Isolation, Purification, and Characterization of

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Biotechnology and Bioprocess Engineering 15: 998-1005 (2010) DOI 10.1007/s12257-010-0116-x

RESEARCH PAPER

Isolation, Purification, and Characterization of Haloalkaline Xylanase from a Marine Bacillus pumilus Strain, GESF-1 Gopalakrishnan Menon, Kalpana Mody, Jitendra Keshri, and Bhavanath Jha

Received: 8 April 2010 / Revised: 4 June 2010 / Accepted: 8 June 2010 © The Korean Society for Biotechnology and Bioengineering and Springer 2010

Abstract A haloalkalitolerant xylanase-producing Bacillus pumilus strain, GESF1 was isolated from an experimental salt farm of CSMCRI. Birch wood xylan and xylose induced maximum xylanase production with considerable activity seen in wheat straw and no activity at all with caboxymethyl cellulose (CMC). A three step purification yielded 21.21-fold purification with a specific activity of 112.42 U/mg protein (unit expressed as µmole of xylose released per min). Xylanase produced showed an optimum activity at pH 8.0, with approximately 50 and 30% relative activity at a pH 6.0 and 10.0, respectively. The temperature optimum was 40°C and kinetic properties such as Km and Vmax were 5.3 mg/mL and 0.42 μmol/min/mL (6593.4 μmol/min/mg protein). Xylanase activity (160 ~ 120%) was considerably enhanced in 2.5 to 7.5% NaCl with 87 and 73% retention of activity in 10 and 15% of NaCl. Enzyme activity was enhanced by Ca2+, Mn2+, Mg2+, and Na+ but strongly inhibited by heavy metals such as Hg2+, Fe3+, Cu2+, Cd2+, and Zn2+. Organic reagents such as βMercaptoethanol enhanced xylanase activity whereas EDTA strongly inhibited its activity. Xylanase, purified from the Bacillus pumilus strain, GESF1 could have potential biotechnological applications. Keywords: birch wood xylan, xylanase, Bacillus pumilus, haloalkalitolerant

Gopalakrishnan Menon, Kalpana Mody*, Jitendra Keshri, Bhavanath Jha Discipline of Marine Biotechnology & Ecology Central Salt & Marine Chemicals Research Institute, Council of Scientific & Industrial Research (CSIR) Gijubhai Badheka Marg, Bhavnagar 364-002, India Tel: +91-278-256-1354; Fax: +91-278-256-7562 E-mail: [email protected]

1. Introduction After cellulose, xylan is the second most abundant natural polysaccharide. It is a major structural polysaccharide in plant cells, accounting for one-third of all renewable organic carbon on earth. Xylan is found in hardwoods (15 ~ 30% of the cell wall content), softwoods (7 ~ 10%), and annual plants (< 30%) and is the major part of hemicellulose, which is a complex of polymeric carbohydrates (xylan, xyloglucan, glucomannan, galactoglucomannan, and arabinogalactan) present in plant cell walls [1-3]. A highlybranched heteropolysaccharide, xylan has a backbone chain of a homopolymer of 1,4-linked β-D-xylopyranosyl units. Xylanases (E.C. 3.2.1.8) are found in both fungi and bacteria [4-6] and it catalyzes the endohydrolysis of 1,4-βD-xylosidic linkages in xylans yielding various 1,4-β-Dxylooligosaccharides. Industrially, xylanase is used in the paper and pulp industry, baking, animal feed, bio-ethanol production, fruit and vegetable processing, etc. [1,7,8]. In combination with enzymes like amylase and lipase, xylanases have been used in biological deinking of paper [9,10]. Enzymatic pre-bleaching of kraft pulp is environment friendly as compared to the traditional chlorinated bleaching, with reduced organo-chlorine compounds being released [11,12]. However, kraft pulp bleaching requires high temperature and alkaline pH and only a few xylanases exhibit stability under such conditions [13]. Xylanases produced from Bacillus are comparatively thermostable, and can tolerate a wide pH range [14]. In the search for xylanases from extremophilic microorganisms, acidophiles, alkaliphiles, and thermophiles have been studied widely. However, halophiles have been inadequately studied. In the present study, a xylanase-producing marine Bacillus pumilus strain, GESF-1 was isolated from

Isolation, Purification, and Characterization of Haloalkaline Xylanase from a Marine Bacillus pumilus Strain, GESF-1

an experimental salt farm at the Central Salt & Marine Chemicals Research Institute. The enzyme has been purified and characterized.

2. Materials and Methods 2.1. Isolation and screening of bacterial culture The culture, GESF-1 was isolated from sediments collected from an experimental salt farm (Lat. 20°79.597’N, Long. 73°12.635E) of CSMCRI. The culture was maintained in Zobell marine agar 2216 (Himedia, India) (in g/ L; peptone 5.0, yeast extract 1.0, FeCl3 0.1, MgCl2 8.8, NaCl 19.45, Na2SO4 3.24, CaCl2·6H2O 1.8, KCl 0.55, NaHCO3 0.16, KBr 0.08, SrCl2 0.034, H3BO3 0.022, Na2SiO3 0.004, NaF 0.0024, NH4NO3 0.0016, Na2HPO4 0.008, agar 15; and pH 7.0 ~ 7.2) slants. Pure colonies obtained were streaked to Zobell marine agar plates supplemented with 0.5% (w/v) birch wood xylan (BioChemika, Fluka) and incubated. After 48 h, the plates were flooded with 0.1% Congo red (kept for 15 min) and were destained with 1 M NaCl for 10 min. The zone of clearance around the bacterial colony on the plates indicated that the culture was xylanase positive. Based on the greater zone of clearance, xylanase from the GESF-1 strain was selected for further study. 2.2. Bacterial identification and phylogenetic analysis The morphological, cultural, and physiological characteristic of the isolated strain was studied according to Bergey’s Manual of Determinative Bacteriology [15]. Salt and pH tolerance of the GESF-1 strain was also determined. Fatty acid methyl ester (FAME) analysis of the culture was performed. For molecular identification using a 16S rDNA sequencing technique, genomic DNA was extracted by the standard chloroform-isoamyl alcohol method [16]. PCR amplification of the 16S rRNA was performed using the following forward and reverse primers: 8f (fD1) 5'-AGA GTT TGA TCC TGG CTC AG-3' and 1495r (rP2) 5'-ACG GCT ACC TTG TTA CGA CTT-3' respectively [17]. The reaction mixture for PCR amplification contained 10X PCR buffer 5 µL, 200 mM dNTPs 5 µL, 2.5 U Taq DNA polymerase, 20 pM of each primers (Sigma, India), and 50 ng of bulk DNA. Amplification was performed in a thermal cycler (Bio-Rad MyCycler, Thermal cycler, California, USA) for an initial denaturation at 94°C for 4 min followed by 35 cycles of 94°C for 1 min, 58°C for 1 min, 72°C for 2 min, and a final extension at 72°C for 5 min. The purified PCR product was sequenced and the 16S rRNA gene sequence was compared with those in the GenBank using the BLASTn program [18]. The 16S rRNA gene sequence was deposited in GenBank with the accession number

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FJ573185. The phylogenetic analysis was done by MEGA 4.1[19] software and the tree was constructed using the neighbor-joining method [20]. 2.3. Xylanase production The culture was inoculated to 500 mL M9 medium [21] containing birch wood xylan (in g/L; xylan 5.0, Yeast extract 2.0, NaCl 2.5, NH4Cl 5.0, KH2PO4 15.0, Na2HPO4 30.0, MgSO4 0.25, final pH adjusted to 8.0) and incubated at 37°C for 48 h. After centrifugation (8,000 g for 10 min), the culture supernatant was used as crude enzyme extract. 2.4. Effect of different carbon sources on xylanase production Effect of carbon source in the medium on xylanase production was determined using birch wood xylan, carboxymethyl cellulose (CMC), sugar cane bagasse, wheat straw, sucrose, maltose, and xylose. The M9 (50 mL in 150 mL Erlenmeyer flasks) medium was supplemented with 0.5% of the above mentioned carbon source, inoculated with the culture, and incubated at 37°C for 48 h. After centrifugation (8,000 g for 10 min), the culture supernatant was considered as the crude enzyme and was checked for xylanase activity as described below. 2.5. Xylanase assay Xylanase activity was determined at 40oC for 60 min in 20 mM Tris-HCl buffer (pH-8.0) by measuring the release of reducing sugar from xylan using the dinitrosalicylic acid (DNS) method [22]. In the blank, the enzyme was added after the addition of DNS reagent. The absorbance was taken at 540 nm. One unit of xylanase activity was defined as the amount of enzyme that produced 1 µmol of xylose equivalent per minute under specified conditions. 2.6. Protein estimation Enzyme protein was estimated using the Folin phenol method [23] with bovine serum albumin as the standard. Protein content of the fractions collected during chromatography was determined measuring the OD at 280 nm. 2.7. Purification of xylanase 2.7.1. Ammonium sulfate precipitation and dialysis Crude xylanase protein was precipitated with 70% saturation of ammonium sulfate and kept overnight at 4oC. The precipitates were centrifuged and the pellet was dissolved in a minimum volume of 20 mM Tris-HCl buffer (pH 8.0). Dialysis was done using dialysis tubing (Sigma, D-0655, molecular weight cut off 12,000) by giving frequent changes with the same buffer at every four hours.

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2.8. Anion exchange chromatography Prior to equilibration, the DEAE-cellulose (SRL, India) was activated by suspending in 0.5 M HCl, degassed for 20 min, and washed with distilled water until it was acid free followed by treatment with 0.5 M NaOH and finally washed with distilled water. This process was repeated three times in order to activate the support. The support was loaded on the column (2 cm × 18 cm) and was equilibrated with 20 mM Tris- HCl buffer of pH 8.0. The ammonium sulfate precipitated and dialysed enzyme solution was loaded on the column and eluted with a stepwise concentration gradient of sodium chloride (0, 0.25, 0.5, 0.75, and 1 M NaCl) in the same buffer. The 3 mL fractions were collected at a flow rate of 0.6 ml/min. Protein concentrations of each fraction were determined at 280 nm. Aliquots of each fraction were assayed for xylanase activity and xylanase active fractions were pooled for subsequent steps. 2.9. Size-exclusion chromatography Five milliliters of pooled fraction, obtained by ion exchange chromatography, was loaded to a Sephadex-G-200 (Sigma, USA) column (1.25 cm × 25 cm) pre-equilibrated with 20 mM of Tris-HCl buffer (pH 8.0) and then eluted with the same buffer. Fractions of 2 mL each were collected at a flow rate of 4 mL/h. Protein concentrations of each fraction were determined by measuring the OD at 280 nm. Aliquots of each fraction were assayed for xylanase activity. The active fractions were pooled, concentrated by lyophilisation, and their purity was checked using SDS-PAGE. 2.10. SDS-PAGE and zymogram analysis SDS-PAGE (12%) [24] was run for determining the molecular weight of the purified enzyme. Standard molecular weight markers (Bangalore Genei, India) were Phosphorylase b (97.4 kDa), bovine serum albumin (66 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), and lactoglobulin (18.4 kDa). Silver staining was done to view the protein bands [16] and zymogram analysis was done by running a native PAGE (7.5%). A 3% agarose plate was prepared into which 0.5% birch wood xylan was incorporated. After electrophoresis, the native PAGE gel was laid over the agarose gel and incubated at 40°C for 3 h. This was then stained with 5% congo red solution for 15 min and then washed with 1 M NaCl for 20 min to visualize the bands. 2.11. Effect of pH and temperature on xylanase activity Optimum pH for xylanase activity was determined at 40°C for 60 min using different buffers with varying pH; citrate buffer (6.0), Mc Levine buffer (7.0), Tris- HCl buffer (8.0), and glycine-NaOH buffer (9.0 ~ 10.0). Stability of the

Biotechnology and Bioprocess Engineering 15: 998-1005 (2010)

purified enzyme with respect to pH was also determined in a pH ranging from 6.0 to 10.0 by incubating the enzyme in respective buffer for 30 min and then the residual activity was determined (assay conditions remaining the same). The optimum temperature required for xylanase activity was determined by incubating the assay system in the temperature, ranging from 30 to 80°C. 2.12. Effect of sodium chloride Effect of sodium chloride on xylanase activity was determined by assaying xylanase in the presence of different concentrations of NaCl from 0 to 15%( w/v) in 20 mM Tris-HCl buffer. 2.13. Effect of Additives Effect of different metal ions and additives at 5 mM concentration was studied using different metal salts such as CaCl2, CoCl2, HgCl2, MnCl2, FeCl3, MgCl2, CuSO4, CdCl2, ZnCl2, NaCl, KCl, β Mercaptoethanol, and EDTA. Activity was measured using the method described above. 2.14. Kinetic studies The kinetic properties of xylanase were determined using varying concentration of the substrate, xylan. Km and Vmax values were calculated by Lineweaver Burk double reciprocal plots.

3. Results and Discussion 3.1. Bacterial identification Most of the enzymes used in biotechnology are generally derived from terrestrial bacteria and fungi. To make them suitable for versatile applications, improved stability of these biocatalysts towards temperature, pH, salinity and other extreme conditions is desired. Enzymes from extremophilic microbes naturally possess such unique properties but represent vastly under-utilized resources [25]. Xylanases from extremophiles like acidophiles, alkaliphiles, and thermophiles have been studied widely [26-33]. Conversely, only a few reports are available on xylanases from halophilic bacteria [25,34-35]. The bacterium, Bacillus pumilus GESF-1 used in the present study was isolated from the sediment sample on an experimental salt farm from CSMCRI, and was a gram positive, rod- shaped and non-motile bacteria, which was catalase and oxidase positive and hydrolysed pectin, gelatin, casein, and tributyrin. When flooded with 0.1% Congo red, it formed a very prominent and clear zone on xylan agar plates. It however did not produce amylase or urease (Table 1). While it was found to show resistance against Ceftazidime and Cloxacillin, it was however highly sensitive to

Isolation, Purification, and Characterization of Haloalkaline Xylanase from a Marine Bacillus pumilus Strain, GESF-1

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Table 1. Morphological and biochemical characteristics of Bacillus pumilus, GESF-1 Tests Colour, size Gram reaction Motility Catalase Oxidase Indole MR VP Simmons citrate Amylase Pectinase Gelatinase Lipase Caesinase Phenylalanine Urease H2S production Nitrate reduction Arginine Lysine Ornithine Adonitol Arabinose Cellobiose Dextrose Dulcitol Fructose Galactose Inositol Inulin Lactose Maltose Mannitol Mannose Melibiose Raffinose Rhamnose Salicin Sorbitol Sucrose Trehalose Xylose

Results Creamy round medium sized Gram positive − + + − + + + − + + + + − − − − + − + − + + + − + − − − + − + − − + − + − + − −

Norfloxacin and Ciprofloxacin. Based on the fact that the strain GESF-1 could tolerate pH 8.0 ~ 10.0 with optimum growth at pH 8.0; and salt concentration of 5 to 15% NaCl with an optimum at 5%, it could be classified as an alkali-tolerant moderately halophilic bacterium (Figs. 1 and 2). The FAME analysis of strain GESF-1 was done using the MIDI Sherlock Microbial Identification System. A Similarity Index (SI) of 0.471 with Bacillus pumilus was seen from the RTSBA library of Sherlock software. A DNA fragment of the 16S rRNA gene having 1,450 bp was amplified and the BLAST search has shown 99% homology with the Bacillus pumilus strain zyj1-3 16S rRNA gene sequence. The phylogenetic tree (Fig. 3) shows

Fig. 1. pH tolerance of the culture GESF-1 with respect to growth after 24 h incubation.

Fig. 2. Salt (NaCl) tolerance of the culture GESF-1 with respect to growth after 24 h incubation.

Fig. 3. Phylogenetic tree showing the taxonomic position of culture GESF-1. Figures within brackets represent the GenBank accession numbers.

the taxonomic position of the GESF-1 isolate. 3.2. Effect of different carbon sources on xylanase production In the present study, Birch wood xylan was proven to be the best carbon source for xylanase production followed by xylose. Sugarcane bagasse also supported xylanase production to some extent while in the presence of sucrose and maltose, xylanase production was very less while CMC did not induce xylanase production (Fig. 4). Appreciable xylanase activity was observed when wheat straw was used as a carbon source making it a promising, alternative, and cheaper process for large scale xylanase production. Xylose

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Biotechnology and Bioprocess Engineering 15: 998-1005 (2010)

Fig. 4. Effect of different carbon sources on xylanase production where S Bagasse is Sugarcane Bagasse and W Straw is Wheat Straw.

was found to be a better inducer for xylanase production as compared to birch wood xylan, in the strain Micrococcus sp AR-135 [27]. In contrast, xylanase production was repressed in Bacillus circulans D1 in xylan medium supplemented with xylose [36]. Similarly xylose had no influence on xylanase production in Bacillus subtilis 168 [37]. The effect of sucrose and maltose on xylanase production was reported to be marginal [38].

Fig. 5. (A) SDS-PAGE of purified xylanase where M denotes molecular marker; lanes 1 and 2, purified xylanase; lane 3, ammonium sulfate precipitated and dialysed xylanase. (B) Zymogram of the purified xylanase.

3.3. Xylanase purification A three step purification of xylanase led to a 21.21-fold purification with a 2.1% yield (Table 2). However, similar low yields of two halotolerant xylanases i.e. 1.5 and 1.7%, respectively, have been reported from a novel halophilic bacterium [25], whereas, a 12-fold purification of xylanase has also been reported from Staphylococcus sp. SG-13 with only a 5% yield [39]. Purified xylanase had a specific activity of 112.42 Units/mg of protein. 3.4. SDS-PAGE and zymogram analysis The purified enzyme migrated as a single band on SDSPAGE suggesting that the purified xylanase was homogeneous. The molecular weight of xylanase was approximately 39.6 kDa (Fig. 5). The zymogram analysis also revealed a single band of xylanolytic activity corresponding to the band obtained in SDS-PAGE. 3.5. Effect of pH and temperature on xylanase activity The xylanase produced by this bacterium showed activity

Fig. 6. Effect of pH on xylanase activity and stability. ( ◆) denotes activity; ( ■) denotes stability after 30 min pre incubation.

in a broad range of pH, ranging from 6.0 to 10.0, though the relative activity was approximately 50 and 30% for pH 6.0 and 10.0, respectively. It was also stable at pH 7.0 and 8.0 after pre-incubating for 30 min, whereas it lost its activity at higher and lower pH (Fig. 6). The pH stability of the xylanase is in concurrence with that of xylanase in Bacillus pumilus ASH [40]. Activity of The purified

Table 2. Purification of xylanase isolated from Bacillus pumillus GESF-1 Enzyme Crude Ammonium sulfate precipitation DEAE column chromatography Gel permeation

Total Vol (mL) 495 30

Total Protein (mg) 190.37 17

Total Activity (units)

Specific activity Units/mg protein

Fold Purification

1,009.8

5.3

, 260.7

15.34

2.89

25.8

1

%Yield 100

20.5

1.94

, 80.56

41.52

7.83

7.9

6

0.19

, 21.36

112.42

21.21

2.1

Isolation, Purification, and Characterization of Haloalkaline Xylanase from a Marine Bacillus pumilus Strain, GESF-1

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Table 3. Effect of metals and organic chemicals (all in 5 mM concentration) on the activity of xylanase from strain GESF-1

Fig. 7. Effect of temperature on xylanase activity.

Fig. 8. Effect of NaCl on xylanase activity.

xylanase exhibited quite good activity at the temperature ranging from 30 to 60°C, with optimum activity at 40°C and retained about 80% activity at 60°C (Fig. 7). Bacillus sp. GRE 7 was reported to have an optimum pH of 7.0, but had good thermal stability, the optimum temperature being 70°C [12]. Based on properties such as alkali tolerance and good thermal stability, this enzyme has potential application in the pulp and paper industry for biobleaching. 3.6. Effect of sodium chloride on xylanase activity Presence of sodium chloride in the assay system significantly increased xylanase activity with 160 ~ 120% activity in the presence of 2.5 ~ 7.5% NaCl. This showed the halophilic nature of the enzyme. Presence of up to 5% sodium chloride in the assay system enhanced the xylanase activity which was retained up to 87 and 73% activity at 10 and 15% NaCl, respectively (Fig. 8). A similar description of the activity of xylanase has also been reported from Bacillus halodurans S7, which remained almost constant at 1 and 5 mM of NaCl [31]. 3.7. Effect of additives on xylanase activity The presence of metal ions like Ca2+, Mn2+, Mg2+, and Na+ enhanced the activity while heavy metals like Hg2+, Fe3+, Cu2+, Cd2+, and Zn2+ strongly inhibited xylanase activity. Similarly, β-mercaptoethanol increased the activity considerably whereas EDTA inhibited enzyme activity to a certain extent which indicated that it was metal ion-dependent

Additives (5 mM)

Relative xylanase activity

Control CaCl2 Co Cl2 Hg Cl2 Mn Cl2 FeCl3 Mg Cl2 CuSO4 Cd Cl2 Zn Cl2 NaCl KCl β Mercaptoethanol EDTA

100 192.81 78.1 0 162.1 0 130.72 0 0 0 130.4 95.75 158.96 42.23

(Table 3). Significant increases in the activity of xylanase was observed in the presence of Mn2+ which is in concurrence with that of Bacillus sp. GRE 7 [12]. In contrast , the presence of Mn2+ has been reported to inhibit xylanase activity of Bacillus sp. strain K-1 and Bacillus halodurans S7, respectively [31,41]. 3.8. Kinetic studies of xylanase The Km and Vmax values were determined by the Lineweaver Burk double reciprocal plot. The substrate, Birchwood xylan was used in different concentrations and maximum activity was observed in 10 mg/mL concentration. The Km value was 5.3 mg/mL and the Vmax was 0.42 μmol/ min/mL (6593.4 μmol/min/mg protein).

4. Conclusion Xylanase, from an extreme archaeon Halorhabdus utahensis, exhibited promising activity in the presence of broad range of NaCl concentration, i.e. from 0 to 30% as well as at high temperature i.e. at 55 and 70oC [35]. While in another report, two halotolerant endo-xylanases [25] from a novel halophilic bacterium exhibited a broad range of pH stability i.e. from 4 to 11, with maximum activity at pH 6.0, in the presence of 5.8% sodium chloride which was reduced to about 20% in the presence of 27% sodium chloride. The activity was stimulated by Ca2+, Mn2+, Mg2+, Ba2+, and Li+. The reported properties of xylanases are comparable to some extent, with xylanase described in the present paper. Xylanases from Bacillus spp. obtained from a diverse ecological niche have been extensively studied. A thermostable xylanases from different Bacillus spp. are studied

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elaborately with respect to their activity and stability at varying pH, temperature, and presence of metal ions, etc. [31,40-42]. Similarly, purification and characterization of alkaliphilic and thermoalkaliphilic Bacillus spp. have been reported [30,32,43]. Bacillus spp. have also been extensively studied by immobilizing the cells for xylanase production [44], improvement in pulp bleaching of kraft pulp using xylanase from Bacillus pumilus [45], expression of endo-β-xylanase gene from B. pumilus PLS in yeast Saccharomyces cerevisiae [46], and use of Bacillus xylanases in increasing bread volume [47,48]. The source of xylanase reported in the present paper is also Bacillus pumilus GESF-1 which exhibited good activity in a broad pH and temperature range and in highly saline condition. The possible application of the xylanase isolated in the present study can be envisaged in the treatment of agricultural waste as well as in the bioremediation of xylan-containing materials for sustainable manufacture of bio-based products. Moreover, it can also be used for bio-bleaching. Thus, this strain could be a good contender for industrial and biotechnological applications under extreme conditions.

Acknowledgements The authors are thankful to Dr P. K. Ghosh, Director, CSMCRI, Bhavnagar for his constant support and encouragement, and the Council of Scientific and Industrial Research (CSIR), New Delhi for financial support to one of the authors, Gopalakrishnan.

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Isolation, Purification, and Characterization of Haloalkaline Xylanase from a Marine Bacillus pumilus Strain, GESF-1

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