characterization of a novel subgroup of extracellular mcl-pha
TRANSCRIPT
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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
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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
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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
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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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