characterization of a novel subgroup of extracellular mcl-pha

1

Characterization of a Novel Subgroup of Extracellular mcl-PHA 1

Depolymerase from Actinobacteria 2

3

Running title: MCL-PHA HYDROLYSIS BY NOVEL PHA DEPOLYMERASE PRODUCERS 4

5

Joana Gangoiti,†, ‡ Marta Santos,†, ‡ M. Auxiliadora Prieto,§ Isabel de la Mata,# 6

Juan L. Serra, †,* and María J. Llama† 7

†Enzyme and Cell Technology Group, Department of Biochemistry and Molecular Biology, 8

Faculty of Science and Technology, University of the Basque Country (UPV/EHU), 9

P.O. Box 644, E-48080 Bilbao, Spain 10 §Department of Environmental Biology, Biological Research Center, CSIC, C/. Ramiro de 11

Maeztu, 9, Madrid E-28040, Spain 12 #Department of Biochemistry and Molecular Biology I, Faculty of Biology, Complutense 13

University of Madrid, C/. José Antonio Nováis, 2, E-28040, Madrid, Spain 14

________________________________ 15

Abbreviations: HA, (R)-3-hydroxyalkanoic acid; mcl, medium-chain-length; P(3HB), poly(3-16

hydroxybutyric acid); P(3HO), poly[3-hydroxyoctanoate-co-3-hydroxyhexanoate (11%)]; 17

P(3HP), poly(3-hydroxypropionic acid); P[3HB-HV(12%)], poly(3-hydroxybutyric acid-co-18

3-hydroxyvaleric acid); PCL, poly(ε-caprolactone); PES, poly(ethylene succinate); PHA, 19

polyhydroxy-alkanoate; PLA, poly(L-lactide); pNP, p-nitrophenyl; scl, short-chain-length; 20

SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; 3-HO, 3-21

hydroxyoctanoic acid; 3-HX, 3-hydroxyhexanoic acid. 22

‡Both researchers share the position of the first author 23

*Corresponding author. Mailing address: Enzyme and Cell Technology Group, Department of 24

Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the 25

Basque Country (UPV/EHU), P.O. Box 644, E-48080 Bilbao, Spain. 26

Phone: (34) 94 601 2541. Fax: (34) 94 601 3500. E-mail: [email protected] 27

28

Copyright © 2012, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.01707-12 AEM Accepts, published online ahead of print on 3 August 2012

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(ABSTRACT) 29

30

Nineteen medium-chain-length (mcl) polyhydroxyalkanoic acid (PHA) degrading 31

microorganisms were isolated from natural sources. From them, seven Gram-positive and 32

three Gram-negative bacteria were identified. The ability of these microorganisms to 33

hydrolyse other biodegradable plastics such as short-chain-length (scl) PHA, poly(ε-34

caprolactone) (PCL), poly(ethylene succinate) (PES) and poly(L-lactide) (PLA) have been 35

studied. Based on the great ability to degrade different polyesters, Streptomyces roseolus SL3 36

was selected, and its extracellular depolymerase was biochemically characterized. The 37

enzyme consisted of one polypeptide chain of 28 kDa, with a pI value of 5.2. Its maximum 38

activity was observed at pH 9.5 with chromogenic substrates. The purified enzyme 39

hydrolyzed mcl-PHA and PCL, but not scl-PHA, PES and PLA. Moreover, the mcl-PHA 40

depolymerase can hydrolyze various substrates for esterases such as tributyrin and p-41

nitrophenyl (pNP)-alkanoates, its maximum activity being measured with pNP-octanoate. 42

Interestingly, when poly[3-hydroxyoctanoate-co-3-hydroxyhexanoate (11%)] was used as 43

substrate the main hydrolysis product was the monomer (R)-3-hydroxyoctanoate. In addition, 44

the genes of several Actinobacteria strains including S. roseolus SL3, were identified based on 45

the peptide de novo-sequencing of the Streptomyces venezuelae SO1 mcl-PHA depolymerase 46

by tandem mass spectrometry. These enzymes did not show significant similarity to mcl-PHA 47

depolymerases characterized previously. Our results suggest that these distinct enzymes might 48

represent a new subgroup of mcl-PHA depolymerases. 49

50

Keywords: extracellular mcl-PHA depolymerase; screening; bioplastic; chiral (R)-3-51

hydroxyoctanoic acid; Streptomyces roseolus, Streptomyces venezuelae, Streptomyces 52

omiyaensis, polyhydroxyalkanoate 53

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(INTRODUCTION) 55

Biodegradability of polymers has drawn much attention as a solution to problems 56

concerning the global environment and biomedical technologies. Several aliphatic polyesters 57

showing properties comparable to conventional plastics have been developed and used as 58

biodegradable plastics, such as poly(3-hydroxyalkanoate) (PHA), poly(ε-caprolactone) (PCL), 59

poly(L-lactide) (PLA), and poly(ethylene succinate) (PES). They can be synthesized from 60

petrochemicals (PES and PCL) or from renewable resources (PLA and PHA) (58). Among 61

these biodegradable plastics, PHA is the only completely synthesized by microorganisms and 62

accumulate intracellularly during unbalanced growth conditions (30). Additionally, PHA is 63

suitable for a broad range of applications in medicine, pharmacy, and industry due to its 64

biocompatibility and biodegradability (2). Moreover, all of the PHA monomers are 65

enantiomerically pure and in R-configuration (3, 40, 44). More than 150 hydroxyalkanoic 66

acids (HAs) have been identified as constituents of these microbial polyesters (6, 57). 67

Interestingly, these monomers are valuable intermediates that can be used as starting materials 68

for the synthesis of antibiotics, vitamins, flavors and pheromones (1). Since chiral (R)-HAs 69

are normally difficult to synthesize by chemical means (2), the study of enzymatic PHA 70

hydrolysis has attracted much attention. 71

The ability to degrade extracellular PHA in the environment depends on the release of 72

extracellular PHA depolymerases (17), that could be specific for either short-chain-length 73

(scl)-PHA (3 to 5 carbon atoms) (EC 3.1.1.75) or medium-chain-length (mcl)-PHA (6 to 14 74

carbon atoms) (EC 3.1.1.76) (17). Depending on the depolymerase activity, the end products 75

are only monomers, both monomers and dimers, or a mixture of oligomers as a result of the 76

enzymatic PHA degradation (17). 77

Extracellular PHA-depolymerase producing microorganisms are widely distributed and 78

have been isolated from various environments (32, 51). Currently, very few mcl-PHA 79


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depolymerases have been characterized in comparison to the number of scl-PHA 80

depolymerases studied (26). To date most of mcl-PHA depolymerases reported belong to 81

Gram negative bacteria, predominantly Pseudomonas species (22, 31). The poly(3-82

hydroxyoctanoate) depolymerase from Pseudomonas fluorescens GK13 (PhaZPflGK13) was the 83

first mcl-PHA depolymerase studied in detail at molecular level (49-51). Additionally, several 84

biotechnological applications of this enzyme have been reported, including the construction of 85

fusion proteins with affinity to mcl-PHAs (13), the production of (R)-3HAs (8) and the 86

synthesis of polyesters (48). Thus, this enzyme is considered as the prototype enzyme of 87

extracellular mcl-PHA depolymerases. In general, these enzymes consist of a signal peptide, 88

an N-terminal substrate binding domain and a C-terminal catalytic domain (15, 22). In a 89

recent study, the identification of a significantly different mcl-PHA depolymerase gene from 90

the thermophilic bacterium Thermus thermophilus HB8 has been reported (36). Recently, the 91

isolation and identification of Streptomyces venezuelae SO1 as a novel mcl-PHA 92

depolymerase (PhaZSveSO1) producer has been reported by our group (47). However, the 93

molecular characteristics of the genes encoding mcl-PHA depolymerases from Streptomyces 94

origins have not been cleared yet. 95

In this paper we report the isolation of several novel extracellular mcl-PHA degrading 96

microorganisms, predominantly Streptomyces species. Two of the isolates, SL3 and SO2, 97

have been identified as Streptomyces roseolus and Streptomyces omiyaensis, respectively. 98

Furthermore, the mcl-PHA depolymerase from S. roseolus SL3 (PhaZSroSL3) has been 99

biochemically characterized. In addition, we provide for the first time information about the 100

primary structure of the mcl-PHA depolymerases from Streptomyces bacteria. 101

102

MATERIALS AND METHODS 103

Chemicals. Poly[3-hydroxyoctanoate-co-3-hydroxyhexanoate (11%)] also named P(3HO) 104

or mcl-PHA, was supplied by Biopolis (Valencia, Spain) and CPI (Newcastle, UK). Accurel 105


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MP-1000 was purchased from Membrana GmbH (Obenburg, Germany). Poly(3-106

hydroxypropionic acid) P(3HP) was donated by CIBA (Manchester, UK). Chromatography 107

media were obtained from GE-Healthcare (Uppsala, Sweden). Molecular weight standards, p-108

nitrophenyl (pNP)-alkanoates, poly(3-hydroxybutyric acid) P(3HB), poly(3-hydroxybutyric 109

acid-co-3-hydroxyvaleric acid) P[3HB-HV(12%)], PCL, PES and PLA most chemicals were 110

obtained from Sigma-Aldrich (St. Louis, MO). All other chemicals were supplied by Merck 111

(Darmstadt, Germany). 112

Preparation of biopolymer suspensions. Latex suspensions of PCL and P(3HO), were 113

prepared according to Schirmer & Jendrossek (50). In the case of PES and PLA, 4 vols of 114

water were poured into 1 vol of polymer suspension in methylene chloride with stirring. The 115

suspensions were emulsified by an ultrasonic homogenizer, and the solvent was then 116

evaporated. P(3HB), P(3HP) and P[3HB-HV(12%)] suspensions of similar concentration (10 117

mg/ml) were prepared by dispersing each polymer powder in water by ultrasonic treatment. 118

Isolation and identification of mcl-PHA-degrading microorganisms. Several mcl-PHA 119

degrading bacterial strains were isolated in our laboratory from natural environmental samples 120

(soil, sludge and water) taken at different places of the Campus of the University of the 121

Basque Country, Vizcaya (Spain). Serial dilutions of the homogenized samples were spread 122

on P(3HO)-mineral agar plates consisting of P(3HO) latex covering Petri plates with mineral 123

media such as M9 (45) and E medium (24). The plates were incubated for 2-3 days at 30ºC. 124

Those strains which showed clearing of the P(3HO) latex were selected and isolated. 125

The bacteria were identified by the sequence analysis of the 16S rRNA gene. The 16S 126

rRNA gene sequences from isolates obtained in this study were deposited in GenBank under 127

the accession numbers JX305978 to JX305987. 128

For further identification, cultural, morphological and physiological characteristics of SL3 129

and SO2 strains were obtained by following the methods given in the international 130

Streptomyces project (ISP) (54). Aerial spore mass color, and substrate mycelium color were 131


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recorded using the ISSC-National Bureau of Standards (NBS) Color Name Charts (18) after 132

incubation for 20 days at 30ºC in oatmeal agar media (ISP medium 3). Morphological 133

observations of spores and mycelia were made by light microscopy (Nikon Eclipse 50i A) and 134

scanning electron microscopy (model JEOL 6100). Carbon utilization test was performed in 135

ISP9 medium with the addition of one of the following sugars: D-glucose (positive control), L-136

arabinose, sucrose, D-xylose, myo-inositol, D-mannitol, D-fructose, rhamnose, raffinose and in 137

the absence of a carbon source (negative control) as described by Shirling & Gottlieb (54). 138

The strains SL3 and SO2 were identified from International Streptomyces Project (ISP) (55, 139

56). The identified mcl-PHA degrading strains have been deposited in the Spanish Type 140

Culture Collection (CECT, Valencia, Spain, www.cect.org) as S. roseolus SL3 CECT 7919 141

and S. omiyaensis SO2 CECT 7923. 142

Microorganisms and growth conditions. The following microorganisms were used in this 143

study: S. venezuelae SO1 CECT 7920, S. omiyaensis SO2 CECT 7923 and S. roseolus SL3 144

CECT 7919. All other strains are listed in Table 1. Polymer-degrading bacteria were routinely 145

grown in solid M9 mineral medium (45) containing 1.5% (wt/vol) agar with the carbon sources 146

indicated in the text. For enzyme production, S. roseolus SL3 and S. venezuelae SO1 cells 147

were grown at 30ºC in 250 ml Erlenmeyer flasks containing 100 ml of mineral medium 148

supplemented with a film (0.15 g) of P(3HO) as the sole carbon and energy source, as 149

described in Santos et al. (47). The strains were maintained as frozen spore suspensions in 150

15% (vol/vol) glycerol at -20ºC according to Kieser et al. (19). 151

For the isolation of genomic DNA, the bacteria were grown for 3 days at 30ºC in 250 ml 152

Erlenmeyer flasks containing 100 ml of S-YEME medium (19) in an orbital incubator shaker 153

at 250 rpm. Cultures were harvested at 4ºC by centrifugation (10,000 ×g for 20 min). The 154

resulting pellet was used for DNA extraction. Genomic DNAs of Streptomyces strains were 155

isolated as described by Kieser et al. (19). 156


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Purification of the mcl-PHA depolymerases. S. roseolus SL3 and S. venezuelae SO1 157

cells were grown in 2-l Erlenmeyer flasks containing 800 ml of mineral medium (23) 158

supplemented with a film (1.2 g) of P(3HO). Flasks were inoculated with 100 ml of a culture 159

grown for 72 h of mineral medium supplemented with glucose (0.4% wt/vol) and the cultures 160

were grown for 3 days in an orbital incubator shaker at 250 rpm and 30ºC. Cells were 161

harvested by filtration, and the enzyme present in the supernatant was purified by adsorption 162

onto porous polypropylene (Accurel MP-1000) as reported by Gangoiti et al. (8). 163

Enzyme assays. Esterase activity was assayed using several pNP-alkanoates as substrate 164

(8). Blanks without enzyme were performed to determine spontaneous hydrolysis not due to 165

enzymatic activity. One unit (U) of esterase activity was the amount of enzyme that released 1 166

μmol of p-nitrophenol per min under standard conditions. The extinction coefficient (ε) for the 167

pNP at pH 9.5 was determined as 16.635 mM-1 · cm-1. 168

Qualitative estimation of the hydrolytic activity toward different polymers of mcl-PHA 169

depolymerase from S. roseolus SL3 was performed by a drop test on indicator plates (14). 170

Briefly, 5 ml of a 1% (wt/vol) polymer emulsion was mixed with 5 ml of 1% (wt/vol) agarose 171

in 200 mM Tris-HCl buffer, pH 8.5, and poured on a glass plate. Samples (20 μl) were loaded 172

in 5-mm-diameter holes made in the gel and incubated at 30°C for 24 h. Similarly, qualitative 173

determination of esterase activity was performed on agarose plates using tributyrin as substrate 174

as described by Gandolfi et al. (7). The diameters of the resulting clearing zones were 175

semiquantitatively correlated with the enzyme activity. 176

Identification of hydrolysis products of P(3HO). The hydrolysis products from P(3HO) 177

substrate catalyzed by PhaZSroSL3 were identified. For this purpose, reaction mixtures 178

containing 250 μg of P(3HO) latex in 20 mM Tris-HCl buffer, pH 8.0, and 50 μg of the 179

purified enzyme were incubated (in 2 ml tubes) at 30ºC and in an orbital shaker at 160 rpm for 180

various time intervals (3 h, 24 h, 48 h and 72 h). The enzymatic reaction was stopped by 181

incubating the tubes for 5 min at 100ºC, and then centrifuged at 4ºC for 60 min at 14,000 ×g. 182


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The degradation products were isolated from supernatants and derivatized using 183

bromophenacyl bromide (BPB) as described by Gebauer & Jendrossek (11). The detection 184

and quantification of the hydrolysis products was performed by HPLC/PDA, and the identity 185

of the 3-HO oligomers peaks detected at 254 nm were determined by HPLC/MS (8). The peak 186

of 26.1 min corresponds to unreacted BPB. 187

Determination of the N-terminal protein sequences. The pure PhaZSroSL3 and PhaZSveSO1 188

were electroblotted from a SDS-PAGE gel to a polyvinylindene difluoride membrane 189

(BiotraceTM PVDF, Pall Corporation, USA). The Edman degradation analysis was carried out 190

in the Proteomics and Bioinformatics facility from UAB, a member of ProteoRed network. 191

Identification of mcl-PHA depolymerases genes. In order to determine the mcl-PHA 192

depolymerase sequences from Streptomyces, PhaZSveSO1 was subjected to de novo peptide 193

sequences analysis by mass spectrometry. For this purpose, a Coomassie Blue stained gel spot 194

corresponding to the enzyme was excised, washed, reduced with DTT and alkylated with 195

iodoacetamide. The in-gel digest with trypsin was carried out on at 37ºC. The resultant 196

peptides were analyzed by Matrix Assisted Laser Desorption Ionisation Tandem Time-of-197

Flight (MALDI-TOF/TOF) mass spectrometer (4700 Proteomics Analyzer, Applied 198

Biosystems) in MS and MS/MS modes. To enhance the quality of tandem mass spectrometry 199

(MS/MS) spectra for the de novo-sequencing, N-terminal chemical modification using 4-200

sulfophenyl isothiocyanate (SPITC) was carried out at 55ºC for 30 min (10). The N-terminal 201

derivatized peptides were desalted and concentrated using μZip-Tips C18 (Millipore) as 202

described by the manufacturer. The sample was spotted onto the MALDI target plate 203

prespotted with alpha-cyano-4-hydroxycinnamic acid matrix. 204

Peptide de novo-sequencing was carried out manually using the program mMass 205

(http://www.mmass.org/). De novo-derived peptides sequences were combined in one search 206

query and analyzed by MS-BLAST (53). Searches were performed against non redundant 207


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proteins with PAM30MS as the search matrix. The sequences obtained were subjected to 208

multiple alignments employing CLUSTALW (28). 209

The PhaZSroSL3, PhaZSveSO1 and PhaZSomSO2 genes were partially amplified by polymerase 210

chain reaction (PCR) using chromosomal DNA as template. The degenerated PCR primers 211

were designed in accordance to the N-terminal protein sequences considering the codon usage 212

in Streptomyces (19), as well as based on the sequence of SVEN_7345 from S. venezuelae 213

ATCC10712 (Accesion No. CCA60631.1). PCR amplifications were performed in Px2 214

Thermal Cycler (Thermo Hybaid, UK) using the TDPfu program, adjusted for the G+C high 215

content of Streptomyces genomes, and employing Pfu DNA polymerase (Promega) (12). The 216

phaZSvenSO1 and phaZSomSO2 genes were partially amplified using primers VN1 (5’-217

CGAGGTGGACGTCGACATCGAGG-3’) and A4R (5’-218

GCGCAGCCACGCCGTGGTCGG-3’), whereas in the case of phaZSroSL3 gene, primers 219

NSL3 (5’-GTSGGSACSGACTGGGACCG-3’) and A4R were used. 220

DNA fragments (~600 bp) amplified in each PCR were purified from the agarose bands. 221

DNA sequences were determined by the dideoxy-chain-termination method (46) with an 222

automated sequencer, DNA Analyzer 3730 (Applied Biosystems, USA). The partial 223

sequences of phaZSroSL3, phaZSvenSO1 and phaZSomSO2 have been deposited in GenBank under 224

the accession numbers JX305988, JX305989 and JX305990, respectively. 225

Enzyme analysis. SDS-PAGE was performed as described by Laemmli (27). Two-226

dimensional electrophoresis was performed by isoelectric focusing using IPG strips (pH 3 to 227

10) (first dimension) and SDS-polyacrylamide gel electrophoresis (second dimension). 228

Protein concentration was determined by the method of Peterson (39) using bovine serum 229

albumin as the standard. 230

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RESULTS 233

Screening of mcl-PHA degrading bacteria and their ability to hydrolyze other 234

aliphatic polyesters. Nineteen bacteria able to grow on P(3HO), as the sole source of carbon 235

and energy, were isolated from samples of soil, sludge and water (first letter(s) in isolated 236

designations, SO, SL and W, respectively). All the isolates produced clearing zone 237

surrounding the colony within 2-3 days of incubation on opaque P(3HO) agar at 30ºC. Ten 238

different bacteria were identified by 16S rRNA gene sequence (Table 1) including three 239

Gram-negative and seven Gram-positive bacteria. The closest relative strain to each isolate 240

has also been included in Table 1. Interestingly, six of these bacteria belonged to 241

Streptomyces genera. 242

The isolated bacteria were screened for polymer degrading capacity using the clear-zone 243

method. None of the Gram-negative bacteria were able to hydrolyze scl-PHA. In contrast, all 244

Streptomyces strains showed rapid growth and degradation of scl-PHA, as well as PCL (Table 245

1). However, none of the isolated bacteria were able to hydrolyze PES and PLA. 246

Characterization of strains SO2 and SL3. Based on their great ability to degrade 247

different polyesters, the mcl-PHA degrading SL3 and SO2 strains isolated from sludge and 248

soil respectively, were selected to study the degradation of P(3HO) in detail. SL3 and SO2 are 249

Gram-positive, aerobic and non-motile filamentous bacteria with branching vegetative hyphae 250

embedded in the substrate and aerial hyphae bearing spores. The spores of both bacteria show 251

smooth surface and occur in rectiflexible chains containing more than 10 spores per chain 252

(Fig. S1 from Supplementary material). The strain SL3 developed an aerial mycelium in the 253

red-color series, and a yellow-brownish substrate mycelium. In contrast, the color of the aerial 254

mycelium of strain SO2 on ISP3 was grey, while that of the substrate mycelium was yellow-255

brownish. These bacteria did not produce diffusible pigments in none of the media tested. SL3 256

and SO2 strains utilized D-glucose, D-xylose, and rhamnose, but were unable to use myo-257

inositol, D-mannitol, sucrose and raffinose. SL3 utilized L-arabinose and D-fructose whereas 258

Table 1


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only a trace of growth was observed in the case of SO2 in presence of these sugars. Based on 259

phylogenetic analyses by the sequence of the 16S rRNA gene, morphological, and 260

physiological characteristics the strains SL3 and SO2, were identified as Streptomyces 261

roseolus and Streptomyces omiyaensis, respectively (see Materials and Methods section for 262

details). The results shown in Table 1 suggest that S. roseolus SL3 and S. omiyaensis SO2 263

may synthesize, at least, two different PHA depolymerases specific for scl- or mcl-PHAs, as it 264

has been suggested for S. exfoliatus (25). 265

Biochemical properties of mcl-PHA depolymerase from S. roseolus SL3. The 266

molecular mass of the purified enzyme from S. roseolus SL3, determined by SDS-PAGE 267

analysis, was approximately 28 kDa (Fig. 1). Non-denaturing (ND)-PAGE analyses showed 268

only one enzyme form with an estimated native molecular mass of 28 kDa, indicating that this 269

native enzyme consists of a single polypeptide chain. Besides, the isoelectric point of 270

PhaZSroSL3 was about 5.2. The effect of pH on the PhaZSroSL3 activity was examined at pH 271

values ranging from 6.0 to 12.0, using pNP-octanoate (pNPO) as the substrate. This enzyme 272

exhibited its maximum activity at pH 9.5, and retained more than 60% of this activity over a 273

pH range from 8.0 to 10.5. The N-terminal amino acid sequence of the mature PhaZSroSL3 was 274

determined by Edman degradation as AIPPVGTDWDRP (Fig. 1). This sequence showed at 275

least 50% identity only to PhaZSspKJ-72 (23). However, it showed low identity to those 276

corresponding to Pseudomonas species (22), indicating that mcl-PHA depolymerases 277

produced by Streptomyces strains may be encoded by a different type of gene. 278

Substrate specificity of the PhaZSroSL3 depolymerase. The PhaZSroSL3 hydrolyzes mcl-279

PHA and PCL (Fig. S2 from supplementary material), forming large clearing zones after 24 h 280

of incubation at 30ºC. These results suggest that the depolymerase is able to hydrolyze ester 281

bonds of β- and ω-polyhydroxyalkanoates with a relatively long side chain. However, as 282

expected, no hydrolytic activity was detected with scl-PHA such as P(3HB), P(3HP) and 283

P[3HB-co-HV(12%)]. Moreover, the enzyme was unable to hydrolyze PES and PLA, a 284

Fig. 1


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poly(alkenedicarboxylate) and a polyester consisting in α-hydroxyalkanoate repeating unit, 285

respectively. Besides, PhaZSroSL3 showed slight activity toward tributyrin, which is a typical 286

substrate for esterases. However, after 3 days of reaction at 30ºC the enzyme was unable of 287

hydrolysing olive oil, which is a suitable substrate for lipases, indicating that this 288

depolymerase does not show lipase activity (data not shown). Similar substrate specificity was 289

observed with PhaZSveSO1 and PhaZSomSO2 (47, and unpublished data, respectively). As 290

described before (14), the prototype PhaZPflGK13 did not hydrolyze scl-PHA and PLA. In 291

addition, similar to Streptomyces enzymes, in this study no hydrolytic activity was observed 292

in the presence of PES and of olive oil using PhaZPflGK13 as catalyst. However, PhaZPflGK13 293

was not able to hydrolyze tributyrin and only a small clearing zone was observed in PCL-294

agarose plates after 24 h at 30ºC (data not shown). 295

Moreover, the esterase activity of PhaZSroSL3 was assayed using several pNP-alkanoates as 296

substrates (Table 2). The enzyme showed the highest esterase activity with pNPO (4.1 U/mg 297

protein) whereas it was unable to hydrolyze pNP-hexadecanoate. On the other hand, its 298

activity with scl pNP-alkanoates was significantly lower. Similar substrate specificities for 299

pNP-alkanoates were described for PhaZSspKJ-72 (23) and PhaZSveSO1 (47). In contrast, 300

PhaZPflGK13 showed maximum esterase activity when pNP-tetradecanoate was used as 301

substrate (8). 302

Products of extracellular mcl-PHA depolymerase from S. roseolus SL3 reaction. 303

Enzymatic degradation of P(3HO) latex catalyzed by PhaZSroSL3 was followed by HPLC-304

PDA, and the identity of the resulting peaks was determined by HPLC-MS. The composition 305

and relative amounts of the hydrolysis products identified were significantly dependent on the 306

time of hydrolysis used (Fig. 2). Thus, during the early enzymatic period (3 h), trimer 3-HO-307

HO-HO (~41%) was the main hydrolysis product detected. However, longer periods of 308

incubation yielded higher concentration of 3-HO monomers, whereas those of trimers 309

markedly decreased. In fact, after 72 h of enzymatic hydrolysis 3-HO monomers were the 310

Fig. 2

Table 2


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main degradation products (~57%) obtained, while the trimers were almost absent (Fig. 2). 311

The trimer 3-HO-HO-HX and dimer 3-HO-HX could also be detected. However, it was 312

difficult to determine the relative amount of the monomer 3-HX since it showed the same 313

retention time than the unreacted BPB (26.1 min). When P(3HO) was incubated at 30°C for 314

72 h, in the absence of the enzyme, no degradation occurred (data not shown). 315

Identification of mcl-PHA depolymerases from Actinobacteria To identify the 316

depolymerase encoding genes from Actinobacteria, the amino acid sequences of four peptides 317

of the purified PhaZSveSO1 (47) were determined by de novo-sequencing analysis as, 318

VDLEHIGSAGHSQGGAAAVNAAIDAR, DSSHLPAVYGEVR, APTTAWIR and 319

RNWHNGDENAR. MS-BLAST analysis of these peptide sequences revealed a best match 320

with a hypothetical protein from Streptosporangium roseum DSM43021 (34; accession No. 321

YP_003340976). The mcl-PHA degrading ability of this bacterium was confirmed by clear 322

zone formation method (data not shown). Furthermore, this protein exhibited high amino acid 323

similarity (more than 69%) with the hypothetical proteins of other Actinobacteria species, 324

including two sequences from Rhodococcus erythropolis strains (Table 3). 325

In parallel with this work, the complete genome of S. venezuelae ATCC 10712 was elucidated 326

(41). Although a putative P(3HB) depolymerase (Accesion No. CCA60573.1) was annotated, 327

none open reading frame (ORF), encoding a mcl-PHA depolymerase, was identified. 328

Interestingly, among the BLAST obtained amino acid sequences, the hypothetical protein 329

SVEN_7345 (Accesion No. CCA60631.1) from this bacterium was found (Table 3). Based on 330

the DNA sequence of this protein, as well as on the N-terminal sequences determined by 331

Edman degradation (see material and methods for details), DNA fragments of ~600 pb of 332

phaZSroSL3, phaZSvenSO1 and phaZSomSO2 genes were amplified using their corresponding 333

isolated chromosomal DNAs as template (Fig. 3). The deduced amino acid sequences shared 334

significantly high similarity (71-94%) with all the hypothetical mcl-PHA depolymerase 335

proteins identified by de novo-sequencing and homology search. 336

Table 3


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The identified mcl-PHA depolymerase gene sequences (overall G+C content ranging from 65 337

to 74% mol) encoded for proteins consisting of ~279-293 amino acids (29.4-30.5 kDa). All 338

these sequences included a classical N-terminal signal peptide of 25 to 35-amino acid-long as 339

predicted by SignalP 4.0 (38) (Fig. 3). The calculated molecular mass of the mature proteins 340

ranged from 26.1 to 27.8 kDa. In addition, the high content of aromatic (7.2-9.4%) and 341

uncharged aliphatic (44.3-50.8%) side chains residues in these amino acid sequences 342

suggested that these enzymes were strongly hydrophobic. In general, these proteins showed a 343

larger number of charged amino acids (17.8-21.8% for E, D, R, K and H) than the mature 344

enzyme of P. fluorescens GK13 (15%). On the other hand, these sequences did not show 345

significant similarity to none of the already known extracellular mcl-PHA depolymerases. In 346

fact, no more than 32.5% and 22.1% of similarity was observed between these proteins and 347

PhaZPflGK13 and PhaZTthHB8, respectively (Table S3 from supplementary material). However, 348

similar to all extracellular PHA depolymerases, the primary structure corresponding to 349

Actinobacteria strains contained strictly conserved amino acids (Ser-Asp-His) that comprise a 350

catalytic triad in the active center (Table 4). Moreover, the catalytic domain of these proteins 351

contained the consensus lipase box pentapeptide of serine hydrolases (G-X1-S-X2-G) in which 352

X1 was a His and X2 was a Gln residue, respectively. Additionally, Table 4 shows those 353

residues identified as possible oxyanion hole amino acids based on the homology modelling 354

of the mcl-PHA depolymerase from S. venezuelae ATCC 10712 (Fig. S4 from supplementary 355

material). 356

357

DISCUSSION 358

In this work, ten mcl-PHA-degrading depolymerase producer bacteria were isolated from 359

natural samples and their ability to degrade different aliphatic biodegradable polyesters were 360

evaluated. Among our identified bacteria, only three of them, P. alcaligenes, S. maltophilia 361

Fig. 3

Table 4


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and R. equi, have already been described as extracellular mcl-PHA degrading bacteria (20, 29, 362

42). Previous works (22, 33) demonstrated that Gram-negative bacteria belonging to 363

Pseudomonas and Stenotrophomonas species are the predominant mcl-PHA-degraders in soil 364

and marine environments. However, among our isolates six Streptomyces strains were 365

identified. Additionally, only those strains belonging to Streptomyces genera showed the 366

ability to degrade not only mcl-PHA, but also scl-PHA and PCL. These results indicated that 367

Streptomycetes may play an important role in the degradation of polyesters. However, none of 368

the isolates can degrade PLA and PES. 369

The greater number of the PHA-degrading microorganisms is known to express only one 370

type of PHA depolymerase that acts upon either scl-PHA or mcl-PHA (17). However, the 371

ability to degrade scl-PHA and mcl-PHA by producing two types of depolymerases is rare 372

and has been reported in only few bacteria (5, 23, 25, 36, 47). In this work, the mcl-PHA 373

degraders, S. roseolus SL3 and S. omiyaensis SO2, were also found to express scl-PHA 374

depolymerase in the presence of P(3HB). Additionally, when S. roseolus SL3 was grown in 375

the presence of P(3HO) as the sole carbon source, it produced one single polypeptide chain of 376

mcl-PHA depolymerase with a mass of ~28 kDa and a pI of ~5.2. These results are similar to 377

those of several MCL-PHA depolymerases characterized from other sources (47), but 378

significantly different from those of the P(3HO) depolymerase from Pseudomonas 379

fluorescens GK13 (dimer, 48 kDa; pI ~7). 380

As previously reported by Santos et al. (47), it is likely that the mcl-PHA depolymerases 381

produced from Streptomyces strains have a wider range of substrate specificity. In this work, 382

the substrate specificity of PhaZSroSL3 confirms this hypothesis. In fact, in contrast to the mcl-383

PHA depolymerases from Pseudomonas, the enzyme degrades PCL and tributyrin but not 384

olive oil. However, none of the mcl-PHA depolymerases reported so far exhibited detectable 385

activities against PLA (14, 20, 23, 47) and PES (47). 386


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The pure PhaZSroSL3 mainly hydrolyzed P(3HO) to the monomeric unit of 3-387

hydroxyoctanoate after 72 h of reaction. In this sense, PhaZSroSL3 behaves as the extracellular 388

MCL-PHA depolymerases of P. alcaligenes LB19 (20) and S. venezuelae SO1 (47). On the 389

other hand, PhaZPflGK13 (49) and PhaZSspKJ-72 (23) mainly hydrolyzed P(3HO) to the dimeric 390

form of 3-hydroxyoctanoate. Thus, PhaZSroSL3 appears to have a promising potential for 391

biotechnological application in the production of enantiomerically pure (R)-3-HO monomers. 392

Several mcl-PHA depolymerases have been biochemically characterized. However, only 393

the PhaZPflGK13-coding gene and other few homologous genes have been cloned and 394

sequenced (21, 29, 37, 50) including a mcl-PHA depolymerase from the predator Bdellovibrio 395

bacteriovorus (31). Additionally, in a recent work, a significant different gene from a 396

thermophilic bacterium, T. thermophilus HB8, has been identified (36). However, no gene 397

sequence of the genus Streptomyces has been reported so far. 398

In this work, de novo-sequencing of PhaZSveSO1 allowed the identification of a novel 399

subgroup of mcl-PHA depolymerases from Actinobacteria. These new type of mcl-PHA 400

depolymerases showed high sequence similarity (more than 60%) to each other (Table S3), as 401

well as with the deduced amino acid sequences of PhaZSroSL3, PhaZSveSO1 and PhaZSomSO2. 402

Inspection of the amino acid sequences revealed no significant similarity to previously 403

characterized mcl-PHA depolymerases (less than 33%). The primary structure of these 404

enzymes showed the signal peptide domain typical of mcl-PHA depolymerases. Besides, as 405

most serine hydrolases, these enzymes showed the catalytic triad amino acids (Ser, Asp, His) 406

and the lipase consensus pentapeptide, Gly-X1-Ser-X2-Gly. In all the enzymes identified in 407

this work, X1 was a His, and X2 was a Gln residue. Similarly, in true lipases X1 residue is 408

generally occupied by His or Tyr, whereas X2 is variable (50). However, in all mcl-PHA 409

depolymerases of Pseudomonas strains analyzed so far, X1 was an Ile and X2 was a Ser 410

residue. Interestingly, contrary to Pseudomonas enzymes, PhaZSroSL3, PhaZSomSO2 and 411

PhaZSveSO1 can degrade PCL and tributyrin as bacterial lipases. The presence of a His residue 412


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in X1 position, instead of a hydrophobic one, would be a possible explanation for the 413

differences observed in substrate specificities between mcl-PHA depolymerases from 414

Pseudomonas and Streptomyces origins. Moreover, the mcl-PHA depolymerases described in 415

this study contained a larger number of charged amino acids (~18-22%) than the mature 416

enzyme from P. fluorescens GK13 (15%). An increased number of intramolecular ion bonds 417

by charged amino acids are known to contribute to the thermal stability of enzymes by 418

conferring them rigidity (36, 43). This fact is in accordance with previous results that 419

demonstrated that PhaZSveSO1 showed higher thermostability than its PhaZPflGK13 counterpart 420

(8, 47). 421

The 3 D model structure of the mcl-PHA depolymerase of S. venezuelae ATCC 10712 was 422

deduced by homology modelling using P. mendocina lipase as template (Fig. S4). This model 423

revealed that the enzyme consisted of a α/β hydrolase core with the catalytic triad (Ser125-424

Asp169-His199) at its surface, being very exposed to the solvent and Gln147 as oxyanion hole 425

amino acid to stabilized the tetrahedral transition estate. Therefore, it is assumed that this 426

enzyme does not undergo the typical phenomenon known as interfacial activation described 427

for several lipases and for the intracellular mcl-PHA depolymerase from P. putida KT2442. 428

Similar conclusions were deduced by de Eugenio et al. (4) based on the 3D model of the 429

PhaZPflGK13. Similarly to that of PhaZPflGK13, S. venezuelae ATCC 10712 does not have a lid 430

domain and shows a similar architecture and catalytic mechanism of ester hydrolysis. 431

Moreover, a disulfide bridge was predicted by the model, explaining the previously observed 432

inhibition of PhaZSveSO1 in presence of DTT (47). 433

Mcl-PHA depolymerases are excellent candidate biocatalysts for environmental, industrial 434

and medical applications. This study provides novel information of mcl-PHA depolymerases 435

from Actinobacteria, in terms of molecular structure, revealing significant differences 436

compared to Pseudomonas enzymes. Additionally, these results offer the possibility of 437


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cloning and expressing these distinct enzymes for their possible exploitation in 438

biotechnological processes. 439

440

ACKNOWLEDGEMENTS 441

This work was carried out in the framework of the IP project “Sustainable Microbial and 442

Biocatalytic Production of Advanced Functional Materials” (BIOPRODUCTION/NMP-2-CT-443

2007-026515) funded by the European Commission and by the Spanish Ministry of Education 444

and Science (BIO2007-28707-E) and UPV/EHU (GIU07/55 and GIU11/25). MS and JG were 445

the recipients of scholarships from the Spanish Ministry of Education. P(3HO) was kindly 446

supplied by Biopolis, S.A. (Valencia, Spain) and CPI (Newcastle, UK). P(3HP) was kindly 447

donated by CIBA (Manchester, UK). 448

449


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TABLE 1. Microbial strains isolated, their closest relative bacteria based on the 16S rRNA analysis and their polyester-degrading abilities.* 626

627

* The ability to degrade different polyesters was determined by the clear zone formation around the colony on the opaque plates after 2-3 days of growth at 30°C. 628

Symbols used: (−), no clearing zone; (+), small clearing zone; (++), medium clearing zone; (+++), large clearing zone. 629

†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, 630

poly(L-lactide); PES, poly(ethylene succinate). 631

‡nd, not determined.632

Strain Closest relative (GenBank accesion Nº) Category Similarity P(3HO)† P(3HP)† P(3HB) P[HB-HV(12%)] PCL PLA PES

SL1 Pseudomonas alcaligenes (Z76653) γ-Proteobacteria 99.6 +++ nd‡ − − + − −

SL2 Streptomyces atratus (DQ026638) Actinobacteria 99.6 + + + + − − −

SL3 Streptomyces roseolus (AB184168) Actinobacteria 99.8 + ++ +++ +++ + − −

SL6 Stenotrophomonas maltophilia (HQ406762.1) γ-Proteobacteria 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) γ-Proteobacteria 99.0 + nd − nd − − −

W2 Rhodococcus equi (X80614) Actinobacteria 99.6 + nd − − − − −

W3 Streptomyces pulveraceus (AB184808) Actinobacteria 99.8 + nd + + + − −

GK13 Pseudomonas fluorescens γ-Proteobacteria - +++ - - - nd − −


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28

TABLE 2. Relative activity of mcl-PHA depolymerase of S. roseolus SL3.* 633

634

635

636

637

638

639

640

641

642

643

644

* The pure enzyme was assayed with the indicated chromogenic substrates at a final concentration of 645

0.3 mM, in all cases. One hundred percent activity corresponded to 4.1 U/mg protein. 646

Substrate

Relative activity (%)

pNP-Acetate

0.5

pNP-Butyrate

3

pNP-Valerate

30

pNP-Octanoate

100

pNP-Decanoate

93

pNP-Dodecanoate

87

pNP-Hexadecanoate

13

pNP-Octadecanoate

4


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TABLE 3. Similarity between amino acid sequences of Sros_5476 and hypothetical proteins identified from sequenced microbial genomes. 647

Protein Source/Microorganism Accesion 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 ZP09405870 78/85 Unpublished

Hypothetical 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 Unpublished

Putative hydrolase RER_58150 Rhodococcus erythropolis PR4 YP_002769262 56/69 Sekine et al. (52)

648

649


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TABLE 4. Alignment of the amino acids sequences of mcl-PHA depolymerases in the neighbourhood of putative active sites. 650

651

The positions (pos.) of Ser, Asp, His and the putative oxyanion hole in the premature depolymerase proteins are indicated. A consensus sequence is written below: amino acids 652

conserved in all the analyzed sequences are indicated by capital letters. The symbols: (*) indicates amino acids with hydrophobic side chains, (+) indicates amino acids with a 653

small side chain, (:) indicates amino acids with charged side chains. The corresponding sequences of the P(3HO) depolymerase of P. fluorescens GK13 is given at the bottom. 654

The consensus sequence of lipases is shown: amino acids conserved in all the analyzed sequences are indicated by capital letters, those which have been conserved in ten or 655

more proteins are marked by low letters. 656

Strain Pos. Ser (S) Pos. Asp (D) Pos. His (H) Pos. Oxyanion hole Reference

S. venezuelae ATCC 10712 154 VDLDHIASAGHSQGGAAA 198 YLAGQRDLTVW 228 RGAGHLSSIGDG 176 DTAVPIQPGPLTDPD Pullan et al. (41)

R. erythropholis PR4 152 VDLEHIGASGHSQGGAAA 196 YLAGQADAIVW 226 RGATHFGTAING 174 DTAVAIQPGPLNDVD Sekine et al. (52)

R. erythropholis SK112 152 VDLEHIGASGHSQGGAAA 196 YLAGQADAIVW 226 RGATHFGTAING 174 DTAVAIQPGPLNDVD Unpublished

Streptosporangium roseum DSM 43021 144 VDLDRIGASGHSQGGAAA 188 ILAGQRDSIVW 218 RGADHFTVVGAP 166 DTVVPIQPGPLADAD Nolan et al. (34)

S. griseus subsp. griseus NCBR 13350 99 VDLEHIGAVGHSQGGSAA 149 LLAGQRDSIVL 173 RGADHFTVVGDP 121 DTVLPIQPGPLADID Ohnishi et al. (35)

S. griseus Xyleb KG-1 156 VDLEHIGAVGHSQGGSAA 200 LLAGQRDSIVL 230 RGADHFTVVGDP 105 DTVLPIQPGPLADID Grubbs et al. (12)

Streptomyces sp. W007 147 VDLEHIGASGHSQGGAAA 191 LLAGQRDSIVF 221 RGADHFTVVGDP 169 DTILPIQPGPLANID Unpublished

S. roseolus SL3 VDLARIGSAGHSQGGAAA YLAGERDLTVW RGAGHLSSIGDG DTAVPIQPGPLTDPD This study

S. venezuelae SO1 VDLEHIGSAGHSQGGAAA YLAGQRDLTVW RGAGHLSSIGDG DTAVPIQPGPLTDPD This study

S. omiyaensis SO2 VDLEHIGSAGHSQGGAAA YLAGQRDLTVW RGAGHLSSIGDG DTAVPIQPGPLTDPD This study

CONSENSUS VDL-:I+--GHSQGG-AA -LAG--D--V* RGA-H*--*-- DT***IQPGPL--*D This study

P. fluorescens GK13 172 LNAQRQYATGISSGGYNT 228 FLHGFVDAVVP 260 PLGGHEWFAASP 111 VQNLLDHGYAVIAP Schirmer & Jendrossek (50)

CONSENSUS LIPASES -V-**GhS-G+--- -----D-*v ---H*------ --***HG*----- Jendrossek (16)


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(LEGENDS TO FIGURES) 657

658

FIG. 1. SDS-PAGE analysis of the purified mcl-PHA depolymerase from S. roseolus SL3. 659

Proteins were separated in a homogeneous 12% (wt/vol) acrylamide gel and revealed by 660

Coomassie brilliant Blue R-250. Molecular mass markers (lane M), purified enzyme (lane 661

1).The N-terminal amino acid sequence of the enzyme, in one letter code, was determined by 662

Edman degradation. 663

664

FIG. 2. Evolution with the hydrolysis time of the abundance of P(3HO) products catalyzed 665

by the S. roseolus SL3 mcl-PHA depolymerase. The products are indicated, respectively, as 666

follows: HO, HO-HO and HO-HO-HO: monomer, dimer and trimer of 3-hydroxyoctanoic 667

acid; HO-HX and HO-HO-HX: dimer and trimer of 3-hydroxyoctanoic acid and 3-668

hydroxyhexanoic acid. The P(3HO) used as a substrate was a copolymer composed by 89% of 669

3-HO and 11% of 3-HX. 670

671

FIG. 3. Alignment of amino acid sequences of mcl-PHA depolymerases. Identical amino 672

acids are indicated shaded in grey. The lipase consensus sequence is marked in bold. Amino 673

acids that might constitute a catalytic triad and the possible oxyanion are indicated in bold and 674

by asterisks. The signal peptides predicted by Signal P 4.0 are boxed. The N-terminal amino 675

acid sequences determined by Edman degradation are indicated in italics. 1, S. venezuelae 676

SO1; 2, S. omiyaensis SO2; 3, S. roseolus SL3; 4, S. venezuelae ATCC 10712 (CCA60631); 677

5, R. erythropolis SK121 (ZP_04385744); 6, R. erythropolis PR4 (YP_002769262); 7, S. 678

griseus Xyleb KG-1 (ZP_08235685); 8, S. griseus subsp. griseus NCBR 13350 679

(YP_001823515); 9, Streptomyces sp. W007 (ZP09405870); 10, St. roseum DSM 43021 680

(YP_003340976). 681


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1MkDa

25

50

100250

75

37

AIPPVGTDWDRP

25

Fig. 1


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30

40

50

60

unda

nce

(%)

HO HO-HX HO-HO HO-HO-HX HO-HO-HO

0

10

20

30

3 h 24 h 48 h 72 hHydrolysis time (h)

Rela

tive

abu

Fig 2Fig. 2


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1 ---------------------------------------T-IPSVGTAWGLP-------- 2 ------------------------------------------------------------ 3 ----------------------------------------AIPPVGTDWDRP-------- 4 MPGRTSRVLGGLLLAAVMAVCTAPGTAGAVAD--AGTTGT-IPSVGTDFGRTGPYEVDVD 57 5 MRKSLKHFVN---AAAAVALCATLGLTGATGT--AAADAPGIPSVGHDWASAGPYTPNVS 55 6 MRKSLKYFAN---AAATVALCATLGLAGPAGP--AAADTPGIPSVGHDWASAGPYTPNVS 55 7 MLWRRNRFALSAALALVLTASGGAGTASASTGTNAVAAA-PAASAAGGFGAPGPYATAVE 59 8 ----------------------------------------------------------ME 2 9 ----------MAALALALTASAGAGTAAAATDTTVVAAASPAASAADDFGAPGPYATAVE 50 10 ----MLRRLLVPLVALVLVLAAAPHAAGADTG---------FPSVGRNWGAAGPYATAVD 47

1 ---VHTFYHPRSMGASGERHPVVIWGNGTGAVPGIYSSLLRHWASHGIIVAAANTPTSNF 57 2 ---VHTFYHPRSMGASGERHPVVIWGNGTGAVPGIYSSLLRHWASHGIIVAAANTPTSNF 57 3 ---VHTFYHPRAMGASGERHPVVIWGNGTGAVPGIYSSLLRHWASHGFIVAAANTPTSNF 57 4 IEAVHTFYYPRTMGRSGERHPVVIWGNGTGAVPGIYSSLLRHWASQGFIVAAANTPTSNF 117 5 IGLVHTLYYPRQLGARGEKHPVVIWGNGTGVLPGAYTSLLRHYASHGFIVVAANTPASNF 115 6 IGLVHTLYYPRQLGARGEKHPAVIWGNGTGVLPGAYTSLLRHYASHGFIVLAANTPASNF 115 7 VGAVTTLYYPRDIADSDRRHPVIVWGNGTGGVPLVYRDLLLHWAGQGFVVAAANTPMSNL 119 8 VGAVTTLYYPRDIADSDRRHPVIVWGNGTGGVPLVYRDLLLHWAGQGFVVAAANTPMSNL 62 9 VGAVTTLYYPRDIADSDRRHPVIVWGNGTGGVPLVYRDLLLHWASQGFVVAAANTPMSNL 1109 VGAVTTLYYPRDIADSDRRHPVIVWGNGTGGVPLVYRDLLLHWASQGFVVAAANTPMSNL 110 10 VGPVTTLYYPRDIAQSPRRHPVIVWGNGTFAFPVVYRDLLLHWASHGFVVAAANTPQSNL 107 1 AISMRAGIDVLERRNADPGSEYFGRVDLEHIGSAGHSQGGAAAVNAAIDARVDTAVPIQP 117 2 AISMRAGIDVLERRNADPGSEYFGRVDLEHIGSAGHSQGGAAAVNAAIDARVDTAVPIQP 117 3 ALSMRAGIDVLERRNADPGSEYFGRVDLARIGSAGHSQGGAAAVNAAVDARVDTAVPIQP 117 4 AISMRAGIDVLEQRNADPSSRFHGKVDLDHIASAGHSQGGAAAVNAAVDPRVDTAVPIQP 177 5 AITMRSGIDLIADKAASPSSVFFGKVDLEHIGAVGHSQGGSAAINAAIDDRVDTAVAIQP 175 6 AITMRSGIDLIADKAASPSSVFYGKVDLEHIGAVGHSQGGSAAINASIDDRVDTAVAIQP 175 Q Q7 GISMRASIDMLTGRNADPGSVFHDRVDLEHIGASGHSQGGAAAIVVGSDPRVDTVLPIQP 179 8 GISMRASIDMLTGRNADPGSVFHDRVDLEHIGASGHSQGGAAAIVVGSDPRVDTVLPIQP 122 9 GISMRASIDMLTGRNADRGSVFFDRVDLEHIGASGHSQGGAAAIVVGSDPRIDTILPIQP 170 10 GISMRAGIELLAQRNADPGSVFHGRVDLDRIGASGHSQGGAAAIVVGGDSRVDTVVPIQP 167 * * 1 GPLTDPDLTDVPMFYLAGQRDLTVWPALVKALYRDSSHLPAVYGEVRGAGHLSSIGDGGD 177 2 GPLTDPDLTDVPMFYLAGQRDLTVWPALVKALYRDSSHLPAVYGEVRGAGHLSSIGDGGD 177 3 GPLTDPDLTGVPVFYLAGERDLTVWPALVKALYRDSDHLPAVYGEVRGAGHLSSIGDGGD 177 4 GPLTDPDLMDEPVFYLAGQRDLTVWPALVKALHRDSDHVPAVYGEVRGAGHLSSIGDGGD 237 5 GPLNDVDLIDEPVLYLAGQADAIVWPAIVRAMYEDADHVPAEYLELRGATHFGTAINGGD 235 6 GPLNDVDLIDEPVLYLAGQADAIVWPAIVRAMYEDADHVPAEYLELRGATHFGTAINGGD 235 7 GPLADIDAVRGPALLLAGQRDSIVLPALVKAFYNAADHIPALYGEVRGADHFTVVGDPGP 239 8 GPLADIDAVRGPALLLAGQRDSIVLPALVKAFYNAADHIPALYGEVRGADHFTVVGDPGP 182 9 GPLANIDAVRVPALLLAGQRDSIVFPALVKAFYNAADHIPALYGEVRGADHFTVVGDPGP 230 10 GPLADADAVHGPMFILAGQRDSIVWPALVKAFYNDADHIPAIYGEVRGADHFTVVGAPGP 227 * * 1 FRAPTTAWIRRNWHNGDENAR---------------------------------- 1981 FRAPTTAWIRRNWHNGDENAR---------------------------------- 198 2 FRAPTT-------------------------------------------------- 183 3 FRAPTT-------------------------------------------------- 183 4 FRAPTTAWLR-YWLLGDENARGMFFGPDCGYCVDSGLWSGWDRNAGALRIPGPTA 291 5 MRGPSTAWLR-YWLLDDPNARTEFFGASCGYCTDTRQFSDFDRNDLALQIPG--- 286 6 MRGPSTAWLR-YWLLDDPNARTEFFGASCGYCTDTQQFSDFDRNDLALQIPG--- 286 7 FAAPTTAWFR-AHLMGDRAAHAQFFGPGCGICADTATWSDVRRNGRALSVPAATP 293 8 FAAPTTAWFR-AHLMGDRAAHAQFFGPGCGICADTATWSDVRRNSRALSVPAATP 236 9 FAAPTTAWFR-AQLMGDRTAGAQFFGPGCGICTDTATWSDVRRNSLALSVPAATP 284

Fig. 3

10 FAGPTTAWFR-FQLMGDEEARGEFSGPGCRVCADTRTWSDVRRNPLALQVPGL-- 279


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characterization of a novel subgroup of extracellular mcl-pha

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