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Isolation and characterization of an extracellular thermoalkanophilic P(3HB-co-3HV) depolymerase from Streptomyces sp. IN1
International Biodeterioration & Biodegradation 65 (2011) 777e785
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Isolation and characterization of an extracellular thermoalkanophilic P(3HB-co-3HV) depolymerase from Streptomyces sp. IN1 Adrian Douglas Allen a, W.A. Anderson a, F. Ayorinde b, B.E. Eribo a, b, * a b
Department of Biology, Graduate School, Howard University, 415 College St. NW, Washington, DC 20059, USA Department of Chemistry, Graduate School, Howard University, Washington, DC 20059, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 October 2010 Received in revised form 9 February 2011 Accepted 10 February 2011
Here, we report on the biodegradation of the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co3HV)] by a novel thermoalkanophilic extracellular esterase from the soil isolate Streptomyces sp. IN1. Preliminary screening and isolation of the bacterium was done using polyhydroxyalkanoate latex medium (PHALM). The isolate was cultured with P(3HB-co-3HV) as the only carbon source and byproducts of degradation were derivatized with [N,O-bis(trimethylsilyl)trifluroacetamide] (BSTFA). These products were identified by gas chromatography/mass spectrometry (GCeMS) as silylated hydroxybutyric acid (3HB) and hydroxyvaleric acid, suggesting extracellular depolymerase activity by the isolate. The depolymerase was isolated by (NH4)2SO4 fractionation, dialyzed and purified using fast protein liquid chromatography (FPLC), and confirmed using P(3HB-co-3HV) as a sole source of carbon. The molecular mass of the FPLC purified enzyme occurred between 45 and 66 kDa (SDSePAGE), but was confirmed by matrix assisted laser desorption ionizationetime of flight mass spectrometry (MALDIeTOF MS) to be 62 kDa. Enzyme activity was significantly inhibited by phenylmethylsulfonyl fluoride (PMSF), dithiothreitol (DTT), and Tween 80, but induced by azide (N3). Sensitivity to PMSF, DTT, and Tween 80 suggests the involvement of serine as an active site amino acid with disulphide bonds contributing to the catalytic activity, as well as the presence of hydrophobic regions in the enzyme. Non-inhibition of activity by azide indicates that metal ions may not be required as cofactors for activity. This observation was further corroborated by the decrease in enzyme activity in the presence of metal ions such as Ca2þ, Mg2þ, Naþ, and Kþ. The kinetic parameters, Vmax and Km, in the presence of p-nitrophenylbutyrate as substrate, were determined to be 5.06 101 mmol min1 and 6.73 101 mM, respectively. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Biodegradation P(3HB-co-3HV) depolymerase Streptomyces sp. Esterase
1. Introduction There is increasing interest in the identification of novel polyhydroxyalkanoate (PHA)-degrading microorganisms because of their biotechnological potential as sources of extracellular esterases. These enzymes could play a significant role in the degradation of industrial pollutants and natural materials such as PHA (Lenz and Marchessault, 2005). Degradation of extracellular PHA and the use of its by-products as a source of carbon and energy is controlled by the secretion of specific extracellular PHA depolymerase(s). These may be specific for medium-chain-length PHA or short-chainlength (scl) PHA, such as poly (3-hydroxybutyrate), P(3HB), or its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate), P(3HBco-3HV) (Ha and Cho, 2002; Kim et al., 2002; Gonçalves and Martins-Franchetti, 2009). * Corresponding author. Department of Biology, 415 College St. NW, Washington, DC 20059, USA. Tel.: þ1 202 806 6937; fax: þ1 202 806 4564. E-mail address: [email protected] (B.E. Eribo). 0964-8305/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2011.02.010
Although significant strides have been made in extracellular PHA depolymerase research, the majority of studies have focused on Gram-negative bacteria having optimum depolymerase activity 65 C and pH 10. Notable exceptions were observed for the depolymerase from Comamonas testosteroni ATSU with an optimum temperature at 70 C (although activity was not retained at this temperature) (Kasuya et al., 1994), and Paucimonas lemoignei with an optimum pH of 12 (Handrick et al., 2001). Fewer reports have addressed the isolation and characterization of depolymerases, particularly thermophilic and/or alkanophilic depolymerases, from Gram-positive bacteria, especially Streptomyces sp., despite their abundance in soil (Klingbeil et al., 1996; Marbrouk and Sabry, 2001; Kim et al., 2003; Calabia and Tokiwa, 2006). However, none of these depolymerases had molecular mass 50 kDa or optimum pH and temperature above 10 and 60 C, respectively. In our previous report, the copolymer P(3HB-co-3HV) was produced from saponified Jatropha curcas oil (Allen et al., 2010). Here, we report on the biodegradation of P(3HB-co-3HV) by a novel
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thermoalkanophilic extracellular esterase from the soil isolate Streptomyces sp. IN1. 2. Materials and methods 2.1. Isolation of PHA-degrading bacteria Soil was collected, dried to constant weight at 30 C, and serially diluted using 0.1% proteose peptone. Inocula were plated onto PHA latex medium (PHALM) and incubated under aerobic and anaerobic conditions at 30 C over 2e10 days or until zones of clearing were visible. PHA degraders were then sub-cultured onto 0.4% (w/v) PHALM, and those that exhibited rapid degradation of the substrate were used for further analyses. Sterilized soil was used as a control. 2.2. Growth medium (PHA latex medium) A 0.4% PHALM (w/v) was prepared by suspending poly-3hydroxybutyrate [P(3HB), Sigma] or P(3HB-co-3HV) into mineral salt medium (MSM) and sonicating (Branson, 2210) for 10e20 min. The constituents of the MSM (Ayorinde et al., 1998) were as follows: macronutrients (g l1): 1.1 g (NH4)2HPO4, 5.8 g K2HPO4, 3.7 g KH2PO4, 10.0 ml of a 100 mM MgSO4 solution, and 1.0 ml of micronutrient solution. The micronutrient solution consisted of (g l1) 2.78 g FeSO4$7H2O, 1.98 g MnCl2$4H2O, 2.81 g CoSO4$7H2O, 1.67 g CaCl2$2H2O, 0.17 g CuCl2$2H2O, and 0.29 g ZnSO4$7H2O. Solid medium was prepared by adding 15 g l1 agar to MSM. All media were sterilized for 15 min at 121 C. 2.3. Phenotypic characterization of the isolate The following tests were used for phenotypic characterization: Gram-stain, oxidase, phenylalanine, nitrate, catalase, cysteine utilization, growth on cornmeal-Tween medium, Emmons medium, 7.5% sodium chloride, glucose, lactose, citrate, and motility. All media were prepared according to the manufacturers’ specifications.
2.6. Growth profile and enzyme activity of isolate The isolate was inoculated into 100 ml PHA latex broth and incubated at 30 C for 2e10 days. Culture broth, 2.0 ml, was removed periodically, and the OD650 determined. Further, esterase activity was evaluated at OD400 with p-nitrophenylbutyrate (PNPB) as substrate (e ¼ 2.75 M1 cm1). Growth responses for the isolate were obtained by interpolating OD650 versus time. Controls contained no substrate (PHA). 2.7. Derivatization of P(3HB-co-3HV) by-products By-products of P(3HB-co-3HV) degradation were derivatized with N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) and analyzed using gas chromatographyemass spectrometry (GCeMS). The following protocol (ThermoScientific) was observed for the reaction: 8.0 mg of dried sample was imbued with 100 ml BSTFA in a clean, dry reaction vial. The mixture was vortexed for 5 min, incubated for 10 min at room temperature, and 1.0 ml of the preparation was used for GCeMS analysis. Alternatively, if by-products were not generated by this method, the preparation (by-product/ BSTFA) was heated for 15 min at 70 C, cooled to room temperature, and analyzed as before. A commercial sample of DL-b-hydroxybutyric acid (Sigma, St. Louis, MO) and valeric acid (Lancaster synthesis Inc., Pelham, NH) were used as controls. 2.8. GCeMS analyses An Agilent Technologies 6890N Network GC System (CA, USA) interfaced directly to an Agilent Technologies 5973 Inert Mass Selective Detector was used to generate data. A Supelco fused-silica SPB-1 (30 m, 0.32 mm i.d., 0.25-mm film) column (Bellefonte, PA, USA) was used to conduct high-resolution capillary gas chromatography. Oven temperature was programmed from 40 to 300 C at 12 C min1, and helium was used as the carrier gas with head pressure of 3 psi. Injector temperature was set at 240 C. A HewlettePackard PC integration program was used to evaluate data. Runs were done in triplicate.
2.4. DNA isolation/PCR/sequencing DNA was isolated using a QIAampÒ DNA mini kit (Qiagen). Briefly, the isolate was grown overnight in trypticase soy broth at 30 C, shaken at 120 rpm (EnvironÒ shaker), pelleted and preincubated in a mixture containing 180 ml of 20 mg ml1 lysozyme, 20 mM TriseHCl, pH 8.0; 2.0 mM EDTA and 1.2% Triton X-100. The DNA was isolated from this mixture, purified, and it’s concentration determined using a NanoDrop 2000C spectrophotometer (Thermo Scientific, Wilmington, DE). Then 16S rDNA amplification was carried out with the following parameters: initial denaturation at 95 C for 5 min followed by 35 cycles of denaturation at 95 C for 45 s, annealing at 51 C for 45 s, extension at 72 C for 2 min, and a final extension at 72 C for 10 min. Amplified 16S rRNA gene was sequenced (Genewiz, Inc., South Plainfield, NJ). 16S rRNA gene sequence was then compared with those in GenBank (http://www.ncbi.nlm.nih.gov).
2.9. Purification and isolation of extracellular PHA depolymerase The culture was incubated as before and broths were centrifuged (Sorvall, refrigerated centrifuge, Dupont Instruments Corp.) at 13,000 rpm for 10 min at 4 C. The supernatant was collected and filtered (Corning 25 mm, 0.22 mm) and ammonium sulfate [(NH4)2SO4] fractions were collected between 30 and 80% saturation at 4 C. Fractions were pooled and dialyzed against 20 mM TriseHCl, pH 7.2, at 4 C using a Slide-A-LyzerÒ dialysis cassette (Rockford, IL) with a 10-kDa molecular weight cutoff (MWCO) or CarolinaÒ dialysis tubing (Burlington, NC) with a MWCO of 13 kDa. Samples were concentrated (Savant, Global Medical Instruments, MN) and a final purification performed using anion exchange chromatography on a BIOCAD fast protein liquid chromatography (FPLC) instrument. 2.10. FPLC analyses
2.5. Evaluation of PHA degradation PHA latex plates (PHALM) and broths were prepared as before with P(3HB-co-3HV) as the sole source of carbon. The isolate was cultured at 30 C, 120 rpm, for 2e10 days and clearing zones on solid medium were measured (in millimeters) every 48 h. Culture broths were then filtered (0.22 mm), and supernatant dried for 48 h at 60 C, and then derivatized. The commercial P(3HB) (Sigma, St. Louis, MO) was used as the control.
A BioCAD 700E perfusion chromatographyÒ workstation (Global Medical Instruments, MN) was used for further purification of proteins. About 1000 ml of filtered (0.22 mm Nalgene filter) sample was automatically loaded into 1.0 ml sample loop. The buffers used for processing were 20 mM TriseHCl, pH 8.0, and 20 mM TriseHCl, pH 7.0, with 1.0 mM NaCl. A HiTrap QFF, 1.0 ml anionic column (GE Healthcare, NJ) was used with a flow rate of 1.0 ml min1. The UV detector was set at 254 and 280 nm with a threshold of 0.5.
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Fractions were collected by peak cutting using an Avantec SF-2120 Super Fraction Collector (Global Medical Instruments, MN) and respective fractions were pooled and concentrated (Savant, GMI, MN) at 4 C. The resulting data were processed using the BioCAD 700E Version 3 Series software. All buffers and samples were sterilized by filtering. 2.11. Protein determination Protein concentrations were determined according to Bradford (1976) using the protein assay reagent (Bio-Rad Laboratories) with bovine serum albumin (BSA) as the standard.
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from 0.0 to 1.0 mg ml1 protein in a total volume of 1.0 ml. Assay was performed as mentioned before with PNPB as substrate. 2.15. Kinetic studies Kinetic studies were performed using 4.8 101 mg ml1 protein and 0.0 to 8.0 101 mM PNPB in a final volume of 1.0 ml. Hydrolytic activity was monitored spectrophotometrically as before. A substrate saturation curve was generated by plotting substrate concentrations against activity values, and Vmax and Km values were estimated from a LineweavereBurk plot. 2.16. Effect of temperature on depolymerase activity and stability
2.12. Enzymatic assay Extracellular PHA depolymerase activity was assayed with a modification of methods outlined previously (Schirmer et al., 1993; Jaeger et al., 1995; Kim et al., 2000). PHA depolymerase was assayed as p-nitrophenylbutyrate (PNPB) esterase activity at 30 C. The reaction mixture contained 0.0e0.80 mM PNPB in ethanol, 10e270 ml enzyme and 50 mM potassium phosphate buffer (pH 8.0) in a final volume of 1.0 ml. Spectral changes for the hydrolysis of PNPB were monitored spectrophotometrically at 400 nm (e ¼ 2.75 M1 cm1). One unit of enzyme activity was defined as the amount of enzyme releasing 1.0 mmol p-nitrophenol min1 (PNP) under assay conditions. Alternatively, the assay was performed with a modification of a method found elsewhere (Shah et al., 2007). Briefly, a mixture was used containing 100 ml enzyme and 900 ml 0.03% PHA latex broth. The mixture was incubated at 30 C for 1.0 h and the decrease in turbidity at OD650 (e ¼ 4.752 105 mM1 cm1) was monitored. One unit of enzyme was defined as the amount of enzyme capable of decreasing the OD650 by 1 unit (mmol min1). Further, to confirm that FPLC purified enzyme was an esterase, about 20 ml of enzyme was incubated with approximately 1.0 ml 0.03% P(3HB-co-3HV) at 30 C, 120 rpm for 24e72 h. Samples were then dehydrated, derivatized, and evaluated by GCeMS analysis. Controls contained no enzyme. 2.13. Protein analysis by SDSePAGE and MALDIeTOF MS A standard protocol was used for SDSe12% PAGE. Samples were electrophoresized at 100 V for about 1.5 h with broad-range molecular marker (Bio-Rad Laboratories) with composition myosin, 200 kDa; b-Galactosidase, 116 kDa; phosphorylase b, 97 kDa; serum albumin, 66 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 31 kDa; and trypsin inhibitor, 21 kDa. Gels were stained with Coomassie Brilliant Blue R-250 and images were obtained (Kodak Gel Logic Imaging System, Rochester, NY). Further, the actual molecular weight of the FPLC purified protein was determined using matrix assisted laser desorption ionizationetime of flight mass spectrometry (MALDIeTOF MS) (Voyager-DEÔ STR workstation). About 1.0 ml FPLC sample was mixed with matrix 2,5 dihydrobenzoic acid (2,5 DHB) in tetrahydrofuran (THF), (10 mg mL1), applied to target, and allowed to dry freely. Data were acquired in reflector mode at 25 kV, grid voltage 94%; extraction delay time was 100 ns, with 150 transients. Runs were done in triplicate. Data analysis was performed with the Applied Biosystems Voyager System 4098 software. 2.14. Effect of protein concentration on extracellular depolymerase activity The relationship between protein concentration and depolymerase activity was measured by varying protein concentrations
The optimum activity of purified extracellular depolymerase was evaluated under various temperatures e 30, 40, 50, 60, 80, and 90 C. Incubations were performed at the indicated temperature and the initial velocity was derived from a progress curve. To evaluate enzyme stability (remaining activity), assay was incubated at the aforementioned temperatures and the remaining activity was ascertained at 5-min intervals over 15 min. The assay conditions were as described before. Reactions were done in triplicate. 2.17. Effect of pH on depolymerase activity and stability The optimum activity of purified extracellular depolymerase was evaluated under variations in pH e 5, 6, 7, 8, 9, 11, and 12. Incubations were performed at the indicated pH and initial velocities were derived from progress curves. Remaining activity (as a percentage) and assay conditions were as mentioned before. The pH was adjusted using 1.0 M hydrochloric acid and 1.0 M sodium hydroxide and reactions were done in triplicate. The molar extinction coefficient (e, M1 cm1) of p-NP at various pHs was determined under assay conditions using phosphate buffer. 2.18. Effect of potential inhibitors on depolymerase activity The potential inhibitors used were as follows: phenylmethylsulfonyl fluoride (PMSF, 5.0 102 mM); dithiothreitol (DTT, 5.0 102 mM); potassium cyanide (KCN, 5 103 mM); sodium azide (NaN3, 5 103 mM); Triton X-100 (0.1%); and Tween 20 (0.1%). The PHA depolymerase activity was evaluated with adjustments in the chemical composition of the assay. Spectral changes were followed over 15 min. The control contained no potential inhibitor other than the compounds required for the standard assay. Reactions were done in duplicate. 2.19. Effect of metal ions on PHA depolymerase activity The effect of metal ions on enzyme activity was determined by the addition of 1.0 mM chloride salts of Ca2þ, Mg2þ, Naþ, and Kþ to the assay mixture. Controls contained no metal ions other than the compounds required for the standard assay. Spectral changes were followed over 15 min. Reactions were done in duplicate. 3. Results 3.1. Isolation of PHA-degrading bacteria PHA-degrading bacteria were isolated based on clear zones of hydrolysis on PHALM plates (Figs. 1 and 2). Although only five degraders were isolated from the soil sample under aerobic conditions, only one, isolate IN1, showed rapid degradation of the substrate over 10 days. Therefore, this isolate was used for all
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Fig. 1. Isolation of Streptomyces sp. IN1 from soil on 0.4% PHA latex medium (a), and degradation of J. curcas PHA by Streptomyces sp. IN1 (b). Zones of degradation are indicated by clearing around isolate (arrows). Degradation occurred between 2 and 10 days at 30 C.
analyses. PHA degraders were not isolated under anaerobic conditions or in the control (autoclaved soil sample). 3.2. Phenotypic characterization of isolate The isolate was a Gram-positive rod, non-motile, catalase positive, halotolerant (7.5% sodium chloride), and produced leathery colonies with non-septate mycelia and yellow spores when cultured on cornmeal-Tween agar. In addition, the bacterium reduced nitrate and cysteine, but was unable to utilize tryptophan, phenylalanine, glucose, or lactose. 3.3. Taxonomic identification of isolate IN1 Based on biochemical and morphological features, isolate IN1 belongs to the genus Streptomyces, and it was designated IN1. Based on its 16S rRNA gene sequence (GenBank accession JF268582) Streptomyces sp. IN1 exhibits 98% homology with Streptomyces sp. C39 (GenBank accession AY741282). 3.4. Evaluation of PHA degradation Semi-quantitative evaluation of P(3HB-co-3HV) degradation over time (250 h) shows that degradation of the material was
Zone off Degradattion (mm)
60
occurring. This was illustrated by the formation of clear zones of hydrolysis (Fig. 1). Degradation was also shown to occur faster with P(3HB-co-3HV) copolymer compared to the control [commercial P(3HB)] (Fig. 2). 3.5. Growth profile and enzyme activity of isolate Growth responses for Streptomyces sp. IN1, in PHA latex broth, were derived by interpolating various time periods against optical density (650 nm) (Fig. 3). Optimum growth was shown to occur at 12 h. An evaluation of extracellular depolymerase activity relative to growth was ascertained by measuring the hydrolysis of p-nitrophenylbutyrate to p-nitrophenol at 400 nm. Optimum extracellular depolymerase activity was shown to occur at 24 h with a decrease thereafter (Fig. 3). 3.6. GCeMS analysis Derivatized by-products of PHA degradation were determined using GCeMS analysis and confirmation with spectra from the National Institute of Standards and Technology (NIST02) database/ library. Silylated by-products were similar for assays using either supernatant or purified enzyme. These by-products were identified as silylated hydroxybutyric and hydroxyvaleric acids (Fig. 4). The spectrum of butanoic acid, 3-[trimethylsilyloxy]-trimethylsilyl ester (Fig. 4a) shows diagnostic ions at m/z 233 (M-15) and m/z73
50 40 30 20 10 0 0
50
100
150
200
250
300
Time (h) Fig. 2. Rate of degradation of Jatropha curcas polymer by Streptomyces sp. IN1 over 250 h incubation at 30 C. J. curcas PHA (C), commercial PHA (B). Zone of degradation as shown in Fig. 1 was measured (in millimeters) every 48 h.
Fig. 3. Growth profile for Streptomyces sp. IN1 in mineral salt medium containing 0.4% (w/v) PHA (C), and variation of extracellular depolymerase activity, during growth over 84 h, 120 rpm, 30 C (B). Activity (mmol min1) is based on p-nitrophenylbutyrate (PNPB) hydrolysis. The control is shown by (A).
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[eOSi(CH3)3]. The spectrum of the commercial silylated hydroxybutyrate (Fig. 4b) is consistent with that of the product in Fig. 4a. Similarly, the silylated hydroxyvalerate with diagnostic ions at m/z 247 (M-15) is shown in Fig. 4c. This was further confirmed by the NIST02 database. 3.7. Purification and isolation of extracellular PHA depolymerase Quantitative evaluation of the results observed for consecutive purification steps (supernatant, ammonium sulfate, fast perfusion liquid chromatography) is shown in Table 1. The protein was purified 14-fold with an activity of 84% by ammonium sulfate precipitation. Several fractions containing three proteins were collected from FLPC using a QFF anion exchange column (data not shown). Individual fractions were evaluated for activity and those with the highest activity were combined and concentrated. This combination accounted for a recovery rate of 91% and a purification fold of 401. Proteins and fractions were consistent over several repetitions.
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3.8. Protein analysis by SDSePAGE and MALDIeTOF MS The SDSePAGE profile for the purified enzyme is shown in Fig. 5. The apparent molecular weight (Mw), as determined by SDSePAGE, occurred between 45 and 66 kDa. However, the actual Mw as determined by matrix assisted laser desorption ionizationetime of flight mass spectrometry (MALDIeTOF MS) was 6.24583 104 Da (Fig. 6).
3.9. Effect of protein concentration on extracellular depolymerase activity The relationship between protein concentration and depolymerase activity exhibited a hyperbolic curve (Fig. 7). Depolymerase activity, measured as p-nitrophenol formation, increased until the protein concentration reached 4.8 101 mg ml1 and remained constant at levels above this value.
Fig. 4. Mass spectra of BSTFA derivatized by-product of J. curcas polymer degradation. These were identified as (a) silylated hydroxybutyrate, (b) silylated hydroxybutyrate (commercial), and (c) silylated hydroxyvalerate. These were confirmed by the NIST database.
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Table 1 Purification of
a
extracellular PHA depolymerase from Streptomyces sp. IN1.
Isolate
Purification step
Volume (ml)
Protein (mg ml1)
Total protein (mg)
Total activity (mmol min1) 105
Specific activity (mmol min1 mg1 protein)
Purification fold
Recovery (%)
Streptomyces sp. IN1
Crude (NH4)2SO4 (30e80%) FPLC (pooled fractions)
250 20 5
0.54 0.37 0.063
128 7.48 0.32
2.31 1.94 2.10
1.79 103 2.59 104 7.22 105
1 14 401
100 84 91
a
Extracellular PHA depolymerase activity was performed in an assay containing 0.03% PHA (see Methods) [OD650 (3 ¼ 4.752 105 mM1 cm1)].
3.10. Kinetic studies The substrate saturation curve was obtained by plotting substrate concentrations against the activity values. Values for Vmax and Km were then determined as 5.06 101 mmol min1 and 6.73 101 mM, respectively, from the LineweavereBurk double reciprocal plot (Fig. 8).
There was significant depolymerase activity in the presence of Triton X-100, 2548%, and azide, 923.24%. However, the activity decreased in activity in the presence of Tween 80, 73.39%; DTT, 72.17%; and PMSF, 57.17%, compared to the control with 100% activity (Vmax 1.25 103 mmol min1). A negligible decrease in activity (92.35%) was shown in the presence of cyanide. 3.14. Effect of metal ions on activity
3.11. Effect of temperature on depolymerase activity and stability The dependence of the purified esterase activity on assay temperature was evaluated (Fig. 9, circular points). The enzyme exhibited activity over all temperatures evaluated, but had an optimum temperature of 80 C, and was partially deactivated at 90 C. The thermal stability profile indicates that the enzyme was stable at 80 C for over 15 min, but was almost completely destabilized at 90 C for the same time period (Fig. 9, bar diagram). 3.12. Effect of pH on depolymerase activity and stability The activity of the depolymerase from Streptomyces sp. IN1 at pH values between 5 and 12 was evaluated (Fig. 10, circular points). The pH profile shows that the enzyme was completely inactivated at pH values below 7, but showed optimum activity at pH 12. The thermal stability profile shows that the enzyme was stable at pH between 7 and 12, but was most stable at pH 12 for the time evaluated (Fig. 10, bar diagram). 3.13. The effect of various chemicals on PHA depolymerase activity The effect of various chemicals and metal ions on the activity of Streptomyces sp. IN1 PHA depolymerase was evaluated (Table 2).
Fig. 5. SDS-PAGE of FPLC purified PHA depolymerase from Streptomyces sp. IN1. Proteins were separated on a SDSe12% polyacrylamide gel and stained with Coomassie Brilliant Blue R-250. Lane 1, molecular weight marker (myosin, 2 00 kDa; b-Galactosidase, 116 kDa; phosphorylase b, 97 kDa; serum albumin, 66 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 31 kDa; trypsin inhibitor, 21 kDa); lane 2, FPLC purified PHA depolymerase (arrow indicates location of protein).
Extracellular depolymerase activity was less than 100% in the presence of all metal ions compared to the control, which had no additional metal ion other than the compounds required for the assay (Table 3). The remaining activities were Mg2þ, 36.59; Ca2þ, 64.64; Naþ, 48.19; and Kþ, 53.7%. 4. Discussion In the current study, a novel thermoalkanophilic P(3HB-co-3HV) esterase from the soil bacterium Streptomyces sp. IN1 (GenBank accession JF268582) was isolated and partially characterized. Presumptive identification was made in accordance with the methods as outlined by the International Streptomyces Project (ISP). Growth responses for Streptomyces sp. IN1 in PHA latex broth and agar (PHA, 0.4%, w/v) were obtained by interpolating optical densities (OD) at 650 nm or zone of clearing (mm) versus different time intervals. Optimum growth and esterase activity was shown to occur at 12 and 24 h of incubation, respectively. Esterase activity, indicated by the hydrolysis of p-nitrophenylbutyrate, was inversely proportional to the growth of Streptomyces sp. IN1 between 36 and 84 h. The decrease in extracellular enzyme activity (36e84 h) may have occurred from changes in culture conditions such as a decrease in pH, from by-products of Streptomyces sp. IN1 metabolism, or from complete hydrolysis of the polymer. Furthermore, initial OD650 values were high since these reflected an environment containing bacteria (viable and dead) and suspended PHA. Subsequently, OD650 decreased because PHA was hydrolyzed (change in nature of PHA granules) by extracellular PHA depolymerase into water-soluble by-products (monomers, dimers, and/or oligomers). Thus, the OD650 at this point (between 12 and 36 h) reflects mostly cells of Streptomyces sp. IN1. Hydrolysis of PHA is vital since bacteria have not been shown to utilize denatured (extracellular) PHA. In essence, the degradative by-products of PHA were then utilized by the viable cells for growth (between 36 and 72 h); therefore the OD650 increased. Interestingly, enzyme activity (indicated by zone of clearing) continued beyond 250 h on solid medium and was shown (semiquantitatively) to occur faster with P(3HB-co-3HV) copolymer compared to the commercial homopolymer, P(3HB). This may have arisen due to steric hindrance and/or other inhibition (noncompetitive) of the enzyme by the agar (solid) or other components in the medium. Thus, the extracellular depolymerase was insufficiently bound to the substrate and degradation occurred more slowly in solid compared to broth. This observation is similar to the previous report (Mergaert et al., 1993) and gives some insight into extracellular depolymerase action and/or conditions which may
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Fig. 6. Matrix assisted laser desorption ionizationetime of flight mass spectrum (MALDIeTOF MS) of FPLC purified extracellular PHA depolymerase from Streptomyces sp. IN1. The actual molecular weight of protein was 6.24583 104 Da. Data were ascertained using 2,5 dihydrobenzoic acid (2,5 DHB) as matrix in reflector mode at 25 kV.
favor optimum degradation of the material. Furthermore, there is evidence to suggest that copolymers of P(3HB-co-3HV) are more susceptible to hydrolysis by extracellular depolymerase than is P(3HB) due to melting point, decreased crystallinity (better access of enzyme to polymer chain), and molecular weight of the polymer (Ha and Cho, 2002; Gonçalves and Martins-Franchetti, 2009). Decreased enzyme activity may also indicate that the extracellular depolymerase from Streptomyces sp. IN1 is more specific for P(3HBco-3HV) than P(3HB). Further confirmation of extracellular esterase secretion by Streptomyces sp. IN1 was obtained by derivatization of P(3HB-co3HV) by-products derived from both FPLC purified enzyme and
Fig. 7. Effect of protein concentration (mg ml1) on extracellular depolymerase activity (mmol min1) of Streptomyces sp. IN1. Optimum activity occurred at 4.8 101 mg ml1 protein. Protein concentration was measured using Bradford assay and activity as PNPB hydrolysis at 400 nm.
culture supernatant. These were confirmed using GCeMS analysis and the National Institute of Standards and Technology (NIST02) database library and were identified as silylated hydroxybutanoic [3-[trimethylsilyloxy]-,trimethylsilyl ester] and hydroxyvaleric acids. These silylated derivatives are unquestionably the products of PHA degradation since they were found only in the assay containing only FPLC purified enzyme and P(3HB-co-3HV) and also the culture supernatant (filtered, 0.22 mm). These by-products had spectra similar to the commercial samples, and were absent from the BSTFA chromatogram. BSTFA is a particularly useful reagent for the derivatization of polar compounds, (such as water-soluble compounds), since it substitutes labile protons on these compounds with a [eSi(CH3)3] (TMS) group, producing a volatile and thermally stable derivative for analysis.
Fig. 8. LineweavereBurk plot for kinetics of Streptomyces sp. IN1 depolymerase activity. Estimates for Vmax and Km are 5.06 101 mmol min1 and 6.73 101 mM, respectively.
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A.D. Allen et al. / International Biodeterioration & Biodegradation 65 (2011) 777e785 Table 2 Effect of potential inhibitors on extracellular depolymerase activity of Streptomyces sp. IN1. Isolate
Relative extracellular depolymerase activity (%)a
Streptomyces sp. IN1 KCNb NaN3c PMSFd DTTe Tween 80 Triton X-100 92.35 923.24 57.17 72.17 73.39 2548 a The extracellular depolymerase activity in the presence of potential inhibitor relative to the control which contained no inhibitor. The activity for control was 6.54 104 mmol min1 and was taken as 100%. b The concentration of the reagents were as follows: Potassium cyanide (KCN), 5 103 mM. c Sodium azide (NaN3), 5 103 mM. d Phenylmethylsulfonyl fluoride (PMSF) 5.0 mM. e Dithiothreitol (DTT), 5.0 mM; Tween 80, 0.1%, Triton X-100, 0.1%.
Fig. 9. Effect of temperature on activity and stability of purified PHA depolymerase from Streptomyces sp. IN1. Depolymerase activity was assayed in 50 mM phosphate buffer, pH 7.9 at [protein], 4.8 101 mg ml1 and [PNPB], 1.11 102 mM at temperatures indicated. The rate of decrease in OD400 was used to determine enzyme activity (C). Depolymerase stability (remaining activity) was evaluated over 15 min at the indicated temperature (bar diagram).
Subsequently, the extracellular protein was purified 14-foldswith a recovery of 84%, and 401-fold with a recovery of 91% by (NH4)2SO4 fractionation and FPLC, respectively. The apparent molecular weight (Mw) of the FPLC purified enzyme as determined by SDSePAGE occurred between 45 and 66 kDa. However, the actual Mw as determined by matrix assisted laser desorption ionizationetime of flight mass spectrometry (MALDIeTOF MS) was 6.24583 104 Da, which differs from previously published data on the extracellular depolymerase from Streptomyces sp. The effect of protein concentration on p-nitrophenylbutyrate esterase activity, estimated by varying protein concentration, was optimum at 4.8 101 mg ml1 protein. Similar observations have been reported for purified depolymerases (Mukai et al., 1993). Further, the depolymerase from Streptomyces sp. IN1 was shown to have estimated Vmax and Km values of 5.06 101 mmol min1 and 6.73 101 mM, respectively. These values are also consistent with previous findings (Schirmer et al., 1993; Sakai et al., 2001; Colak and Guner, 2004). Surprisingly, the enzyme exhibited activity at all temperatures evaluated, but showed optimum activity and stability for over 15 min at 80 C. Similarly, enzyme activity was completely inhibited at pH values below 7, but was optimum at pH 12 with a decrease in activity after 15 min. It is notable that these particular properties of the esterase could be used in diverse industrial processes requiring high pH and/or temperatures such as degradation of polymers and
industrial wastes, for food production, and to replace mesophilic enzymes. Enzyme functions under these conditions are advantageous since solubility of polymeric substrates increases, and microbial contamination is reduced. Previous reports indicate optimum depolymerase activity at 70 C and pH 12 (Mukai et al., 1993; Klingbeil et al., 1996; Çolak et al., 2005). Further evaluation of Streptomyces sp. IN1 depolymerase in the presence of potential inhibitors PMSF, DTT, cyanide, and Tween 80 indicates a considerable decrease in activity. PMSF inhibition suggests the involvement of serine residues in the active site of the enzyme. These residues are known to interact selectively and irreversibly with the compound. Thus, this depolymerase may belong to the serine hydrolase family. Sensitivity to DTT suggests the apparent requirement of disulphide bonds or reduced thiol groups for enzyme activity. Sensitivity to PMSF and DTT are characteristic properties that may be conserved among short-chainlength PHA depolymerases (Jendrossek et al., 1996). A negligible decrease in enzyme activity (6.7%) was shown to occur with cationchelating reagents such as cyanide. This may indicate the dependence of enzyme activity on metal ions. However, this observation contrasts with that for azide, another cation chelator, which showed a 10-fold increase in enzyme activity. This particular observation was further corroborated by enzyme activity in the presence of metal ions such as Ca2þ, Mg2þ, Naþ, and Kþ. These findings differ from previous reports involving esterases and cations at similar concentrations (Kasuya et al., 2000; Handrick et al., 2001; Colak and Guner, 2004). The inhibitory effect of nonionic detergent, Tween 80, suggests that hydrophobic regions may exist near or at the active site of the enzyme. This, however, is not supported by Triton X-100, wherein activity was induced 9fold. Altogether, these observations suggest that this enzyme may be structurally different from other depolymerases. Therefore, it would be interesting to clarify the molecular aspects of this novel thermoalkanophilic depolymerase from Streptomyces sp. IN1. It was of interest to find that the soil isolate, Streptomyces sp. IN1, produced a thermoalkanophilic P(3HB-co-3HV) depolymerase with molecular weight >62 kDa. This enzyme could play a significant role in diverse industrial processes requiring high temperatures and pH. Further studies are underway to further characterize and optimize production of the esterase.
Table 3 Effect of metal ions on extracellular depolymerase activity of Streptomyces sp. IN1.
Fig. 10. Effect of pH on activity and stability of purified PHA depolymerase from Streptomyces sp. IN1. Depolymerase activity was assayed in 50 mM phosphate buffer at pH indicated with [protein], 4.8 101 mg ml1 and [PNPB], 1.11 102 mM. The rate of decrease in OD400 was used to determine enzyme activity (C). Depolymerase stability (remaining activity) was evaluated over 15 min at the indicated pH (bar diagram).
Isolate
Relative extracellular depolymerase activity (%)a
Streptomyces sp. IN1
CaCl2 64.64
MgCl2 36.59
NaCl 48.19
KCl 53.7
a The extracellular depolymerase activity in the presence of the metal ions relative to control which contained no additional metal ion in the assay medium. Control’s activity, 6.54 104 mmol min1, was taken as 100%. All salts had concentration of 1.0 mM.
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Acknowledgments We would like to express our sincere gratitude to Dr. Oumar Diall from the University of Mali for the gift of the Jatropha curcas oil. Some aspects of this work were supported through the Howard Hughes Core Laboratory at Howard University. References Allen, A.D., Anderson, W.A., Ayorinde, F.O., Eribo, B.E., 2010. Biosynthesis and characterization of copolymer poly(3HB-co-3HV) from saponified Jatropha curcas oil by Pseudomonas oleovorans. Journal of Industrial Microbiology and Biotechnology 37, 849e856. Ayorinde, F.O., Saeed, K.A., Price, E., Morrow, A., Collins, W.E., McInnis, F., Pollack, S.K., Eribo, B.E., 1998. Production of poly-(b-hydroxybutyrate) from saponified Vernonia galamensis oil by Alcaligenes eutrophus. Journal of Industrial Microbiology and Biotechnology 21, 46e50. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248e254. Calabia, B.P., Tokiwa, Y., 2006. A novel PHB depolymerase from a thermophilic Streptomyces sp. Biotechnology Letters 28, 383e388. Colak, A., Guner, S., 2004. Polyhydroxyalkanoate degrading hydrolase-like activities by Pseudomonas sp. isolated from soil. International Biodeterioration and Biodegradation 53, 103e109. Çolak, A., S¸is¸ik, O., Saglam, N., Güner, S., Çanakçi, S., Beldüz, A.O., 2005. Characterization of thermoalkalophilic esterase from a novel thermophilic bacterium, Anoxybacillus gonensis G2. Bioresource Technology 96, 625e631. Gonçalves, S.P.C., Martins-Franchetti, S.M., 2009. Biodegradation of the films of PP, PHBV and its blends in soil. Journal of Polymers and the Environment 17, 280e285. Ha, C.S., Cho, W.J., 2002. Miscibility, properties, and degradability of microbial polyester containing blends. Progress in Polymer Science 27, 759e809. Handrick, R., Reinhardt, S., Focarete, M.L., Scandola, M., Adams, G., Kawalcuk, M., Jendrossek, D., 2001. A new type of thermoalkalophilic hydrolase of Paucimonas lemoignei with high specificity for amorphous polyester of short chain length hydroxyalkanoic acids. The Journal of Biological Chemistry 276, 36215e36224. Jaeger, K.E., Steinbüchel, A., Jendrossek, D., 1995. Substrate specificities of bacterial polyhydroxyalkanoates depolymerases and lipases: Bacterial lipases hydrolyze poly(w-hydroxyalkonoates). Applied Environmental Microbiology 61, 3113e3118.
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Jendrossek, D., Schirmer, A., Schegel, H.G.,1996. Biodegradation of polyhydroxyalkanoic acids. Applied Microbiology and Biotechnology 46, 451e463. Kasuya, K.I., Mitomo, H., Nakahara, M., Akiba, A., Kudo, T., Doi, Y., 2000. Identification of a marine benthic P(3HB)-degrading bacterium isolate and characterization of its P(3HB) depolymerase. Biomacromolecules 1, 194e201. Kasuya, K., Doi, Y., Yao, T., 1994. Enzymatic degradation of poly[(R)-3hydroxybutyrate] by Comamonas testosteroni ATSU of soil bacterium. Polymer Degradation and Stability 45, 379e386. Kim, H.J., Kim, D.Y., Nam, J.S., Bae, K.S., Rhee, Y.H., 2003. Characterization of an extracellular medium-chain-length poly(3-hydroxyalkanoate) depolymerase from Streptomyces sp. KJ-72. Antonie van Leeuwenhoek 83, 183e189. Kim, D.Y.S., Nam, J.S., Rhee, Y.H., 2002. Characterization of an extracellular mediumchain-length poly(3-hydroxyalkanoate) depolymerase from Pseudomonas alcaligenes LB. Biomacromolecules 3, 291e296. Kim, H.M., Ryu, K.E., Bae, K.-S., Rhee, Y.H., 2000. Purification and characterization of extracellular medium chain length polyhydroxyalkanoate depolymerase from Pseudomonas sp. RY-1. Journal of Bioscience and Bioengineering 89, 196e198. Klingbeil, B., Kroppenstedt, R., Jendrossek, D., 1996. Taxonomical identification of Streptomyces exfoliates K10 and characterization of its poly(3-hydroxybutyrate) depolymerase gene. FEMS Microbiology Letters 142, 215e221. Lenz, R.W., Marchessault, R.H., 2005. Bacterial polyesters: Biosynthesis, biodegradable plastics and biotechnology. Biomacromolecules 6, 1e8. Marbrouk, M.M., Sabry, S.A., 2001. Degradation of poly(3-hydroxybutyrate) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by a marine Streptomyces sp. SNG9. Microbiological Research 156, 323e335. Mergaert, J., Webb, A., Anderson, C., Wouters, A., Swings, J., 1993. Microbial degradation of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3hydroxyvalerate) in soils. Applied Environmental Microbiology 59, 3233e3238. Mukai, K., Yamada, K., Doi, Y.,1993. Enzymatic degradation of poly(hydroxyalkanoate) by a marine bacterium. Polymer Degradation and Stability 41, 85e91. Sakai, K., Kawson, H., Iwami, A., Nakamura, M., Moriguchi, M., 2001. Isolation of a thermophilic poly-L-lactide degrading bacterium from compost and its enzymatic characterization. Journal of Bioscience and Bioengineering 92, 298e300. Schirmer, A., Jendrossek, D., Schlegel, H.S., 1993. Degradation of poly(3hydroxyoctanoic acid) [P(3HO)] by bacteria: Purification and properties of a P(3HO) depolymerase from Pseudomonas fluorescens GK13. Applied and Environmental Microbiology 59, 1220e1227. Shah, A.A., Hasan, F., Hameed, A., Ahmed, S., 2007. Isolation and characterization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) degrading bacteria and purification of PHBV depolymerase from newly isolated Bacillus sp. AF3. International Biodeterioration and Biodegradation 60, 109e115.
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International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Isolation and characterization of an extracellular thermoalkanophilic P(3HB-co-3HV) depolymerase from Streptomyces sp. IN1 Adrian Douglas Allen a, W.A. Anderson a, F. Ayorinde b, B.E. Eribo a, b, * a b
Department of Biology, Graduate School, Howard University, 415 College St. NW, Washington, DC 20059, USA Department of Chemistry, Graduate School, Howard University, Washington, DC 20059, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 October 2010 Received in revised form 9 February 2011 Accepted 10 February 2011
Here, we report on the biodegradation of the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co3HV)] by a novel thermoalkanophilic extracellular esterase from the soil isolate Streptomyces sp. IN1. Preliminary screening and isolation of the bacterium was done using polyhydroxyalkanoate latex medium (PHALM). The isolate was cultured with P(3HB-co-3HV) as the only carbon source and byproducts of degradation were derivatized with [N,O-bis(trimethylsilyl)trifluroacetamide] (BSTFA). These products were identified by gas chromatography/mass spectrometry (GCeMS) as silylated hydroxybutyric acid (3HB) and hydroxyvaleric acid, suggesting extracellular depolymerase activity by the isolate. The depolymerase was isolated by (NH4)2SO4 fractionation, dialyzed and purified using fast protein liquid chromatography (FPLC), and confirmed using P(3HB-co-3HV) as a sole source of carbon. The molecular mass of the FPLC purified enzyme occurred between 45 and 66 kDa (SDSePAGE), but was confirmed by matrix assisted laser desorption ionizationetime of flight mass spectrometry (MALDIeTOF MS) to be 62 kDa. Enzyme activity was significantly inhibited by phenylmethylsulfonyl fluoride (PMSF), dithiothreitol (DTT), and Tween 80, but induced by azide (N3). Sensitivity to PMSF, DTT, and Tween 80 suggests the involvement of serine as an active site amino acid with disulphide bonds contributing to the catalytic activity, as well as the presence of hydrophobic regions in the enzyme. Non-inhibition of activity by azide indicates that metal ions may not be required as cofactors for activity. This observation was further corroborated by the decrease in enzyme activity in the presence of metal ions such as Ca2þ, Mg2þ, Naþ, and Kþ. The kinetic parameters, Vmax and Km, in the presence of p-nitrophenylbutyrate as substrate, were determined to be 5.06 101 mmol min1 and 6.73 101 mM, respectively. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Biodegradation P(3HB-co-3HV) depolymerase Streptomyces sp. Esterase
1. Introduction There is increasing interest in the identification of novel polyhydroxyalkanoate (PHA)-degrading microorganisms because of their biotechnological potential as sources of extracellular esterases. These enzymes could play a significant role in the degradation of industrial pollutants and natural materials such as PHA (Lenz and Marchessault, 2005). Degradation of extracellular PHA and the use of its by-products as a source of carbon and energy is controlled by the secretion of specific extracellular PHA depolymerase(s). These may be specific for medium-chain-length PHA or short-chainlength (scl) PHA, such as poly (3-hydroxybutyrate), P(3HB), or its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate), P(3HBco-3HV) (Ha and Cho, 2002; Kim et al., 2002; Gonçalves and Martins-Franchetti, 2009). * Corresponding author. Department of Biology, 415 College St. NW, Washington, DC 20059, USA. Tel.: þ1 202 806 6937; fax: þ1 202 806 4564. E-mail address: [email protected] (B.E. Eribo). 0964-8305/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2011.02.010
Although significant strides have been made in extracellular PHA depolymerase research, the majority of studies have focused on Gram-negative bacteria having optimum depolymerase activity 65 C and pH 10. Notable exceptions were observed for the depolymerase from Comamonas testosteroni ATSU with an optimum temperature at 70 C (although activity was not retained at this temperature) (Kasuya et al., 1994), and Paucimonas lemoignei with an optimum pH of 12 (Handrick et al., 2001). Fewer reports have addressed the isolation and characterization of depolymerases, particularly thermophilic and/or alkanophilic depolymerases, from Gram-positive bacteria, especially Streptomyces sp., despite their abundance in soil (Klingbeil et al., 1996; Marbrouk and Sabry, 2001; Kim et al., 2003; Calabia and Tokiwa, 2006). However, none of these depolymerases had molecular mass 50 kDa or optimum pH and temperature above 10 and 60 C, respectively. In our previous report, the copolymer P(3HB-co-3HV) was produced from saponified Jatropha curcas oil (Allen et al., 2010). Here, we report on the biodegradation of P(3HB-co-3HV) by a novel
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thermoalkanophilic extracellular esterase from the soil isolate Streptomyces sp. IN1. 2. Materials and methods 2.1. Isolation of PHA-degrading bacteria Soil was collected, dried to constant weight at 30 C, and serially diluted using 0.1% proteose peptone. Inocula were plated onto PHA latex medium (PHALM) and incubated under aerobic and anaerobic conditions at 30 C over 2e10 days or until zones of clearing were visible. PHA degraders were then sub-cultured onto 0.4% (w/v) PHALM, and those that exhibited rapid degradation of the substrate were used for further analyses. Sterilized soil was used as a control. 2.2. Growth medium (PHA latex medium) A 0.4% PHALM (w/v) was prepared by suspending poly-3hydroxybutyrate [P(3HB), Sigma] or P(3HB-co-3HV) into mineral salt medium (MSM) and sonicating (Branson, 2210) for 10e20 min. The constituents of the MSM (Ayorinde et al., 1998) were as follows: macronutrients (g l1): 1.1 g (NH4)2HPO4, 5.8 g K2HPO4, 3.7 g KH2PO4, 10.0 ml of a 100 mM MgSO4 solution, and 1.0 ml of micronutrient solution. The micronutrient solution consisted of (g l1) 2.78 g FeSO4$7H2O, 1.98 g MnCl2$4H2O, 2.81 g CoSO4$7H2O, 1.67 g CaCl2$2H2O, 0.17 g CuCl2$2H2O, and 0.29 g ZnSO4$7H2O. Solid medium was prepared by adding 15 g l1 agar to MSM. All media were sterilized for 15 min at 121 C. 2.3. Phenotypic characterization of the isolate The following tests were used for phenotypic characterization: Gram-stain, oxidase, phenylalanine, nitrate, catalase, cysteine utilization, growth on cornmeal-Tween medium, Emmons medium, 7.5% sodium chloride, glucose, lactose, citrate, and motility. All media were prepared according to the manufacturers’ specifications.
2.6. Growth profile and enzyme activity of isolate The isolate was inoculated into 100 ml PHA latex broth and incubated at 30 C for 2e10 days. Culture broth, 2.0 ml, was removed periodically, and the OD650 determined. Further, esterase activity was evaluated at OD400 with p-nitrophenylbutyrate (PNPB) as substrate (e ¼ 2.75 M1 cm1). Growth responses for the isolate were obtained by interpolating OD650 versus time. Controls contained no substrate (PHA). 2.7. Derivatization of P(3HB-co-3HV) by-products By-products of P(3HB-co-3HV) degradation were derivatized with N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) and analyzed using gas chromatographyemass spectrometry (GCeMS). The following protocol (ThermoScientific) was observed for the reaction: 8.0 mg of dried sample was imbued with 100 ml BSTFA in a clean, dry reaction vial. The mixture was vortexed for 5 min, incubated for 10 min at room temperature, and 1.0 ml of the preparation was used for GCeMS analysis. Alternatively, if by-products were not generated by this method, the preparation (by-product/ BSTFA) was heated for 15 min at 70 C, cooled to room temperature, and analyzed as before. A commercial sample of DL-b-hydroxybutyric acid (Sigma, St. Louis, MO) and valeric acid (Lancaster synthesis Inc., Pelham, NH) were used as controls. 2.8. GCeMS analyses An Agilent Technologies 6890N Network GC System (CA, USA) interfaced directly to an Agilent Technologies 5973 Inert Mass Selective Detector was used to generate data. A Supelco fused-silica SPB-1 (30 m, 0.32 mm i.d., 0.25-mm film) column (Bellefonte, PA, USA) was used to conduct high-resolution capillary gas chromatography. Oven temperature was programmed from 40 to 300 C at 12 C min1, and helium was used as the carrier gas with head pressure of 3 psi. Injector temperature was set at 240 C. A HewlettePackard PC integration program was used to evaluate data. Runs were done in triplicate.
2.4. DNA isolation/PCR/sequencing DNA was isolated using a QIAampÒ DNA mini kit (Qiagen). Briefly, the isolate was grown overnight in trypticase soy broth at 30 C, shaken at 120 rpm (EnvironÒ shaker), pelleted and preincubated in a mixture containing 180 ml of 20 mg ml1 lysozyme, 20 mM TriseHCl, pH 8.0; 2.0 mM EDTA and 1.2% Triton X-100. The DNA was isolated from this mixture, purified, and it’s concentration determined using a NanoDrop 2000C spectrophotometer (Thermo Scientific, Wilmington, DE). Then 16S rDNA amplification was carried out with the following parameters: initial denaturation at 95 C for 5 min followed by 35 cycles of denaturation at 95 C for 45 s, annealing at 51 C for 45 s, extension at 72 C for 2 min, and a final extension at 72 C for 10 min. Amplified 16S rRNA gene was sequenced (Genewiz, Inc., South Plainfield, NJ). 16S rRNA gene sequence was then compared with those in GenBank (http://www.ncbi.nlm.nih.gov).
2.9. Purification and isolation of extracellular PHA depolymerase The culture was incubated as before and broths were centrifuged (Sorvall, refrigerated centrifuge, Dupont Instruments Corp.) at 13,000 rpm for 10 min at 4 C. The supernatant was collected and filtered (Corning 25 mm, 0.22 mm) and ammonium sulfate [(NH4)2SO4] fractions were collected between 30 and 80% saturation at 4 C. Fractions were pooled and dialyzed against 20 mM TriseHCl, pH 7.2, at 4 C using a Slide-A-LyzerÒ dialysis cassette (Rockford, IL) with a 10-kDa molecular weight cutoff (MWCO) or CarolinaÒ dialysis tubing (Burlington, NC) with a MWCO of 13 kDa. Samples were concentrated (Savant, Global Medical Instruments, MN) and a final purification performed using anion exchange chromatography on a BIOCAD fast protein liquid chromatography (FPLC) instrument. 2.10. FPLC analyses
2.5. Evaluation of PHA degradation PHA latex plates (PHALM) and broths were prepared as before with P(3HB-co-3HV) as the sole source of carbon. The isolate was cultured at 30 C, 120 rpm, for 2e10 days and clearing zones on solid medium were measured (in millimeters) every 48 h. Culture broths were then filtered (0.22 mm), and supernatant dried for 48 h at 60 C, and then derivatized. The commercial P(3HB) (Sigma, St. Louis, MO) was used as the control.
A BioCAD 700E perfusion chromatographyÒ workstation (Global Medical Instruments, MN) was used for further purification of proteins. About 1000 ml of filtered (0.22 mm Nalgene filter) sample was automatically loaded into 1.0 ml sample loop. The buffers used for processing were 20 mM TriseHCl, pH 8.0, and 20 mM TriseHCl, pH 7.0, with 1.0 mM NaCl. A HiTrap QFF, 1.0 ml anionic column (GE Healthcare, NJ) was used with a flow rate of 1.0 ml min1. The UV detector was set at 254 and 280 nm with a threshold of 0.5.
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Fractions were collected by peak cutting using an Avantec SF-2120 Super Fraction Collector (Global Medical Instruments, MN) and respective fractions were pooled and concentrated (Savant, GMI, MN) at 4 C. The resulting data were processed using the BioCAD 700E Version 3 Series software. All buffers and samples were sterilized by filtering. 2.11. Protein determination Protein concentrations were determined according to Bradford (1976) using the protein assay reagent (Bio-Rad Laboratories) with bovine serum albumin (BSA) as the standard.
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from 0.0 to 1.0 mg ml1 protein in a total volume of 1.0 ml. Assay was performed as mentioned before with PNPB as substrate. 2.15. Kinetic studies Kinetic studies were performed using 4.8 101 mg ml1 protein and 0.0 to 8.0 101 mM PNPB in a final volume of 1.0 ml. Hydrolytic activity was monitored spectrophotometrically as before. A substrate saturation curve was generated by plotting substrate concentrations against activity values, and Vmax and Km values were estimated from a LineweavereBurk plot. 2.16. Effect of temperature on depolymerase activity and stability
2.12. Enzymatic assay Extracellular PHA depolymerase activity was assayed with a modification of methods outlined previously (Schirmer et al., 1993; Jaeger et al., 1995; Kim et al., 2000). PHA depolymerase was assayed as p-nitrophenylbutyrate (PNPB) esterase activity at 30 C. The reaction mixture contained 0.0e0.80 mM PNPB in ethanol, 10e270 ml enzyme and 50 mM potassium phosphate buffer (pH 8.0) in a final volume of 1.0 ml. Spectral changes for the hydrolysis of PNPB were monitored spectrophotometrically at 400 nm (e ¼ 2.75 M1 cm1). One unit of enzyme activity was defined as the amount of enzyme releasing 1.0 mmol p-nitrophenol min1 (PNP) under assay conditions. Alternatively, the assay was performed with a modification of a method found elsewhere (Shah et al., 2007). Briefly, a mixture was used containing 100 ml enzyme and 900 ml 0.03% PHA latex broth. The mixture was incubated at 30 C for 1.0 h and the decrease in turbidity at OD650 (e ¼ 4.752 105 mM1 cm1) was monitored. One unit of enzyme was defined as the amount of enzyme capable of decreasing the OD650 by 1 unit (mmol min1). Further, to confirm that FPLC purified enzyme was an esterase, about 20 ml of enzyme was incubated with approximately 1.0 ml 0.03% P(3HB-co-3HV) at 30 C, 120 rpm for 24e72 h. Samples were then dehydrated, derivatized, and evaluated by GCeMS analysis. Controls contained no enzyme. 2.13. Protein analysis by SDSePAGE and MALDIeTOF MS A standard protocol was used for SDSe12% PAGE. Samples were electrophoresized at 100 V for about 1.5 h with broad-range molecular marker (Bio-Rad Laboratories) with composition myosin, 200 kDa; b-Galactosidase, 116 kDa; phosphorylase b, 97 kDa; serum albumin, 66 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 31 kDa; and trypsin inhibitor, 21 kDa. Gels were stained with Coomassie Brilliant Blue R-250 and images were obtained (Kodak Gel Logic Imaging System, Rochester, NY). Further, the actual molecular weight of the FPLC purified protein was determined using matrix assisted laser desorption ionizationetime of flight mass spectrometry (MALDIeTOF MS) (Voyager-DEÔ STR workstation). About 1.0 ml FPLC sample was mixed with matrix 2,5 dihydrobenzoic acid (2,5 DHB) in tetrahydrofuran (THF), (10 mg mL1), applied to target, and allowed to dry freely. Data were acquired in reflector mode at 25 kV, grid voltage 94%; extraction delay time was 100 ns, with 150 transients. Runs were done in triplicate. Data analysis was performed with the Applied Biosystems Voyager System 4098 software. 2.14. Effect of protein concentration on extracellular depolymerase activity The relationship between protein concentration and depolymerase activity was measured by varying protein concentrations
The optimum activity of purified extracellular depolymerase was evaluated under various temperatures e 30, 40, 50, 60, 80, and 90 C. Incubations were performed at the indicated temperature and the initial velocity was derived from a progress curve. To evaluate enzyme stability (remaining activity), assay was incubated at the aforementioned temperatures and the remaining activity was ascertained at 5-min intervals over 15 min. The assay conditions were as described before. Reactions were done in triplicate. 2.17. Effect of pH on depolymerase activity and stability The optimum activity of purified extracellular depolymerase was evaluated under variations in pH e 5, 6, 7, 8, 9, 11, and 12. Incubations were performed at the indicated pH and initial velocities were derived from progress curves. Remaining activity (as a percentage) and assay conditions were as mentioned before. The pH was adjusted using 1.0 M hydrochloric acid and 1.0 M sodium hydroxide and reactions were done in triplicate. The molar extinction coefficient (e, M1 cm1) of p-NP at various pHs was determined under assay conditions using phosphate buffer. 2.18. Effect of potential inhibitors on depolymerase activity The potential inhibitors used were as follows: phenylmethylsulfonyl fluoride (PMSF, 5.0 102 mM); dithiothreitol (DTT, 5.0 102 mM); potassium cyanide (KCN, 5 103 mM); sodium azide (NaN3, 5 103 mM); Triton X-100 (0.1%); and Tween 20 (0.1%). The PHA depolymerase activity was evaluated with adjustments in the chemical composition of the assay. Spectral changes were followed over 15 min. The control contained no potential inhibitor other than the compounds required for the standard assay. Reactions were done in duplicate. 2.19. Effect of metal ions on PHA depolymerase activity The effect of metal ions on enzyme activity was determined by the addition of 1.0 mM chloride salts of Ca2þ, Mg2þ, Naþ, and Kþ to the assay mixture. Controls contained no metal ions other than the compounds required for the standard assay. Spectral changes were followed over 15 min. Reactions were done in duplicate. 3. Results 3.1. Isolation of PHA-degrading bacteria PHA-degrading bacteria were isolated based on clear zones of hydrolysis on PHALM plates (Figs. 1 and 2). Although only five degraders were isolated from the soil sample under aerobic conditions, only one, isolate IN1, showed rapid degradation of the substrate over 10 days. Therefore, this isolate was used for all
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Fig. 1. Isolation of Streptomyces sp. IN1 from soil on 0.4% PHA latex medium (a), and degradation of J. curcas PHA by Streptomyces sp. IN1 (b). Zones of degradation are indicated by clearing around isolate (arrows). Degradation occurred between 2 and 10 days at 30 C.
analyses. PHA degraders were not isolated under anaerobic conditions or in the control (autoclaved soil sample). 3.2. Phenotypic characterization of isolate The isolate was a Gram-positive rod, non-motile, catalase positive, halotolerant (7.5% sodium chloride), and produced leathery colonies with non-septate mycelia and yellow spores when cultured on cornmeal-Tween agar. In addition, the bacterium reduced nitrate and cysteine, but was unable to utilize tryptophan, phenylalanine, glucose, or lactose. 3.3. Taxonomic identification of isolate IN1 Based on biochemical and morphological features, isolate IN1 belongs to the genus Streptomyces, and it was designated IN1. Based on its 16S rRNA gene sequence (GenBank accession JF268582) Streptomyces sp. IN1 exhibits 98% homology with Streptomyces sp. C39 (GenBank accession AY741282). 3.4. Evaluation of PHA degradation Semi-quantitative evaluation of P(3HB-co-3HV) degradation over time (250 h) shows that degradation of the material was
Zone off Degradattion (mm)
60
occurring. This was illustrated by the formation of clear zones of hydrolysis (Fig. 1). Degradation was also shown to occur faster with P(3HB-co-3HV) copolymer compared to the control [commercial P(3HB)] (Fig. 2). 3.5. Growth profile and enzyme activity of isolate Growth responses for Streptomyces sp. IN1, in PHA latex broth, were derived by interpolating various time periods against optical density (650 nm) (Fig. 3). Optimum growth was shown to occur at 12 h. An evaluation of extracellular depolymerase activity relative to growth was ascertained by measuring the hydrolysis of p-nitrophenylbutyrate to p-nitrophenol at 400 nm. Optimum extracellular depolymerase activity was shown to occur at 24 h with a decrease thereafter (Fig. 3). 3.6. GCeMS analysis Derivatized by-products of PHA degradation were determined using GCeMS analysis and confirmation with spectra from the National Institute of Standards and Technology (NIST02) database/ library. Silylated by-products were similar for assays using either supernatant or purified enzyme. These by-products were identified as silylated hydroxybutyric and hydroxyvaleric acids (Fig. 4). The spectrum of butanoic acid, 3-[trimethylsilyloxy]-trimethylsilyl ester (Fig. 4a) shows diagnostic ions at m/z 233 (M-15) and m/z73
50 40 30 20 10 0 0
50
100
150
200
250
300
Time (h) Fig. 2. Rate of degradation of Jatropha curcas polymer by Streptomyces sp. IN1 over 250 h incubation at 30 C. J. curcas PHA (C), commercial PHA (B). Zone of degradation as shown in Fig. 1 was measured (in millimeters) every 48 h.
Fig. 3. Growth profile for Streptomyces sp. IN1 in mineral salt medium containing 0.4% (w/v) PHA (C), and variation of extracellular depolymerase activity, during growth over 84 h, 120 rpm, 30 C (B). Activity (mmol min1) is based on p-nitrophenylbutyrate (PNPB) hydrolysis. The control is shown by (A).
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[eOSi(CH3)3]. The spectrum of the commercial silylated hydroxybutyrate (Fig. 4b) is consistent with that of the product in Fig. 4a. Similarly, the silylated hydroxyvalerate with diagnostic ions at m/z 247 (M-15) is shown in Fig. 4c. This was further confirmed by the NIST02 database. 3.7. Purification and isolation of extracellular PHA depolymerase Quantitative evaluation of the results observed for consecutive purification steps (supernatant, ammonium sulfate, fast perfusion liquid chromatography) is shown in Table 1. The protein was purified 14-fold with an activity of 84% by ammonium sulfate precipitation. Several fractions containing three proteins were collected from FLPC using a QFF anion exchange column (data not shown). Individual fractions were evaluated for activity and those with the highest activity were combined and concentrated. This combination accounted for a recovery rate of 91% and a purification fold of 401. Proteins and fractions were consistent over several repetitions.
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3.8. Protein analysis by SDSePAGE and MALDIeTOF MS The SDSePAGE profile for the purified enzyme is shown in Fig. 5. The apparent molecular weight (Mw), as determined by SDSePAGE, occurred between 45 and 66 kDa. However, the actual Mw as determined by matrix assisted laser desorption ionizationetime of flight mass spectrometry (MALDIeTOF MS) was 6.24583 104 Da (Fig. 6).
3.9. Effect of protein concentration on extracellular depolymerase activity The relationship between protein concentration and depolymerase activity exhibited a hyperbolic curve (Fig. 7). Depolymerase activity, measured as p-nitrophenol formation, increased until the protein concentration reached 4.8 101 mg ml1 and remained constant at levels above this value.
Fig. 4. Mass spectra of BSTFA derivatized by-product of J. curcas polymer degradation. These were identified as (a) silylated hydroxybutyrate, (b) silylated hydroxybutyrate (commercial), and (c) silylated hydroxyvalerate. These were confirmed by the NIST database.
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Table 1 Purification of
a
extracellular PHA depolymerase from Streptomyces sp. IN1.
Isolate
Purification step
Volume (ml)
Protein (mg ml1)
Total protein (mg)
Total activity (mmol min1) 105
Specific activity (mmol min1 mg1 protein)
Purification fold
Recovery (%)
Streptomyces sp. IN1
Crude (NH4)2SO4 (30e80%) FPLC (pooled fractions)
250 20 5
0.54 0.37 0.063
128 7.48 0.32
2.31 1.94 2.10
1.79 103 2.59 104 7.22 105
1 14 401
100 84 91
a
Extracellular PHA depolymerase activity was performed in an assay containing 0.03% PHA (see Methods) [OD650 (3 ¼ 4.752 105 mM1 cm1)].
3.10. Kinetic studies The substrate saturation curve was obtained by plotting substrate concentrations against the activity values. Values for Vmax and Km were then determined as 5.06 101 mmol min1 and 6.73 101 mM, respectively, from the LineweavereBurk double reciprocal plot (Fig. 8).
There was significant depolymerase activity in the presence of Triton X-100, 2548%, and azide, 923.24%. However, the activity decreased in activity in the presence of Tween 80, 73.39%; DTT, 72.17%; and PMSF, 57.17%, compared to the control with 100% activity (Vmax 1.25 103 mmol min1). A negligible decrease in activity (92.35%) was shown in the presence of cyanide. 3.14. Effect of metal ions on activity
3.11. Effect of temperature on depolymerase activity and stability The dependence of the purified esterase activity on assay temperature was evaluated (Fig. 9, circular points). The enzyme exhibited activity over all temperatures evaluated, but had an optimum temperature of 80 C, and was partially deactivated at 90 C. The thermal stability profile indicates that the enzyme was stable at 80 C for over 15 min, but was almost completely destabilized at 90 C for the same time period (Fig. 9, bar diagram). 3.12. Effect of pH on depolymerase activity and stability The activity of the depolymerase from Streptomyces sp. IN1 at pH values between 5 and 12 was evaluated (Fig. 10, circular points). The pH profile shows that the enzyme was completely inactivated at pH values below 7, but showed optimum activity at pH 12. The thermal stability profile shows that the enzyme was stable at pH between 7 and 12, but was most stable at pH 12 for the time evaluated (Fig. 10, bar diagram). 3.13. The effect of various chemicals on PHA depolymerase activity The effect of various chemicals and metal ions on the activity of Streptomyces sp. IN1 PHA depolymerase was evaluated (Table 2).
Fig. 5. SDS-PAGE of FPLC purified PHA depolymerase from Streptomyces sp. IN1. Proteins were separated on a SDSe12% polyacrylamide gel and stained with Coomassie Brilliant Blue R-250. Lane 1, molecular weight marker (myosin, 2 00 kDa; b-Galactosidase, 116 kDa; phosphorylase b, 97 kDa; serum albumin, 66 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 31 kDa; trypsin inhibitor, 21 kDa); lane 2, FPLC purified PHA depolymerase (arrow indicates location of protein).
Extracellular depolymerase activity was less than 100% in the presence of all metal ions compared to the control, which had no additional metal ion other than the compounds required for the assay (Table 3). The remaining activities were Mg2þ, 36.59; Ca2þ, 64.64; Naþ, 48.19; and Kþ, 53.7%. 4. Discussion In the current study, a novel thermoalkanophilic P(3HB-co-3HV) esterase from the soil bacterium Streptomyces sp. IN1 (GenBank accession JF268582) was isolated and partially characterized. Presumptive identification was made in accordance with the methods as outlined by the International Streptomyces Project (ISP). Growth responses for Streptomyces sp. IN1 in PHA latex broth and agar (PHA, 0.4%, w/v) were obtained by interpolating optical densities (OD) at 650 nm or zone of clearing (mm) versus different time intervals. Optimum growth and esterase activity was shown to occur at 12 and 24 h of incubation, respectively. Esterase activity, indicated by the hydrolysis of p-nitrophenylbutyrate, was inversely proportional to the growth of Streptomyces sp. IN1 between 36 and 84 h. The decrease in extracellular enzyme activity (36e84 h) may have occurred from changes in culture conditions such as a decrease in pH, from by-products of Streptomyces sp. IN1 metabolism, or from complete hydrolysis of the polymer. Furthermore, initial OD650 values were high since these reflected an environment containing bacteria (viable and dead) and suspended PHA. Subsequently, OD650 decreased because PHA was hydrolyzed (change in nature of PHA granules) by extracellular PHA depolymerase into water-soluble by-products (monomers, dimers, and/or oligomers). Thus, the OD650 at this point (between 12 and 36 h) reflects mostly cells of Streptomyces sp. IN1. Hydrolysis of PHA is vital since bacteria have not been shown to utilize denatured (extracellular) PHA. In essence, the degradative by-products of PHA were then utilized by the viable cells for growth (between 36 and 72 h); therefore the OD650 increased. Interestingly, enzyme activity (indicated by zone of clearing) continued beyond 250 h on solid medium and was shown (semiquantitatively) to occur faster with P(3HB-co-3HV) copolymer compared to the commercial homopolymer, P(3HB). This may have arisen due to steric hindrance and/or other inhibition (noncompetitive) of the enzyme by the agar (solid) or other components in the medium. Thus, the extracellular depolymerase was insufficiently bound to the substrate and degradation occurred more slowly in solid compared to broth. This observation is similar to the previous report (Mergaert et al., 1993) and gives some insight into extracellular depolymerase action and/or conditions which may
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Fig. 6. Matrix assisted laser desorption ionizationetime of flight mass spectrum (MALDIeTOF MS) of FPLC purified extracellular PHA depolymerase from Streptomyces sp. IN1. The actual molecular weight of protein was 6.24583 104 Da. Data were ascertained using 2,5 dihydrobenzoic acid (2,5 DHB) as matrix in reflector mode at 25 kV.
favor optimum degradation of the material. Furthermore, there is evidence to suggest that copolymers of P(3HB-co-3HV) are more susceptible to hydrolysis by extracellular depolymerase than is P(3HB) due to melting point, decreased crystallinity (better access of enzyme to polymer chain), and molecular weight of the polymer (Ha and Cho, 2002; Gonçalves and Martins-Franchetti, 2009). Decreased enzyme activity may also indicate that the extracellular depolymerase from Streptomyces sp. IN1 is more specific for P(3HBco-3HV) than P(3HB). Further confirmation of extracellular esterase secretion by Streptomyces sp. IN1 was obtained by derivatization of P(3HB-co3HV) by-products derived from both FPLC purified enzyme and
Fig. 7. Effect of protein concentration (mg ml1) on extracellular depolymerase activity (mmol min1) of Streptomyces sp. IN1. Optimum activity occurred at 4.8 101 mg ml1 protein. Protein concentration was measured using Bradford assay and activity as PNPB hydrolysis at 400 nm.
culture supernatant. These were confirmed using GCeMS analysis and the National Institute of Standards and Technology (NIST02) database library and were identified as silylated hydroxybutanoic [3-[trimethylsilyloxy]-,trimethylsilyl ester] and hydroxyvaleric acids. These silylated derivatives are unquestionably the products of PHA degradation since they were found only in the assay containing only FPLC purified enzyme and P(3HB-co-3HV) and also the culture supernatant (filtered, 0.22 mm). These by-products had spectra similar to the commercial samples, and were absent from the BSTFA chromatogram. BSTFA is a particularly useful reagent for the derivatization of polar compounds, (such as water-soluble compounds), since it substitutes labile protons on these compounds with a [eSi(CH3)3] (TMS) group, producing a volatile and thermally stable derivative for analysis.
Fig. 8. LineweavereBurk plot for kinetics of Streptomyces sp. IN1 depolymerase activity. Estimates for Vmax and Km are 5.06 101 mmol min1 and 6.73 101 mM, respectively.
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A.D. Allen et al. / International Biodeterioration & Biodegradation 65 (2011) 777e785 Table 2 Effect of potential inhibitors on extracellular depolymerase activity of Streptomyces sp. IN1. Isolate
Relative extracellular depolymerase activity (%)a
Streptomyces sp. IN1 KCNb NaN3c PMSFd DTTe Tween 80 Triton X-100 92.35 923.24 57.17 72.17 73.39 2548 a The extracellular depolymerase activity in the presence of potential inhibitor relative to the control which contained no inhibitor. The activity for control was 6.54 104 mmol min1 and was taken as 100%. b The concentration of the reagents were as follows: Potassium cyanide (KCN), 5 103 mM. c Sodium azide (NaN3), 5 103 mM. d Phenylmethylsulfonyl fluoride (PMSF) 5.0 mM. e Dithiothreitol (DTT), 5.0 mM; Tween 80, 0.1%, Triton X-100, 0.1%.
Fig. 9. Effect of temperature on activity and stability of purified PHA depolymerase from Streptomyces sp. IN1. Depolymerase activity was assayed in 50 mM phosphate buffer, pH 7.9 at [protein], 4.8 101 mg ml1 and [PNPB], 1.11 102 mM at temperatures indicated. The rate of decrease in OD400 was used to determine enzyme activity (C). Depolymerase stability (remaining activity) was evaluated over 15 min at the indicated temperature (bar diagram).
Subsequently, the extracellular protein was purified 14-foldswith a recovery of 84%, and 401-fold with a recovery of 91% by (NH4)2SO4 fractionation and FPLC, respectively. The apparent molecular weight (Mw) of the FPLC purified enzyme as determined by SDSePAGE occurred between 45 and 66 kDa. However, the actual Mw as determined by matrix assisted laser desorption ionizationetime of flight mass spectrometry (MALDIeTOF MS) was 6.24583 104 Da, which differs from previously published data on the extracellular depolymerase from Streptomyces sp. The effect of protein concentration on p-nitrophenylbutyrate esterase activity, estimated by varying protein concentration, was optimum at 4.8 101 mg ml1 protein. Similar observations have been reported for purified depolymerases (Mukai et al., 1993). Further, the depolymerase from Streptomyces sp. IN1 was shown to have estimated Vmax and Km values of 5.06 101 mmol min1 and 6.73 101 mM, respectively. These values are also consistent with previous findings (Schirmer et al., 1993; Sakai et al., 2001; Colak and Guner, 2004). Surprisingly, the enzyme exhibited activity at all temperatures evaluated, but showed optimum activity and stability for over 15 min at 80 C. Similarly, enzyme activity was completely inhibited at pH values below 7, but was optimum at pH 12 with a decrease in activity after 15 min. It is notable that these particular properties of the esterase could be used in diverse industrial processes requiring high pH and/or temperatures such as degradation of polymers and
industrial wastes, for food production, and to replace mesophilic enzymes. Enzyme functions under these conditions are advantageous since solubility of polymeric substrates increases, and microbial contamination is reduced. Previous reports indicate optimum depolymerase activity at 70 C and pH 12 (Mukai et al., 1993; Klingbeil et al., 1996; Çolak et al., 2005). Further evaluation of Streptomyces sp. IN1 depolymerase in the presence of potential inhibitors PMSF, DTT, cyanide, and Tween 80 indicates a considerable decrease in activity. PMSF inhibition suggests the involvement of serine residues in the active site of the enzyme. These residues are known to interact selectively and irreversibly with the compound. Thus, this depolymerase may belong to the serine hydrolase family. Sensitivity to DTT suggests the apparent requirement of disulphide bonds or reduced thiol groups for enzyme activity. Sensitivity to PMSF and DTT are characteristic properties that may be conserved among short-chainlength PHA depolymerases (Jendrossek et al., 1996). A negligible decrease in enzyme activity (6.7%) was shown to occur with cationchelating reagents such as cyanide. This may indicate the dependence of enzyme activity on metal ions. However, this observation contrasts with that for azide, another cation chelator, which showed a 10-fold increase in enzyme activity. This particular observation was further corroborated by enzyme activity in the presence of metal ions such as Ca2þ, Mg2þ, Naþ, and Kþ. These findings differ from previous reports involving esterases and cations at similar concentrations (Kasuya et al., 2000; Handrick et al., 2001; Colak and Guner, 2004). The inhibitory effect of nonionic detergent, Tween 80, suggests that hydrophobic regions may exist near or at the active site of the enzyme. This, however, is not supported by Triton X-100, wherein activity was induced 9fold. Altogether, these observations suggest that this enzyme may be structurally different from other depolymerases. Therefore, it would be interesting to clarify the molecular aspects of this novel thermoalkanophilic depolymerase from Streptomyces sp. IN1. It was of interest to find that the soil isolate, Streptomyces sp. IN1, produced a thermoalkanophilic P(3HB-co-3HV) depolymerase with molecular weight >62 kDa. This enzyme could play a significant role in diverse industrial processes requiring high temperatures and pH. Further studies are underway to further characterize and optimize production of the esterase.
Table 3 Effect of metal ions on extracellular depolymerase activity of Streptomyces sp. IN1.
Fig. 10. Effect of pH on activity and stability of purified PHA depolymerase from Streptomyces sp. IN1. Depolymerase activity was assayed in 50 mM phosphate buffer at pH indicated with [protein], 4.8 101 mg ml1 and [PNPB], 1.11 102 mM. The rate of decrease in OD400 was used to determine enzyme activity (C). Depolymerase stability (remaining activity) was evaluated over 15 min at the indicated pH (bar diagram).
Isolate
Relative extracellular depolymerase activity (%)a
Streptomyces sp. IN1
CaCl2 64.64
MgCl2 36.59
NaCl 48.19
KCl 53.7
a The extracellular depolymerase activity in the presence of the metal ions relative to control which contained no additional metal ion in the assay medium. Control’s activity, 6.54 104 mmol min1, was taken as 100%. All salts had concentration of 1.0 mM.
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Acknowledgments We would like to express our sincere gratitude to Dr. Oumar Diall from the University of Mali for the gift of the Jatropha curcas oil. Some aspects of this work were supported through the Howard Hughes Core Laboratory at Howard University. References Allen, A.D., Anderson, W.A., Ayorinde, F.O., Eribo, B.E., 2010. Biosynthesis and characterization of copolymer poly(3HB-co-3HV) from saponified Jatropha curcas oil by Pseudomonas oleovorans. Journal of Industrial Microbiology and Biotechnology 37, 849e856. Ayorinde, F.O., Saeed, K.A., Price, E., Morrow, A., Collins, W.E., McInnis, F., Pollack, S.K., Eribo, B.E., 1998. Production of poly-(b-hydroxybutyrate) from saponified Vernonia galamensis oil by Alcaligenes eutrophus. Journal of Industrial Microbiology and Biotechnology 21, 46e50. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248e254. Calabia, B.P., Tokiwa, Y., 2006. A novel PHB depolymerase from a thermophilic Streptomyces sp. Biotechnology Letters 28, 383e388. Colak, A., Guner, S., 2004. Polyhydroxyalkanoate degrading hydrolase-like activities by Pseudomonas sp. isolated from soil. International Biodeterioration and Biodegradation 53, 103e109. Çolak, A., S¸is¸ik, O., Saglam, N., Güner, S., Çanakçi, S., Beldüz, A.O., 2005. Characterization of thermoalkalophilic esterase from a novel thermophilic bacterium, Anoxybacillus gonensis G2. Bioresource Technology 96, 625e631. Gonçalves, S.P.C., Martins-Franchetti, S.M., 2009. Biodegradation of the films of PP, PHBV and its blends in soil. Journal of Polymers and the Environment 17, 280e285. Ha, C.S., Cho, W.J., 2002. Miscibility, properties, and degradability of microbial polyester containing blends. Progress in Polymer Science 27, 759e809. Handrick, R., Reinhardt, S., Focarete, M.L., Scandola, M., Adams, G., Kawalcuk, M., Jendrossek, D., 2001. A new type of thermoalkalophilic hydrolase of Paucimonas lemoignei with high specificity for amorphous polyester of short chain length hydroxyalkanoic acids. The Journal of Biological Chemistry 276, 36215e36224. Jaeger, K.E., Steinbüchel, A., Jendrossek, D., 1995. Substrate specificities of bacterial polyhydroxyalkanoates depolymerases and lipases: Bacterial lipases hydrolyze poly(w-hydroxyalkonoates). Applied Environmental Microbiology 61, 3113e3118.
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