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Oct 13, 2010 - be considered as a waste of feather protein (Onifade et al. A. M. Mazotto 4 A. C. N. de Melo 4 A. Macrae 4. A. S. Rosado 4 R. Peixoto 4 S. M. L. ...

World J Microbiol Biotechnol (2011) 27:1355–1365 DOI 10.1007/s11274-010-0586-1

ORIGINAL PAPER

Biodegradation of feather waste by extracellular keratinases and gelatinases from Bacillus spp. Ana Maria Mazotto • Ana Cristina N. de Melo • Andrew Macrae • Alexandre Soares Rosado • Raquel Peixoto • Sabrina M. L. Cedrola • Soˆnia Couri • Russolina B. Zingali • Ana Lu´cia V. Villa • Leon Rabinovitch Jeane Q. Chaves • Alane B. Vermelho



Received: 30 April 2010 / Accepted: 20 September 2010 / Published online: 13 October 2010 Ó Springer Science+Business Media B.V. 2010

Abstract In this study, three feather degrading bacterial strains were isolated from agroindustrial residues from a Brazilian poultry farm. Three Gram-positive, spore-forming, rod-shaped bacteria and were identified as B. subtilis 1271, B. licheniformis 1269 and B. cereus 1268 using biochemical, physiologic and molecular methods. These Bacillus spp. strains grew and produced keratinases and peptidases using chicken feather as the sole source of nitrogen and carbon. B. subtilis 1271 degraded feathers

A. M. Mazotto  A. C. N. de Melo  A. Macrae  A. S. Rosado  R. Peixoto  S. M. L. Cedrola  A. B. Vermelho (&) Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Go´es (IMPPG), Bloco I, Centro de Cieˆncias da Sau´de (CCS), Universidade Federal do Rio de Janeiro (UFRJ), Cidade Universita´ria, Ilha do Funda˜o, Rio de Janeiro, RJ 21941-590, Brazil e-mail: [email protected] S. Couri Instituto Federal de Educac¸a˜o Cieˆncia e Tecnologia do Rio de janeiro, Campus Rio de Janeiro, Rua Senador Furtado no 121, Maracana˜, Rio de Janeiro, Brazil R. B. Zingali Departamento de Bioquı´mica, Instituto de Cieˆncias Biome´dicas, Bloco H, Centro de Cieˆncias da Sau´de (CCS), Universidade Federal do Rio de Janeiro (UFRJ), Cidade Universita´ria, Ilha do Funda˜o, Rio de Janeiro, RJ 21941-590, Brazil A. L. V. Villa Universidade federal do Rio de Janeiro, Campus Macae´, R. Aloı´sio Gomes da Silva no 50, Granja do Cavaleiros, Macae´, RJ 27930, Brazil L. Rabinovitch  J. Q. Chaves Departamento de Bacteriologia, Instituto Oswaldo Cruz, Fundac¸a˜o Oswaldo Cruz, Av. Brasil 4365, Rio de Janeiro, RJ 21045-900, Brazil

completely after 7 days at room temperature and produced the highest levels of keratinase (446 U ml-1). Feather hydrolysis resulted in the production of serine, glycine, glutamic acid, valine and leucine as the major amino acids. Enzymography and zymography analyses demonstrated that enzymatic extracts from the Bacillus spp. effectively degraded keratin and gelatin substrates as well as, casein, hemoglobin and bovine serum albumin. Zymography showed that B. subtilis 1271 and B. licheniformis 1269 produced peptidases and keratinases in the 15–140 kDa range, and B. cereus produced a keratinase of *200 kDa using feathers as the carbon and nitrogen source in culture medium. All peptidases and keratinases observed were inhibited by the serine specific peptidase inhibitor phenylmethylsulfonyl fluoride (PMSF). The optimum assay conditions of temperature and pH for keratinase activity were 40–50°C and pH 10.0 for all strains. For gelatinases the best temperature and pH ranges were 50–70°C and pH 7.0–11. These isolates have potential for the biodegradation of feather wastes and production of proteolytic enzymes using feather as a cheap and eco-friendly substrate. Keywords Bacillus spp.  Feather degradation  Feather keratin  Keratinase  Peptidase

Introduction In poultry processing industries all over the world, chicken feathers are generally an unwanted waste byproduct (Suzuki et al. 2006). In Brazil 800,000 tons/year of feathers are discarded by this sector. The accumulation of feathers can eventually lead to environmental pollution and can also be considered as a waste of feather protein (Onifade et al.

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1998). Traditional ways to degrade feathers such as alkali hydrolysis and steam pressure cooking to produce feather meal may destroy amino acids and they also consume large amounts of energy (Dalev et al. 1997; Cai et al. 2008). In addition to the conventional methods, the incineration of feathers has ecological disadvantages including the release of large amounts of carbon dioxide and there is an apparent protein wastage and not to mention energy losses (Matsui et al. 2009). Feathers are comprised essentially of keratin, an insoluble structural protein, tightly packed in a b-sheet polypeptide chain, extensively cross-linked with disulfide, hydrogen and hydrophobic bonds (Riffel et al. 2003; Fraser and Parry 2008). Keratinous wastes are not degraded by commonly known proteases like trypsin, pepsin and papain due to presence of the disulfide bonds, but are easily degraded by keratinases (Papadopoulos 1986, Gupta and Ramnani 2006). These peptidases are largely serine or metallopeptidases (EC 3.4.21/24) found in several microorganisms and have attracted a great deal of attention due to their multiple applications in industry for the development of nonpolluting processes (Onifade et al. 1998; Gupta and Ramnani 2006). Keratinolytic microorganisms and their enzymes may have important applications in biotechnological and industrial processes involving keratin-containing wastes from poultry and leather industries. In the pharmaceutical industry, they have a role in personal care products involving hair removal and as peeling agents (Grazziotin et al. 2006; Brandelli 2008; Macedo et al. 2008; Pillai and Archana 2008). Biotechnological applications involving keratin and keratinases include bio-hydrogen production, biodegradable films and keratin composites (Ba´lint et al. 2005; Barone et al. 2005). In medicine, recent studies indicate that keratinases may have an important role in deactivating prions and could increase ungual drug delivery (Mohorcic et al. 2007; Yoshioka et al. 2007). Biodegradation of feathers by keratinase from microorganisms may provide a viable alternative to produce a digestible keratin through peptide production. These enzymes are produced by some species of Bacillus genus, actinomycetes and fungi (Gupta and Ramnani 2006). Keratinases, from B. licheniformis and B. subtilis have been studied and shown to be effective at feather degradation (Rozs et al. 2001; Thys and Brandelli 2006). These microorganisms are a source of Versazyme and Valkerase, commercial keratinases which have been used for feather meal improvement (Odetallah et al. 2005). Here we describe the isolation and identification of three keratin degrading Bacillus strains isolated from a poultry industry. Keratinases and peptidases produced by Bacillus spp. during submerse cultivation on feather medium were investigated by qualitative and quantitative analyses using zymography, spectrophotometry and enzymography. We

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also report the partial characterization of these enzymes produced by three novel Bacillus spp.

Materials and methods Chemicals Media constituents were obtained from Oxoid Ltd. (Cambridge, England). Reagents used in electrophoresis and molecular mass standards were acquired from Amersham Life Science (Little Chalfont, England). Polyethyleneglycol 4000 (PEG 4000) was purchased from Vetec (Rio de Janeiro, Brazil). The peptidase inhibitors (trans-epoxysuccinyl l-leucylamido- (4-guanidino) butane [E-64], phenylmethylsulphonyl fluoride [PMSF], 1,10-phenanthroline, pepstatin A and EDTA) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The proteinaceous substrates gelatin and casein were purchased from Merck (Darmstadt, Germany), bovine serum albumin and hemoglobin from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents were analytical grade. Isolation and selection of keratinolytic microorganism Feather-degrading Bacillus spp. strains were isolated from industrial residues of a local poultry industry (Rio de Janeiro, Brazil). One gram of feather wastes was added to a 100 ml aqueous solution comprising 0.5% yeast extract, 0.5% peptone, 2% sucrose and 2% KCl and stored at 28°C for 48 h. The suspension was streaked on agar plates containing the same medium composition and incubated at 28°C for 72 h. The single colonies obtained were cultivated in tubes containing saline solution (0.85% NaCl) supplemented with a whole feather for 28 days at 28°C in order to select keratinolytic microorganisms based on their ability to hydrolyze feather. Phenotypic and molecular identification of the keratinolytic microorganisms The feather degrading strains were evaluated by specific biochemical, physiologic and cytomorphologic tests for to identify bacteria belonging to the Genus Bacillus (Claus and Berkeley 1986; Vasconcelos and Rabinovitch 1994). Strains were preserved under refrigeration as spores in solid Nutrient Agar medium. The strains were lyophilized and deposited in the Colec¸a˜o de Culturas do Geˆnero Bacillus e Geˆneros Correlatos-CCGB (which is affiliated to the World Federation of Culture Collections) located in the Laborato´rio de Fisiologia Bacteriana, Instituto Oswaldo Cruz/FIOCRUZ.

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Genomic DNA of the bacterial isolates was extracted using the Wizard Genomic DNA Purification Kit (Promega, Madison, USA). Concentrations were determined by using of a Qubit fluorometer (Invitrogen/Molecular Probes California, USA). Amplification of the 16S rDNA region was performed by PCR using universal primers pA and pH as previously described (Massol-Deya et al. 1995). The amplified products were purified with Ilustra GFX PCR DNA and Gel band Purification Kit (Ge Healthcare, Buckinghamshire, UK) and then directly sequenced in both directions by using pA and pH primers with a MEGABACE system (Ge Healthcare, Buckinghamshire, UK). Preliminary identification of sequences was performed by blastn against the Genbank database 14/08/2009 (www. ncbi.nlm.nih.gov/blast). The sequence was submitted to the GenBank using Sequin Application Version 9.50 (ftp:// ftp.ncbi.nih.gov/sequin/sequin.win.exe). Growth medium and enzyme extract preparation The Bacillus sp. strains were cultivated on phosphatebuffered medium (0.06 M Na2HPO47H2O and 0.04 M KH2PO4, pH 7.2) supplemented with 1% native feather as the only nitrogen and carbon source for 7 days at 28°C on a rotary shaker (300 rpm). After incubation, the media were centrifuged at 2,000g for 20 min at 4°C and the supernatant solutions were used as enzyme extracts to analyze keratinase and gelatinase activities. Feather keratin substrate Chicken feathers obtained from poultry waste were washed extensively with water and detergent, dried at 60°C overnight, delipidated with chloroform:methanol (1:1, v/v) and dried again at 60°C. The Wawrzkiewicz et al. (1991) method was modified to produce keratin powder from the lipid free dried feathers. Briefly, 10 g of feathers were heated with a reflux condenser at 100°C for 80–120 min with 500 ml of DMSO. Keratin was then precipitated by the addition of two volumes of acetone and maintained at 4°C for 24–48 h. The keratin precipitates were collected by centrifugation (2,000g/15 min), washed twice with distilled water and dried at 4°C. A white powder was obtained for qualitative and quantitative biochemical analyses related to keratinases activity and as a keratin standard in feather degradation studies (Vermelho et al. 2009). SDS–PAGE Feather keratin powder was analyzed in 15% SDS–PAGE using the method of Laemmli (1970). Electrophoresis was carried out at 170 V. A keratin solution (1 mg/ml) was added to the SDS–PAGE sample buffer in the proportion of

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6:4 (v/v). The gels were silver and coomassie blue stained. Phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa) and a-lactalbumin (14.4 kDa) were used as molecular mass standards (Pharmacia Biotech). Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) In order to verify the homogeneity of the keratin substrate obtained from the feathers, the keratin powder was analyzed using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Immediately prior to mass spectrometry, acetonitrile/water (5:95, v/v) and trifluoroacetic acid were added to the samples. A C18 zip tip was hydrated, the sample loaded, and water used to wash the sample. An elution solution of acetonitrile/water (60:40, v/v), and 0.1% (w/v) trifluoroacetic acid was then loaded three times, a-cyano-4- Hydroxycinnamic acid (CCA) matrix was added, and 1 ll of the sample mixture was spotted directly on a MALDI target for analysis. Peptide mass spectrometry was carried out with a Bruker Biflex III MALDI-TOF mass spectrometer in the reflectron mode. The experiments were performed in triplicate using three independent experimental sets. Feather keratin enzymography Twenty microliter samples of concentrated culture supernatant were mixed with an equal volume of feather keratin powder diluted in phosphate buffer (0.06 M Na2HPO47H2O and 0.04 M KH2PO4, pH 7.2) to obtain a final concentration of 0.15 mg/ml. The reaction mixtures were incubated for 1 h at 37°C. Reactions were terminated by freezing and the mixtures stored at -20°C until analyzed. Reaction mixtures (20 ll) were added to 20 ll SDS–PAGE sample buffer [125 mM Tris, pH 6.8, 4% (w/v) SDS, 20% (v/v) glycerol, 0.002% (w/v) bromophenol blue] supplemented with 5% (v/v) b-mercaptoethanol, and boiled at 100°C for 5 min. Keratin hydrolysis was then analyzed on 15% SDS–PAGE. Electrophoresis was carried out at 170 V for 2 h at 28°C, and protein bands were silver stained. As a control of enzymatic activity, aliquots of the concentrated supernatant were heat-inactivated with the substrates before incubation. In addition, a second control for keratin substrate was made by replacing concentrated supernatants with the same volume of buffer (De Melo et al. 2007). Quantifying keratinase and gelatinase activities Enzymatic activity was evaluated at different pH values (3.0–11.0) and temperatures (10–80°C). Enzyme extract

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(250 ll) was added to 375 ll of feather keratin powder (6.67%) in a citric acid buffer (0.05 M citric acid pH 3.0, 4.0 and 5.0), phosphate buffer (0.06 M Na2HPO47H2O and 0.04 M KH2PO4 pH 6.0, 7.0 and 8.0) or aminoacetic acid buffer (0.1 M aminoacetic acid pH 9.0, 10.0 and 11.0). The reaction mixture was incubated for 1 h at 37°C and then stopped by the addition of 250 ll of 10% trichloroacetic acid. Samples were then put into a refrigerator at 4°C for 30 min. The supernatant was collected after centrifugation (15 min at 2,500g) and activity measured at 280 nm. One unit of keratinolytic activity was defined as the amount of enzyme required to produce an increase of 0.01 absorbance unit at 280 nm, under standard assay conditions (1 h at 37°C). Gelatinase activity was measured according to the method of Jones et al. (1998). Briefly, 100 ll of the enzyme extract and 900 ll of the same buffer solutions above were added to 1.5 ml of the substrate solution (gelatin in distilled water, 1% w/v) and the mixture was incubated at 37°C for 30 min. A 375 ll sample was then removed from the reaction mixture and added to 500 ll of isopropanol. This was centrifuged at 2,500g for 15 min and the supernatant was collected and the absorbance was measured as described by Lowry et al. (1951). One unit of gelatinolytic activity was defined as the amount of enzyme required to produce an increase of 0.01 absorbance unit at 660 nm, under standard assay conditions (30 min at 37°C). High-performance thin-layer chromatography (HPTLC) An aliquot of culture supernatant (5 ll) was analyzed by silica gel 60 HPTLC plates for amino acid detection. The HPLC plates were run for approximately 1 h at room temperature in a TLC tank using butanol/acetic acid/distilled water (4:1:1 v/v/v) as solvent. The amino acid specific ninhydrin reagent (7.5% in butanol/acetone 1:1 v/v) was used for development. Commercial amino acids were used as standard. Zymography Culture supernatants were concentrated 20-fold by dialysis (cut off 9 kDa) against PEG 4000 overnight at 4°C. The concentrated culture supernatants were mixed with sample buffer for zymography (125 mM Tris, pH 6.8, 4% SDS, 20% glycerol and 0.002% bromophenol blue) in a sample:buffer ratio 6:4 (De Melo et al. 2007; Vermelho et al. 2009). Keratinases and gelatinases were assayed and characterized by 12.5% SDS–PAGE with co-polymerized keratin feather powder and gelatin (0.1%). Additionally other substrates such as casein, bovine serum albumin (BSA) and hemoglobin (0.1%) were incorporated in gel for substrate specificity studies. Gels were loaded with 30 ll of

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concentrated culture supernatant per slot for keratin-SDS– PAGE, and 20 ll of concentrated culture supernatant per slot for other substrates co-polymerized in SDS–PAGE. After electrophoresis at 170 V for 2 h at 4°C the gels were soaked for 1 h at 28°C in 2.5% Triton X-100. Afterwards, the gels were then incubated for 48 h at 37°C in proteolysis buffer Tris–HCl buffer, pH 7.4 (0.5 M Tris). Then, the gels were stained for 1 h with 0.2% Coomassie brilliant blue R250 in methanol-acetic acid–water (50:10:40) and destained in the same solvent (Lopes et al. 2008). For enzymatic classification 3 mM phenylmethylsulfonyl fluoride (PMSF), 0.26 mM ethylenediaminetetraacetic acid (EDTA), 10 mM 1,10 phenanthroline (Phenan), 10 lM pepstatin A (Peps) and 5 lM trans-epoxysuccinyl L-leucylamido-(4-guanidino) butane (E-64) were used in proteolysis buffer. To quantify the inhibition the bands were analyzed by ImageJ software.

Results Selection and identification of keratinolytic Bacillus spp. Three aerobic, mesophilic, Gram-positive, and sporeforming bacilli were selected as keratinolytic after demonstrating complete feather degradation in whole feather broth. They were identified by biochemical, physiological and cytomorphological characterization as Bacillus licheniformis (LFB-FIOCRUZ 1269), Bacillus subtilis (LFB-FIOCRUZ 1271) and Bacillus cereus (LFB-FIOCRUZ 1268) at the Laborato´rio de Fisiologia Bacteriana, Instituto Oswaldo Cruz/FIOCRUZ, Brazil. The analyses of their 16S rDNA sequences confirmed the biochemical results indicating the identification of Bacillus subtilis, Bacillus licheniformis and Bacillus cereus. The 16S rDNA sequences of isolates 1268, 1269 and 1271 showed 99% sequence similarity with Bacillus cereus, Bacillus licheniformis and Bacillus subtilis, respectively. The nucleotide sequences were deposited at GenBank and their accession numbers are: GQ482980 (B. subtilis 1271), GQ482981 (B. licheniformis 1269) and GQ482979 (B. cereus 1268). Production of keratinases and peptidases by Bacillus species in feather medium The three Bacillus species were further tested for keratinolytic and proteolytic activity. All the strains grew and produced keratinase and peptidase using chicken feather as the sole source of nitrogen and carbon. B. subtilis 1271 degraded the feathers completely after 7 days at room temperature and produced the highest level of keratinase (446 U/ml), Fig. 1. B. licheniformis 1269 produced lower

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three strains showed amino acids bands migrating at the serine, glycine, glutamic acid, valine and leucine positions (Fig. 2a), indicating that these amino acids are present in high concentrations. The highest concentration of soluble protein (4.0 mg/ml) was observed in the B. subtilis 1271 supernatant (Fig. 2b), while B. licheniformis 1269 had the lowest concentration of soluble protein (1.42 mg/ml).

Activity (U/ml)

500 400 300 200 100 0 68

2 s1

i

reu

ce

rm

ifo

B.

71

69

2 s1

B.

1359

lic

n he

B.

li

bti

su

2 s1

Fig. 1 Keratinolytic and proteolytic activity of Bacillus strains grown in feather medium. The black bar represents the keratinolytic activity and the gray bars the proteolytic activity

Fig. 2 Degradation of feather in culture medium, demonstrating protein concentration and TLC of amino acids from culture supernatant of Bacillus strains. a TLC of amino acid produced during degradation of feather by B. cereus 1268, B. licheniformis 1269 and B. subtilis 1271. b Concentration of soluble proteins in culture medium (bars) and feather degradation (filled diamond) after incubation with B. cereus 1268, B. licheniformis 1269 and B. subtilis 1271 for 7 days at 28°C

keratinolytic activity and a higher level of proteolytic activity (394.1 U/ml), as shown in Fig. 1. B. licheniformis 1269 presented only 29.5 U/ml of keratinolytic activity but was able to degrade 44.2% of the feathers in the medium (Figs. 1 and 2). The soluble proteins and free amino acids produced during feather hydrolysis by Bacillus strains were analyzed. HTLC analyses of the culture supernatants of the

Feather keratin degradation by keratinases of Bacillus species Keratin was successfully extracted from feathers using the method described and resulted in a white, homogenous keratin powder. MALDI-TOF and SDS–PAGE analyses (Fig. 3a, b, respectively) confirmed the presence of pure keratin. The major fragments were in the m/z 9,000–10,000 range, confirming the presence of a b-keratin (Fig. 3a). Fragments in m/z 2,000–8,000 range corresponded to the a-cyno-4-hydroxycinnamic acid matrix. SDS–PAGE analyses resulted in a single band migrating at *10 kDa (Fig. 3b). Cell-free supernatants from the three Bacillus species were then incubated with feather keratin for 1 h at 37°C. After incubation, the single *10 kDa band, characteristic of feather keratin, was no longer observed and

Fig. 3 Feather keratin degradation by Bacillus strains. a MALDI TOF spectrum: major fragments of 9,819.85 and 10,301.27 Da (m/z) were detected indicating the presence of keratin. b SDS–PAGE of keratin feather powder: first column from top to bottom Phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa) and a-lactalbumin (14.4 kDa) were used as molecular mass standards (Pharmacia Biotech), second column a single *10 kDa band. c Enzymography of keratin degradation by extracellular keratinases of B. cereus 1268, B. licheniformis 1269 and B. subtilis 1271. Feather keratin degradation profiles were analyzed by 15% SDS–PAGE, and gels were silver stained. In the gel strips on the left, the enzyme extract solution immediately after the addition of feather keratin (note 10 kDa band); in strips on the right, the same solution incubated for 1 h at 37°C (note lack of 10 kDa band)

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considered degraded (Fig. 3c), confirming the presence of keratinases in the supernatants. Effect of different substrates on the extracellular peptidases of Bacillus spp. The ability of the extracellular peptidases to degrade different proteinaceous substrates was evaluated using keratin, gelatin, casein, BSA and hemoglobin co-polymerized with 12.5% sodium dodecyl sulfate–polyacrylamide gels (SDS–PAGE). The gels showed that B. cereus 1268 produced a single peptidase of *200 kDa able to degrade all substrates tested (Fig. 4). B. subtilis 1271 and B. licheniformis 1269 produced multiple peptidases with the ability to degrade gelatin (Fig. 4). In B. licheniformis, bands with an apparent molecular mass of 60 and 100 kDa presented keratinase activity but these enzymes were more prominent in B. subtilis 1271 which presented bands migrating in a range of 15–140 kDa. A broad range of other protein substrates like casein, BSA and hemoglobin were hydrolyzed by B. subtilis and B licheniformis. A peptidase migrating at 30 kDa in B. licheniformis showed strong activity with casein, BSA and hemoglobin (Fig. 4). Effect of temperature and pH on enzyme activity The effect of temperature and pH on extracellular keratinase and gelatinase activities of the three Bacillus spp. was determined using gelatin and feather keratin as substrates, respectively. High gelatinase activity was observed at 60°C for B. subtilis 1271 and 70°C for B. licheniformis 1269 (Fig. 5a). Meanwhile, the gelatinase of B. cereus 1268 was more active at a broader temperature range (50–70°C). Both keratinases of B. cereus 1268 and B. licheniformis 1269 were optimally active at 40°C. The keratinase of B. subtilis exhibited maximal activity at 50°C (Fig. 5a). B. subtilis strain 1271 presented maximum gelatinase and keratinase activity at pH 9.0 and 10, respectively (Fig. 5b) and in B. cereus the optimum pH was 10 for both substrates (Fig. 5a). In B. licheniformis, the gelatinases were active in the range of 7.0–11 and keratinolytic activity was highest at pH 10 (Fig. 5b), approximately 75% of the activity was lost at pH 9.0 and 11.0. Effect of inhibitors on enzyme activity

Fig. 4 Zymograms with co-polymerized gelatin, feather keratin, casein, BSA or hemoglobin. Enzyme extracts of B. subtilis 1271, B. cereus 1268 and B. licheniformis 1269 were prepared as described in material and methods. Gel strips containing 30 ll of concentrated culture supernatant were incubated for 48 h at 37°C in 0.5 M Tris– HCl, pH 7.4. The molecular masses of the peptidases, expressed in kDa, are shown on the left

To characterize the extracellular proteolytic activities of Bacillus spp. isolates, zymogram gels containing keratin or gelatin as substrates were incubated in the absence and in the presence of proteolytic inhibitors of the four major peptidase classes (aspartic, cysteine, serine and metallopeptidase). The proteolytic inhibition results showed that the profiles of the extracellular peptidases expressed by

these Bacillus spp. are composed of serine peptidases (Fig. 6). As shown in Table 1, keratinases and peptidases were strongly inhibited by PMFS. E-64 (a cysteine peptidase inhibitor), pepstatin A (an aspartic peptidase inhibitor), EDTA and 1,10-phenanthroline (metallopeptidase inhibitor or metal-dependent enzyme) did not alter significantly the behavior of the enzymes.

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Fig. 5 Effect of temperature (a) and pH (b) on keratinolytic (filled square) and gelatinolytic (filled diamond) activity of extract enzymatic from B. cereus 1268, B. licheniformis 1269 and B. subtilis 1271

Fig. 6 Effects of proteolytic inhibitors on the extracellular peptidases and keratinases of B. cereus 1268, B. licheniformis 1269 and B. subtilis 1271, in gelatin zymogram (a) and keratin zymogram (b). Gel strips were incubated separately in the absence (control) or in the presence of different proteolytic inhibitors: 3 mmol l-1

phenylmethylsulfonyl fluoride (PMSF), 0.26 mmol l-1 ethylenediaminetetraacetic acid (EDTA), 10 mmol l-1 1,10 phenanthroline (Phenan), 10 lmol l-1 pepstatin A (Peps) and 5 lmol l-1 M transepoxysuccinyl L-leucylamido-(4-guanidino) butane (E-64)

Discussion

amino acids such as methionine, lysine, and tryptophan are lost and other non-nutritive amino acids, such as lysinoalanine and lanthionine are formed (Dalev et al. 1997; Matsui et al. 2009). In spite of its limitations, this meal is already incorporated into the diet, as feed, for chicken poultry, rainbow trout, shrimp and salmon. However this type of feed needs an amino acid supplement (Bertsch and Coello 2005). The use of microorganisms capable of

Here we have demonstrated the degradation of intact feathers and amino acid production by submerse fermentation, using three Bacillus spp. isolated from poultry waste. Currently some of the feather waste produced by the poultry industry is transformed into feather meal, however the final product is not very digestible and some essential

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Table 1 Effect of various inhibitors on keratinases and peptidases of B. cereus 1268, B. licheniformis 1269 and B. subtilis 1271 Inhibitor

Residual activity (%) B. cereus 1268

Control PMSF EDTA 1,10-phenanthroline Pepstatin A E-64

B. subtilis 1271

Keratinase

Gelatinase

Keratinase

Gelatinase

Keratinase

Gelatinase

100

100

100

100

100

100

0

2.18

1.86

14.64

14.44

23.74

77.33 87.21

100.63 92.24

97.96 77.45

78.15 80.34

100.1 87.66

101.38

78.58

105.47

76.78

73.96

99.01

74.17

105.45

82.48

85.6

75.96 99.90

producing extracellular keratinases is a possible alternative and eco-friendly method to convert this abundant waste into low-cost amino acids and peptides to be used in animal feed and foodstuff as a supplement (Khardenavis et al. 2009). Feathers are generated in large quantities by poultry processing industries and their accumulation in nature could lead to environmental problems. Consequently efforts to recycle this waste product are desirable. New biotechnological processes for the reuse of keratin feathers with a better yield are needed (Gupta and Ramnani 2006; Daroit et al. 2009). Biodegradable films and keratin composites are other ways to reuse feather keratin (Barone et al. 2005; Gosh et al. 2008). Also hydrolyzed keratin can be used in cosmetic formulations following a reconstructive capillary methodology (Mazotto et al. 2010). Bacterial keratinases are of particular interest because of their potential in biotechnological methods for hydrolysis of proteinaceous solid wastes coming from poultry. With this in mind many Bacillus species have been reported to produce keratinolytic proteases (Nilegaonkar et al. 2007; Giongo et al. 2007; Sousa et al. 2007; Son et al. 2008; Cai and Zheng 2009). Most of these strains were isolated from poultry waste (Park and Son 2009; Cai and Zheng 2009) and other sources such as sediment samples from a hot spring (Pillai and Archana 2008) and the Amazon basin (Giongo et al. 2007). Our results corroborate with the broad distribution of keratinase producers in this genus. This study detected keratinolytic activity in the isolates B. subtilis, B. cereus and B. licheniformis and indicated the production of alkaline keratinases and peptidases. For the first time peptidases were shown to be involved in feather degradation with a broad substrate degradation profile as demonstrated by zymograms incorporated with feather keratin, gelatin, bovine serum albumin, hemoglobin and casein. Due to different methodologies and substrates employed to detect and analyze keratinases, studies on keratinolytic activity cannot be directly compared. However, some parameters such as degradation of feathers can be:

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B. licheniformis 1269

96.87 107.6

B. subtilis 1271 completely degraded feathers in medium, B. licheniformis degraded 44% and B. cereus 80%. B. pumilus FH9 was able to degrade 96% and B. lichenimormis SA1 hydrolyzed 87.2% (El-Refai et al. 2005). Some isolates have been described completely degrading feathers in culture medium such as B. megaterium F7-1 (Park and Son 2009), Bacillus pseudofirmus FA30-01 (Kojima et al. 2006) and B. licheniformis PWD-1 (Williams et al. 1990). The ability of a microorganism to degrade keratin, and the resulting levels of keratinase produced, varies according to species, keratin substrates and culture conditions (Cai and Zheng 2009). In the culture supernatant of the three Bacillus strains of this study, the amino acids present in high concentration were serine, glycine, glutamic acid, valine and leucine. The product of feather hydrolysis by Vibrio sp. kr2 was rich in serine, leucine and glutamic acid (Grazziotin et al. 2006), and in feather hydrolysate obtained from B. cereus DCUW high concentrations of lysine, glutamic acid, histidine and threonine were found (Gosh et al. 2008). Keratin amino acid produced by Streptomyces sp. MS-2 included valine, leucine, isoleucine and alanine (Mabrouk 2008). Free amino acid contents of the culture supernatants of Meiothermus ruber were investigated and significant amounts of leucine, valine glycine, alanine and serine were detected (Matsui et al. 2009). The amino acids obtained from feather hydrolysis by microbial keratinase can be used as a feed supplement for poultry and cattle. The discrepancy between the amino acid content of degraded feather with that expected from pure keratins probably results from microbial metabolism and conversion of free amino acids (Matsui et al. 2009). Keratinases with molecular masses ranging from 18 to 240 kDa have been reported (Gupta and Ramnani 2006) and in this study the three Bacillus species secreted peptidases ranging from 15 to 200 kDa. From the literature, a purified keratinase from the B. subtilis strain KS-1 was described as a single polypeptide of 25.4 kDa (Suh and Lee 2001). Kojima et al. (2006) characterized a keratinase from the B. pseudofirmus strain FA30-01 at 27 kDa. Strains from

World J Microbiol Biotechnol (2011) 27:1355–1365

the genus Bacillus, in particular the B. licheniformis strains, have been shown to secrete extracellular keratinases in the 33 to 42 kDa range (Lin et al. 1992; Rozs et al. 2001). B. lichenifornis 1269 produced a 60 kDa keratinase that degraded keratin and gelatin, but not the other substrates. This keratinase has the potential to be used in the dehairing process in the leather industry. This specific keratinolytic activity is advantageous in the leather industry, because collagen, the major leather-forming protein, is not significantly degraded by the keratinase (Pillai and Archana 2008). Interestingly, the B. cereus strain used in this investigation produced an extracellular keratinase with an apparent molecular mass of 200 kDa. This result is similar to the results found for Kocuria rosea and Fervidobacterium islandicum keratinases with 240 and 200 kDa, respectively (Bernal et al. 2006; Nam et al. 2002), but unknown until now within the genus Bacillus. Other studies have analyzed extracellular keratinase production using zymography with non specific substrates such as gelatin (Nam et al. 2002; Giongo et al. 2007; Gosh et al. 2008), casein (Nilegaonkar et al. 2007; Sousa et al. 2007) with feather meal (Kojima et al. 2006) incorporated in the gel. These studies detected only one or two peptidases in the culture supernatant of keratinolytic microorganisms. In contrast, in this study a large number of peptidases were detected in the culture supernatant of B. licheniformis 1269 and B. subtilis 1271 by gelatin zymography. Additionally five keratinases were detected by keratin zymography in the B. subtilis 1271 culture supernatant. This is the first study reporting multiple keratinase production. The results suggest that feather degradation is not due to a single keratinase. Most keratinases from Bacillus spp. described belong to the serine peptidase class. Therefore, phenylmethanesulfonyl fluoride (PMSF) is the potential inhibitor of these enzymes. However, some Bacillus keratinases can be partially inhibited by EDTA due the importance of cations as stabilizing agents in these keratinases (Ramnani and Gupta 2006). Keratinases of B. cereus DCUW, Bacillus sp. P13 and B. pseudofirmus FA30-01 were completely inhibited by PMSF (Kojima et al. 2006; Gosh et al. 2008; Pillai and Archana 2008), as well as the keratinases produced by B. subtilis 1271, B. cereus 1268 and B. licheniformis 1269. In this work, we have shown that feathers are a cheap and environmentally friendly substrate for the production of multiple alkaline peptidases by Bacillus strains. Peptidases have been routinely used in industry for various purposes including bioremediation processes, in the pharmaceutical industry, cheese making, baking, preparation of soy hydrolysates, debittering of protein hydrolysates, leather treatment and mainly in laundry (Rao et al. 1998). Among these peptidases, alkaline peptidases are the most appropriate as detergent additives because they digest

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various proteinaceous stains (Saeki et al. 2007). The peptidases produced by Bacillus spp. isolated in this study were able to degrade keratin and other proteins, such as casein, BSA, hemoglobin and gelatin. Currently, a large proportion of the commercially available alkaline peptidases are produced by Bacillus species because of their high pH and temperature stability (Tari et al. 2006). Bacteria of the genus Bacillus, usually secrete two types of extracellular peptidases, a neutral and an alkaline peptidase (Park et al. 2004; Tang et al. 2004). Significant keratinolytic and proteolytic activity was observed for all three species studied in this work and this activity responded to changes in pH and temperature. The best pH was in the 7.0–11.0 range and the best temperature in the 40–70°C range. The keratinase from B. cereus was had optimum activity at pH 7.0 and 45°C (Sousa et al. 2007), whereas keratinases from B. pseudofirmus FA30-01, B. licheniformis AP-1 and B. cereus MCM B-326 had optimum values at pH between 9.0 and 11.0 and temperature from 50 to 70°C (Nilegaonkar et al. 2007; Tang et al. 2004; Kojima et al. 2006). In conclusion we have isolated and described three keratinolytic Bacillus species that produced several peptidases and keratinases with molecular masses between 15 and 200 kDa. These enzymes were active over a wide pH and temperature range making them of interest in industrial processes. High levels of simultaneous proteolytic and keratinolytic activity from Bacilllus strains are new. The fact they were isolated from feather waste, although not necessarily surprising, reinforces their potential application in processes using feather derivatives and all biotechnological processes involving keratin hydrolysis. Acknowledgments We would like to thank the technical assistance of Denise da Rocha de Souza supported by fellowships grants from MCT/CNPq. This study was supported by grants from Coordenac¸a˜o de Aperfeic¸oamento Pessoal de Nı´vel Superior (CAPES), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (MCT/ CNPq), Conselho de Ensino para Graduados e Pesquisas (CEPG/ UFRJ), Fundac¸a˜o Oswaldo Cruz (FIOCRUZ), Fundac¸a˜o Carlos Chagas Filho de Amparo a` Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Fundac¸a˜o Universita´ria Jose´ Bonifa´cio (FUJB).

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