

ean

alyzedsequ

ences

arein

dicatedby

capitalletters,those

wh

ichh

avebeen

conserved

in10

orm

oreprotein

sare

marked

bylow

ercaseletters,an

dth

esym

bolsare

asdefi

ned

forth

econ

sensu

ssequ

ences.

bA

min

oacids

that

constitu

teth

ecatalytic

triad,the

possibleoxyan

ion,an

dth

econ

servedam

ino

acidsofth

elipase

consen

sus

sequen

ceare

indicated

inbold.

mcl PHA Hydrolysis by Novel PHA Depolymerase Producers

October 2012 Volume 78 Number 20 aem.asm.org 7235

on April 11, 2018 by guest

http://aem.asm

.org/D

ownloaded from

to PhaZPflGK13, S. venezuelae ATCC 10712 PhaZ does not have a liddomain, and the two enzymes show a similar architecture andcatalytic mechanism of ester hydrolysis. Moreover, a disulfidebridge was predicted by the model, explaining the previously ob-served inhibition of PhaZSveSO1 in the presence of DTT (47).

mcl PHA depolymerases are excellent candidate biocatalystsfor environmental, industrial, and medical applications. Thisstudy provides novel information on mcl PHA depolymerasesfrom Actinobacteria, in terms of molecular structure, revealingsignificant differences from Pseudomonas enzymes. Additionally,these results offer the possibility of cloning and expression of thesedistinct enzymes for their possible exploitation in biotechnologi-cal processes.

ACKNOWLEDGMENTS

This work was carried out in the framework of the IP project SustainableMicrobial and Biocatalytic Production of Advanced Functional Materials(BIOPRODUCTION/NMP-2-CT-2007-026515), funded by the Euro-pean Commission and by the Spanish Ministry of Education and Science(BIO2007-28707-E and CTQ2011-25052), and UPV/EHU (GIU07/55and GIU11/25). M.S. and J.G. were the recipients of scholarships from theSpanish Ministry of Education.

P(3HO) was kindly supplied by Biopolis, S.A. (Valencia, Spain), andCPI (Newcastle, United Kingdom). P(3HP) was kindly donated by CIBA(Manchester, United Kingdom).

REFERENCES1. Chen GQ, Wu Q. 2005. Microbial production and applications of chiral

hydroxyalkanoates. Appl. Microbiol. Biotechnol. 67:592–599.2. Chen GQ. 2011. Biofunctionalization of polymers and their applications.

Adv. Biochem. Eng. Biotechnol. 125:29 – 45.3. de Eugenio LI, et al. 2010. The turnover of medium-chain-length poly-

hydroxyalkanoates in Pseudomonas putida KT2442 and the fundamentalrole of PhaZ depolymerase for the metabolic balance. Environ. Microbiol.12:207–221.

4. de Eugenio LI, García JL, García PL, Prieto MA, Sanz JM. 2008.Comparative analysis of the physiological and structural properties of amedium chain length polyhydroxyalkanoate depolymerase from Pseu-domonas putida KT2442. Eng. Life Sci. 8:260 –267.

5. Elbanna K, Lutke-Eversloh T, Jendrossek D, Luftmann H, SteinbüchelA. 2004. Studies on the biodegradability of polythioester copolymers andhomopolymers by polyhydroxyalkanoate (PHA)-degrading bacteria andPHA depolymerases. Arch. Microbiol. 182:212–225.

6. Escapa IF, et al. 2011. Disruption of �-oxidation pathway in Pseudomo-nas putida KT2442 to produce new functionalized PHAs with thioestergroups. Appl. Microbiol. Biotechnol. 89:1583–1598.

7. Gandolfi R, Gaspari F, Franzetti L, Molinari F. 2000. Hydrolytic andsynthetic activities of esterases and lipases of non-starter bacteria isolatedfrom cheese surface. Ann. Microbiol. 50:183–189.

8. Gangoiti J, Santos M, Llama MJ, Serra JL. 2010. Production of chiral(R)-3-hydroxyoctanoic acid monomers catalyzed by Pseudomonas fluore-scens GK13 poly(3-hydroxyoctanoic acid) depolymerase. Appl. Environ.Microbiol. 76:3554 –3560.

9. García-Hidalgo J, Hormigo D, Prieto MA, Arroyo M, de la Mata I.2012. Extracellular production of Streptomyces exfoliatus poly(3-hydroxybutyrate) depolymerase in Rhodococcus sp. T104: determinationof optimal biocatalyst conditions. Appl. Microbiol. Biotechnol. 93:1975–1988.

10. García-Murria MJ, Valero ML, Sánchez del Pino MM. 2011. Simplechemical tools to expand the range of proteomics applications J. Proteom-ics 74:137–150.

11. Gebauer B, Jendrossek D. 2006. Assay of poly(3-hydroxybutyrate) de-polymerase activity and product determination. Appl. Environ. Micro-biol. 72:6094 – 6100.

12. Grubbs KJ, et al. 2011. Genome sequence of Streptomyces griseus strainXylebKG-1, an ambrosia beetle-associated actinomycete. J. Bacteriol. 193:2890 –2891.

13. Ihssen J, Magnani D, Thöny-Meyer L, Ren Q. 2009. Use of extracellular

medium chain length polyhydroxyalkanoate depolymerase for targetedbinding of proteins to artificial poly[(3-hydroxyoctanoate)-co-(3-hydroxyhexanoate)] granules. Biomacromolecules 10:1854 –1864.

14. Jaeger KE, Steinbüchel A, Jendrossek D. 1995. Substrate specificities ofbacterial polyhydroxyalkanoate depolymerases and lipases: bacteriallipases hydrolyze poly(omega-hydroxyalkanoates). Appl. Environ. Micro-biol. 61:3113–3118.

15. Jendrossek D, Schirmer A, Handrick R. 1997. Recent advances in char-acterization of bacterial PHA depolymerases, p 89 –101. In Eggink G,Steinb̈uchel A, Poirier Y, Witholt B (ed), 1996 International Symposiumon Bacterial Polyhydroxyalkanoates. NRC Research Press, Ottawa, ON,Canada.

16. Jendrossek D. 2001. Extracellular polyhydroxyalkanoate depolymerases:the key enzymes of PHA degradation, p 41– 83. In Doi Y, Steinb̈uchel A(ed), Biopolymers, vol 386 3b. Polyesters II. Wiley-VCH, Weinheim, Ger-many.

17. Jendrossek D, Handrick R. 2002. Microbial degradation of polyhydroxy-alkanoates. Annu. Rev. Microbiol. 56:403– 432.

18. Kelly KL. 1964. Color-name charts illustrated with centroid colors. Inter-Society Color Council, National Bureau of Standards, Chicago, IL.

19. Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA. 2000.Practical Streptomyces genetics. John Innes Foundation, Norwich, UnitedKingdom.

20. Kim DY, Nam JS, Rhee YH. 2002. Characterization of an extracellularmedium-chain-length poly(3-hydroxyalkanoate) depolymerase fromPseudomonas alcaligenes LB19. Biomacromolecules 3:291–296.

21. Kim DY, Kim HC, Kim SY, Rhee YH. 2005. Molecular characterizationof extracellular medium-chain-length poly(3-hydroxyalkanoate) depoly-merase genes from Pseudomonas alcaligenes strains. J. Microbiol. 43:285–294.

22. Kim DY, Kim HW, Chung MG, Rhee YH. 2007. Biosynthesis, modifi-cation, and biodegradation of bacterial medium-chain-length polyhy-droxyalkanoates. J. Microbiol. 45:87–97.

23. Kim HJ, Kim DY, Nam JS, Bae KS, Rhee YH. 2003. Characterization ofan extracellular medium-chain-length poly(3-hydroxyalkanoate) depoly-merase from Streptomyces sp. KJ-72. Antonie Van Leeuwenhoek 83:183–189.

24. Kim O, Gross RA, Hammar WJ, Newmark RA. 1996. Microbial synthe-sis of poly(beta-hydroxyalkanoates) containing fluorinated side-chainssubstituents. Macromolecules 29:4572– 4581.

25. Klingbeil B, Kroppenstedt R, Jendrossek D. 1996. Taxonomical identi-fication of Streptomyces exfoliatus K10 and characterization of its poly(3-hydroxybutyrate) depolymerase gene. FEMS Microbiol. Lett. 142:215–221.

26. Knoll M, Hamm T, Wagner F, Martinez V, Pleiss J. 2009. The PHAdepolymerase engineering database: a systematic analysis tool for the di-verse family of polyhydroxyalkanoate (PHA) depolymerases. BMC Bioin-formatics 10:89 –90. doi:10.1186/1471-2105-10-89.

27. Laemmli UK. 1970. Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature 227:680 – 685.

28. Larkin MA, et al. 2007. ClustalW and ClustalX version 2. Bioinformatics23:2947–2948.

29. Lim JH. 2006. Expression, purification and characterization of Rhodococ-cus equi P2 MCL-PHA depolymerase in Escherichia coli. MS thesis. Chun-gnam National University, Daejeon, South Korea.

30. Madison LL, Huisman GW. 1999. Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic. Microbiol. Mol. Biol. Rev. 63:21–53.

31. Martínez V, et al. 2012. Identification and biochemical evidence of amedium-chain-length polyhydroxyalkanoate depolymerase in theBdellovibrio bacteriovorus predatory hydrolytic arsenal. Appl. Environ.Microbiol. 78:6017– 6026.

32. Mergaert J, Swings J. 1996. Biodiversity of microorganisms that degradebacterial and synthetic polyesters. J. Ind. Microbiol. 17:463– 469.

33. Nam JS, Kim HC, Kim DY, Rhee YH. 2002. Distribution and diversity ofmicrobial communities relating to biodegradation of medium-chain-length poly(3-hydroxyalkanoates) in soils, p 192. Proceedings of the 9thInternational Symposium on the Genetics of Industrial Microorganisms,Gyeongju, South Korea.

34. Nolan M, et al. 2010. Complete genome sequence of Streptosporangiumroseum type strain (NI 9100). Stand. Genomic Sci. 2:29 –37.

35. Ohnishi Y, et al. 2008. Genome sequence of the streptomycin-producing

Gangoiti et al.

7236 aem.asm.org Applied and Environmental Microbiology

on April 11, 2018 by guest

http://aem.asm

.org/D

ownloaded from

microorganism Streptomyces griseus IFO 13350 J. Bacteriol. 190:4050 –4060.

36. Papaneophytou CP, Velalia EE, Pantazaki AA. 2011. Purification andcharacterization of an extracellular medium-chain length polyhydroxy-alkanoate depolymerase from Thermus thermophilus HB8. Polym. De-grad. Stab. 96:670 – 678.

37. Park IJ, Rhee YH, Cho NY, Shin KS. 2006. Cloning and analysis ofmedium-chain-length poly(3-hydroxyalkanoate) depolymerase gene ofPseudomonas luteola M13-4. J. Microbiol. Biotechnol. 16:1935–1939.

38. Petersen TN, Brunak S, von Heijne G, Nielsen H. 2011. SignalP 4.0:discriminating signal peptides from transmembrane regions. Nat. Meth-ods 8:785–786.

39. Peterson GL. 1983. Determination of total protein. Methods Enzymol.91:95–119.

40. Prieto MA, et al. 1999. Engineering of stable recombinant bacteria forproduction of chiral medium-chain-length poly-3-hydroxyalkanoates.Appl. Environ. Microbiol. 65:3265–3271.

41. Pullan ST, Chandra G, Bibb MJ, Merrick M. 2011. Genome-wide anal-ysis of the role of GlnR in Streptomyces venezuelae provides new insightsinto global nitrogen regulation in actinomycetes. BMC Genomics 12:175.doi:10.1186/1471-2164-12-175.

42. Ramsay BA, Saracovan I, Ramsay JA, Marchessault RH. 1994. A methodfor the isolation of microorganisms producing extracellular long-side-chain poly (�-hydroxyalkanoate) depolymerase. J. Environ. Polym. Deg.2:1–7.

43. Romen F, Reinhardt S, Jendrossek D. 2004. Thermotolerant poly(3-hydroxybutyrate)-degrading bacteria from hot compost and characteriza-tion of the PHB-depolymerase of Schlegelella sp. KB1a. Arch. Microbiol.182:157–164.

44. Ruth K, et al. 2007. Efficient production of (R)-3-hydroxycarboxylicacids by biotechnological conversion of polyhydroxyalkanoates and theirpurification. Biomacromolecules 8:279 –286.

45. Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: a labora-tory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, NY.

46. Sanger F, Nicklen S, Coulson AR. 1977. DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. U. S. A. 74:5463–5467.

47. Santos M, et al. Polyester hydrolytic and synthetic activity catalysed by themedium-chain-length poly(3-hydroxyalkanoate) depolymerase fromStreptomyces venezuelae SO1. Appl. Microbiol. Biotechnol., in press. doi:10.1007/s00253-012-4210-1.

48. Santos M, et al. 2012. Poly(3-hydroxyoctanoate) depolymerase fromPseudomonas fluorescens GK13: catalysis of ester-forming reactions innon-aqueous media. J. Mol. Catal. B Enzym. 77:81– 88.

49. Schirmer A, Jendrossek D, Schlegel HG. 1993. Degradation of poly(3-hydroxyoctanoic acid) [P(3HO)] by bacteria: purification and propertiesof a P(3HO) depolymerase from Pseudomonas fluorescens GK13. Appl.Environ. Microbiol. 59:1220 –1227.

50. Schirmer A, Jendrossek D. 1994. Molecular characterization of the ex-tracellular poly(3-hydroxyoctanoic acid) [P(3HO)] depolymerase gene ofPseudomonas fluorescens GK13 and of its gene product. J. Bacteriol. 176:7065–7073.

51. Schirmer A, Matz C, Jendrossek D. 1995. Substrate specificities of poly-(hydroxyalkanoate)-degrading bacteria and active site studies on the ex-tracellular poly(3-hydroxyoctanoic acid) depolymerase of Pseudomonasfluorescens GK13. Can. J. Microbiol. 41(Suppl 1):170 –179.

52. Sekine M, et al. 2006. Sequence analysis of three plasmids harboured inRhodococcus erythropolis strain PR4. Environ. Microbiol. 8:334 –346.

53. Shevchenko A, et al. 2001. Charting the proteomes of organisms withunsequenced genomes by MALDI-quadrupole time-of-flight massspectrometry and BLAST homology searching. Anal. Chem. 73:1917–1926.

54. Shirling EB, Gottlieb D. 1966. Method for classification of Streptomycesspecies. Int. J. Syst. Bacteriol. 16:313–340.

55. Shirling EB, Gottlieb D. 1968. Cooperative description of type cultures ofStreptomyces. 11. Species descriptions from first study. Additional descrip-tions. Int. J. Syst. Bacteriol. 18:69 –189.

56. Shirling EB, Gottlieb D. 1972. Cooperative description of type strains ofStreptomyces V. Additional descriptions. Int. J. Syst. Bacteriol. 22:265–394.

57. Steinbüchel A, Valentin HE. 1995. Diversity of bacterial polyhydroxy-alkanoic acids. FEMS Microbiol. Lett. 128:219 –228.

58. Vroman I, Tighzert L. 2009. Biodegradable polymers. Materials 2:307–334.

mcl PHA Hydrolysis by Novel PHA Depolymerase Producers

October 2012 Volume 78 Number 20 aem.asm.org 7237

on April 11, 2018 by guest

http://aem.asm

.org/D

ownloaded from