ORIGINAL PAPER

Biodegradation of feather waste by extracellular keratinasesand gelatinases from Bacillus spp.

Ana Maria Mazotto • Ana Cristina N. de Melo • Andrew Macrae •

Alexandre Soares Rosado • Raquel Peixoto • Sabrina M. L. Cedrola •

Sonia Couri • Russolina B. Zingali • Ana Lucia V. Villa • Leon Rabinovitch •

Jeane Q. Chaves • Alane B. Vermelho

Received: 30 April 2010 / Accepted: 20 September 2010 / Published online: 13 October 2010

� 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-form-

ing, 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

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 phen-

ylmethylsulfonyl 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 biodegrada-

tion 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 (Su-

zuki 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.

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 Goes (IMPPG), Bloco I, Centro de

Ciencias da Saude (CCS), Universidade Federal do Rio de

Janeiro (UFRJ), Cidade Universitaria, Ilha do Fundao, Rio de

Janeiro, RJ 21941-590, Brazil

e-mail: abvermelho@micro.ufrj.br

S. Couri

Instituto Federal de Educacao Ciencia 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 Bioquımica, Instituto de Ciencias Biomedicas,

Bloco H, Centro de Ciencias da Saude (CCS), Universidade

Federal do Rio de Janeiro (UFRJ), Cidade Universitaria, Ilha do

Fundao, Rio de Janeiro, RJ 21941-590, Brazil

A. L. V. Villa

Universidade federal do Rio de Janeiro, Campus Macae,

R. Aloısio Gomes da Silva no 50, Granja do Cavaleiros, Macae,

RJ 27930, Brazil

L. Rabinovitch � J. Q. Chaves

Departamento de Bacteriologia, Instituto Oswaldo Cruz,

Fundacao Oswaldo Cruz, Av. Brasil 4365, Rio de Janeiro,

RJ 21045-900, Brazil

123

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

DOI 10.1007/s11274-010-0586-1

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 micro-

organisms and have attracted a great deal of attention due

to their multiple applications in industry for the develop-

ment 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 (Balint 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 deliv-

ery (Mohorcic et al. 2007; Yoshioka et al. 2007). Biodeg-

radation 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 keratin-

ases 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

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. (Cam-

bridge, England). Reagents used in electrophoresis and

molecular mass standards were acquired from Amersham

Life Science (Little Chalfont, England). Polyethylenegly-

col 4000 (PEG 4000) was purchased from Vetec (Rio de

Janeiro, Brazil). The peptidase inhibitors (trans-epox-

ysuccinyl l-leucylamido- (4-guanidino) butane [E-64],

phenylmethylsulphonyl fluoride [PMSF], 1,10-phenan-

throline, pepstatin A and EDTA) were obtained from

Sigma Chemical Co. (St. Louis, MO, USA). The protein-

aceous 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) supple-

mented 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 Colecao de Culturas do Genero

Bacillus e Generos Correlatos-CCGB (which is affiliated

to the World Federation of Culture Collections) located in

the Laboratorio de Fisiologia Bacteriana, Instituto

Oswaldo Cruz/FIOCRUZ.

1356 World J Microbiol Biotechnol (2011) 27:1355–1365

123

Genomic DNA of the bacterial isolates was extracted

using the Wizard Genomic DNA Purification Kit (Pro-

mega, 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 MEGAB-

ACE 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 phosphate-

buffered medium (0.06 M Na2HPO4�7H2O 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 kera-

tinase and gelatinase activities.

Feather keratin substrate

Chicken feathers obtained from poultry waste were washed

extensively with water and detergent, dried at 60�C over-

night, 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

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 ana-

lyzed using matrix assisted laser desorption/ionization time

of flight mass spectrometry (MALDI-TOF MS). Immedi-

ately 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 acetoni-

trile/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 anal-

ysis. Peptide mass spectrometry was carried out with a

Bruker Biflex III MALDI-TOF mass spectrometer in the

reflectron mode. The experiments were performed in trip-

licate using three independent experimental sets.

Feather keratin enzymography

Twenty microliter samples of concentrated culture super-

natant were mixed with an equal volume of feather keratin

powder diluted in phosphate buffer (0.06 M Na2H-

PO4�7H2O 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] supple-

mented 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

World J Microbiol Biotechnol (2011) 27:1355–1365 1357

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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 Na2HPO4�7H2O

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% trichloro-

acetic acid. Samples were then put into a refrigerator at 4�C

for 30 min. The supernatant was collected after centrifu-

gation (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 absor-

bance 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/dis-

tilled water (4:1:1 v/v/v) as solvent. The amino acid spe-

cific 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 sam-

ple: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

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 R-

250 in methanol-acetic acid–water (50:10:40) and de-

stained in the same solvent (Lopes et al. 2008). For enzy-

matic 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-leu-

cylamido-(4-guanidino) butane (E-64) were used in prote-

olysis 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 spore-

forming bacilli were selected as keratinolytic after dem-

onstrating complete feather degradation in whole feather

broth. They were identified by biochemical, physiological

and cytomorphological characterization as Bacillus li-

cheniformis (LFB-FIOCRUZ 1269), Bacillus subtilis

(LFB-FIOCRUZ 1271) and Bacillus cereus (LFB-FIO-

CRUZ 1268) at the Laboratorio 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 licheni-

formis 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 keratin-

olytic 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

1358 World J Microbiol Biotechnol (2011) 27:1355–1365

123

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 ana-

lyzed. HTLC analyses of the culture supernatants of the

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).

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, char-

acteristic of feather keratin, was no longer observed and

0

100

200

300

400

500

B. cereus 1268

B. lichenifo

rmis 1269

B. subtilis 1271

Act

ivit

y (U

/ml)

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 super-

natant 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. subtilis1271 for 7 days at 28�C

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 Phosphor-

ylase 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 extracel-

lular 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)

World J Microbiol Biotechnol (2011) 27:1355–1365 1359

123

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 dif-

ferent proteinaceous substrates was evaluated using kera-

tin, gelatin, casein, BSA and hemoglobin co-polymerized

with 12.5% sodium dodecyl sulfate–polyacrylamide gels

(SDS–PAGE). The gels showed that B. cereus 1268 pro-

duced a single peptidase of *200 kDa able to degrade all

substrates tested (Fig. 4). B. subtilis 1271 and B. licheni-

formis 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 hydro-

lyzed 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 kera-

tinase and gelatinase activities of the three Bacillus spp.

was determined using gelatin and feather keratin as sub-

strates, 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. li-

cheniformis 1269 were optimally active at 40�C. The ker-

atinase of B. subtilis exhibited maximal activity at 50�C

(Fig. 5a). B. subtilis strain 1271 presented maximum gel-

atinase and keratinase activity at pH 9.0 and 10, respec-

tively (Fig. 5b) and in B. cereus the optimum pH was 10

for both substrates (Fig. 5a). In B. licheniformis, the gela-

tinases 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

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 metallo-

peptidase). 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 pepti-

dase inhibitor), pepstatin A (an aspartic peptidase inhibi-

tor), EDTA and 1,10-phenanthroline (metallopeptidase

inhibitor or metal-dependent enzyme) did not alter signif-

icantly the behavior of the enzymes.

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

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123

Discussion

Here we have demonstrated the degradation of intact

feathers and amino acid production by submerse fermen-

tation, 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

amino acids such as methionine, lysine, and tryptophan are

lost and other non-nutritive amino acids, such as lysino-

alanine 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

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 ethylenedia-

minetetraacetic acid (EDTA), 10 mmol l-1 1,10 phenanthroline

(Phenan), 10 lmol l-1 pepstatin A (Peps) and 5 lmol l-1 M trans-

epoxysuccinyl L-leucylamido-(4-guanidino) butane (E-64)

World J Microbiol Biotechnol (2011) 27:1355–1365 1361

123

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 com-

posites 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 reconstruc-

tive 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:

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 pep-

tidases 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

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 B. licheniformis 1269 B. subtilis 1271

Keratinase Gelatinase Keratinase Gelatinase Keratinase Gelatinase

Control 100 100 100 100 100 100

PMSF 0 2.18 1.86 14.64 14.44 23.74

EDTA 75.96 77.33 100.63 97.96 78.15 100.1

1,10-phenanthroline 99.90 87.21 92.24 77.45 80.34 87.66

Pepstatin A 101.38 78.58 105.47 76.78 73.96 96.87

E-64 99.01 74.17 105.45 82.48 85.6 107.6

1362 World J Microbiol Biotechnol (2011) 27:1355–1365

123

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 signifi-

cantly 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 Fervidobacte-

rium 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, phenylmethane-

sulfonyl fluoride (PMSF) is the potential inhibitor of these

enzymes. However, some Bacillus keratinases can be par-

tially 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. Pepti-

dases have been routinely used in industry for various

purposes including bioremediation processes, in the phar-

maceutical industry, cheese making, baking, preparation of

soy hydrolysates, debittering of protein hydrolysates, lea-

ther treatment and mainly in laundry (Rao et al. 1998).

Among these peptidases, alkaline peptidases are the most

appropriate as detergent additives because they digest

various proteinaceous stains (Saeki et al. 2007). The pep-

tidases 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 pepti-

dases are produced by Bacillus species because of their

high pH and temperature stability (Tari et al. 2006). Bac-

teria 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 keratino-

lytic 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. lichenifor-

mis 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 pepti-

dases 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 biotechno-

logical 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 Coordenacao

de Aperfeicoamento Pessoal de Nıvel Superior (CAPES), Conselho

Nacional de Desenvolvimento Cientıfico e Tecnologico (MCT/

CNPq), Conselho de Ensino para Graduados e Pesquisas (CEPG/

UFRJ), Fundacao Oswaldo Cruz (FIOCRUZ), Fundacao Carlos

Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro

(FAPERJ) and Fundacao Universitaria Jose Bonifacio (FUJB).

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