APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1992, p.

3271-3275

0099-2240/92/103271-05$02.00/0
Copyright 1992, American Society for Microbiology

Purification and Characterization of a Keratinase from

Feather-Degrading Bacillus licheniformis Strain

Vol. 58, No. 10

XIANG LIN,t CHUNG-GINN LEE,4 ELLEN S. CASALE, AND JASON C. H. SHIH*

Department of Poultry Science and University Biotechnology Program, North Carolina State University,
Raleigh, North Carolina 27695-7608

Received 27 April 1992/Accepted 21 July 1992

The major component of feathers is keratin. Because of a

high degree of cross-linking by cystine disulfide bonds,
hydrogen bonding, and hydrophobic interactions, keratin is
insoluble and not degradable by proteolytic enzymes, such
as trypsin, pepsin, and papain (7, 8, 14). Despite the unusual
stability of keratin, feathers do not accumulate in nature.
Our laboratory has reported the isolation and characterization of a feather-degrading bacterium, Bacillus licheniformis
PWD-1 (23, 24). The bacterium grows on feathers as the
primary organic substrate for supplying carbon, sulfur, and
energy. Biodegradation of feathers by this bacterium represents a method for improving the utilization of feathers as,
for example, a feed protein. Recently, feeding experiments
with chickens demonstrated a significantly better growth
response when the bacterial fermentation product feather
lysate (18) was added to the diet than when untreated
feathers or commercial feather meal was added (18, 22). This
study reports the purification and characterization of the
keratinase secreted by feather-degrading B. lichenifornis
PWD-1.

containing 10 ml of culture medium. After 4 days of incubation, 10 ml of the culture medium was transferred to a 3-liter
Fernbach flask containing 1.0 liter of medium. After 4 days
of incubation, the medium was collected for keratinase
purification. All incubations were done at 50C with shaking
at 120 rpm in a controlled-environment shaker (New Brunswick Scientific Co., New Brunswick, N.J.).
Enzyme purification. The culture medium was prefiltered
through glass wool to remove residual undegraded feathers.
The medium was then filtered through a 0.45-,um-pore-size
membrane with a Pellicon cassette system (Millipore Corp.,
Bedford, Mass.) to remove bacterial cells and other particles. The filtrate was concentrated by membrane ultrafiltration (molecular weight cutoff, >10,000) with the same (Pellicon) system. The crude concentrated keratinase solution
was applied to a column of carboxymethyl cellulose (CMcellulose) (2.5 by 60 cm) at 4C. The column was equilibrated
with buffer A (25 mM potassium phosphate buffer [pH 5.8]).
Approximately 50 mg of protein was loaded on the column.
Elution was begun with 500 ml of buffer A. The eluted
proteins were monitored by measuring the A280 of the
fractions. When the 280-nm reading was a continuous baseline, elution with buffer B (25 mM potassium phosphate
buffer [pH 6.2], 20 mM NaCl) was begun. Approximately 300
ml was used for this elution. Finally, buffer C (25 mM
potassium phosphate buffer [pH 6.8], 20 mM NaCl) was
used. The elution flow rate was 0.4 ml/min, and 5-ml
fractions were collected. Fractions were screened with the
milk-agarose plate assay. Fractions exhibiting proteolytic
activity were assayed for keratinase activity with the azokeratin hydrolysis test. Keratinase active fractions were
pooled and concentrated with an Amicon stirred cell filtration system with Diaflo ultrafilters (molecular weight cutoff,
>10,000) (Amicon Div., W. R. Grace and Co., Beverly,
Mass.) at 4C. Purification of keratinase was continued with
a Sephadex G-75 column (1.5 by 90 cm) at 4C. Equilibration
and elution were carried out at 4C with 50 mM potassium
phosphate buffer (pH 7.0) at a flow rate of 0.3 ml/min.
Fraction volumes of 3 ml were collected. Protein elution was
followed by monitoring of the A280 of each fraction. Fractions were screened with the milk-agarose plate assay, and

MATERIALS AND METHODS

Organism and growth conditions. The bacterium used in
this study was a patented strain of B. licheniformis PWD-1
isolated in our laboratory (19, 23). All culture conditions and
the feather culture medium were as previously described.
The medium contained, per liter, the following: 0.5 g of
NH4Cl, 0.5 g of NaCl, 0.3 g of K2HPO4, 0.4 g of KH2PO4,
0.1 g of MgCl2 6H20, 0.1 g of yeast extract, and 10 g of
hammer-milled chicken feathers. The pH was adjusted to
7.5. Feathers were washed, dried, and hammer milled prior
to being added to the medium. The medium was sterilized by
autoclaving.
The bacterium was cultured in a test tube (20 by 1.5 cm)
Corresponding author.
t Permanent address: Shenyang Agricultural University, Shenyang, Liaoning, People's Republic of China.
*

t Present address: Department of Animal Production, Council of

Agriculture, Taipei, Taiwan, Republic of China.
3271

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A keratinase was isolated from the culture medium of feather-degrading Bacilus licheniformis PWD-1 by use
of an assay of the hydrolysis of azokeratin. Membrane ultrafiltration and carboxymethyl cellulose ion-exchange
and Sephadex G-75 gel chromatographies were used to purify the enzyme. The specific activity of the purified
keratinase relative to that in the original medium was approximately 70-fold. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis and Sephadex G-75 chromatography indicated that the purified
keratinase is monomeric and has a molecular mass of 33 kDa. The optimum pH and the pI were determined
to be 7.5 and 7.25, respectively. Under standard assay conditions, the apparent temperature optimum was
50C. The enzyme is stable when stored at -20C. The purified keratinase hydrolyzes a broad range of
substrates and displays higher proteolytic activity than most proteases. In practical applications, keratinase is
a useful enzyme for promoting the hydrolysis of feather keratin and improving the digestibility of feather meal.

3272

LIN ET AL.

Separations, Inc., Westboro, Mass.) or centrifuged to reinsoluble protein, and a 0.5-ml aliquot was removed
reaction with ninhydrin reagent to determine the
production of free amino groups. The standard curve was
move
for a

determined with leucine at 0.1 to 0.5 ,umol.

Both the free amino group assay and the azokeratin
hydrolysis assay were used to compare the keratinolytic
activities of a variety of proteases (Sigma). The enzymes
tested were papain (EC 3.4.22.2), bovine trypsin (EC
3.4.4.4), porcine elastase (EC 3.4.21.36), Clostridium histolyticum collagenase (EC 3.4.24.3), Tntirachium proteinase
K (EC 3.4.21.14), and keratinase.
Protein determination. Protein concentrations were determined by the Bio-Rad (Richmond, Calif.) protein assay
method as described by Bradford (3). The microassay pro-

cedure was performed, and BSA was used as the standard

protein.
Enzyme properties. The native molecular weight of keratinase was estimated by Sephadex G-75 column chromatography; calibration was done with standard proteins from a kit
obtained from Pharmacia Fine Chemicals (Piscataway,
N.J.). To examine the purity and determine the subunit
molecular weight, we performed discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) as described by Laemmlli (9). The protein bands
were stained with a solution of Coomassie blue R-250.
Isoelectric focusing, as described in Bio-Rad Instruction
Manual 161-0310, was used to determine the isoelectric point
of keratinase. The experiment was conducted with an LKB
horizontal electrophoresis unit (2117 Multiphor) and LKB
Ampholines. The protein bands were stained with a Bio-Rad
silver stain kit by use of the method developed by Mehta and
Patrick (11) and Confavreux et al. (4). The pH and apparent
temperature optimum for keratinase activity were assayed
by the azokeratin hydrolysis assay. To determine whether a
prosthetic group is an integral part of keratinase, we scanned
the enzyme solution with a Shimadzu UV-160 UV-visible
recording spectrophotometer. A BSA solution of the same
protein concentration was used for comparison.
Determination of the production of sulfhydryl groups. Reduction of disulfides to sulfhydryls in keratin may be part of
the mechanism of enzymatic keratinolysis. To test this
hypothesis, we determined the production of sulfhydryl
groups by use of the Ellman reaction (5) during the enzymatic reaction, while hydrolysis was monitored as the increase in the production of free amino groups as described
above. One hundred milligrams of feather keratin was incubated at 50C with 50 ,ug of keratinase in 15 ml of 50 mM
phosphate buffer (pH 7.5). Every 10 min, 1.6 ml of reaction
mixture was removed for analysis, 1.0 ml for sulfhydryls and
0.6 ml for free amino groups.

RESULTS
Enzymatic hydrolysis of azokeratin. Insoluble azokeratin
as the chromogenic substrate was incubated with the keratinase solution to produce a soluble colored product. The
supernatant demonstrated an increase in the A450 in a linear
fashion in the first 30 min because of the release of azopeptide derivatives into the solution. This assay, in conjunction
with prescreening by the milk-agarose plate assay, was used
for the purification of keratinase.
Purification of keratinase. A summary of the purification of
keratinase from the culture medium of B. licheniformis
PWD-1 is presented in Table 1. Membrane ultrafiltration and
CM-cellulose ion-exchange and Sephadex G-75 gel chromatographics yielded a purified keratinase fraction having an
overall purification factor of 70-fold. The final product had a
specific activity of about 6,000 U/mg. The ultrafiltration step
removed approximately 60% of the total protein while maintaining 70% of the enzyme activity. CM-cellulose column
chromatography purified the enzyme 43-fold and removed
98% of the original total protein. Elution from the Sephadex
G-75 column yielded a homogeneous protein, as shown by a
single protein band in SDS-PAGE (Fig. 1A).
Enzyme properties. The purified PWD-1 keratinase is a
monomeric protein with a molecular mass of 33 kDa, as
estimated by both Sephadex G-75 chromatography (data not
shown) and SDS-PAGE (Fig. 1). The pH optimum and
apparent temperature optimum of keratinase activity, as
determined by the azokeratin hydrolysis assay, are 7.5 and

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then the active fractions were assayed for keratinase activity

with the azokeratin hydrolysis test. Keratinase active fractions were pooled and concentrated with the Amicon stirred
cell unit.
Enzyme hydrolysis of azokeratin. Azokeratin was prepared
by reacting ball-milled feather powder (24) with sulfanilic
acid and NaNO2 by use of a method similar to that described
by Tomarelli et al. for azoalbumin (21). For a standard assay,
5 mg of azokeratin was added to a 1.5-ml centrifuge tube
along with 0.8 ml of 50 mM potassium phosphate buffer (pH
7.5). This mixture was agitated until the azokeratin was
completely suspended. A 0.2-ml aliquot of an appropriately
diluted enzyme solution was mixed with the azokeratin, and
the mixture was incubated for 15 min in a 50C water bath.
The reaction was terminated by the addition of 0.2 ml of 10%
trichloroacetic acid, and the mixture was filtered. The A450
of the filtrate was measured with a UV-160 spectrophotometer (Shimadzu, Columbia, Md.). A control was prepared by
adding trichloroacetic acid to a reaction mixture before
adding the enzyme solution. Without knowing the molar
extinction coefficient of azopeptides, we defined 1 U of
keratinase activity as an increase in the A450 of 0.01 after 15
min in the test reaction compared with the control reaction.
Milk-agarose plate assay. A method that was an alternative
to the azokeratin hydrolysis assay was used to screen large
numbers of fractions collected from CM-cellulose and Sephadex G-75 columns. One gram of agarose was dissolved in 98
ml of 50 mM potassium phosphate buffer (pH 7.5). After the
agarose solution was cooled to 60 to 70C, 2 ml of evaporated
whole milk was added with gentle agitation. The mixture was
poured rapidly onto a clean glass plate (20 by 20 cm) and
spread evenly. After the agarose had solidified, 81 (9 by 9)
small wells (5 mm in diameter) were punched in the agarose.
A 25-,l aliquot of a sample was applied to each well. The
plate was incubated in a moist atmosphere either overnight
at 37C or for 2 h at 50C. Clear zones around wells were
indicative of proteolytic activity. A similar method was
previously published (17). Keratinase activity was further
identified by azokeratin hydrolysis as described above.
Free amino group assay. For the native feather keratin and
other protein substrates, including casein, elastin, collagen,
and bovine serum albumin (BSA) (all from Sigma Chemical
Co., St. Louis, Mo.), the proteolytic activities of keratinase
were determined on the basis of the production of free amino
groups. A modified ninhydrin method was used for detecting
free amino groups (16). Five milligrams of a protein substrate
was placed in a 1.5-ml tube with 0.8 ml of buffer and 0.2 ml
of enzyme solution containing 8 ,ug of purified keratinase.
After incubation at 50C for 30 min, the reaction was stopped
by the addition of trichloroacetic acid. The reaction mixture
was filtered with a 5.0-,um-pore-size syringe filter (Micron

APPL. ENVIRON. MICROBIOL.

B. LICHENIFORMIS KERATINASE

VOL. 58, 1992

TABLE 1. Purification of keratinase from the medium of

B. licheniforinis PWD-1
Step

Medium
Membrane concentration
CM-cellulose chromatography
Sephadex G-75
chromatography

Sp act
(U/mg of
protein)

M.W(KDa) 1

% of U

Purification
(fold)

100
69.3

1.0
1.8

Total
protein
(mg)

Total
ua

142
54.5

12,200
8,450

86
150

3.3

12,080

3,720

99

43

1.5

8,910

5,990

73.1

70

3273

3 4

4-

97.4
66.2

a One unit is defined as an increase in the A450 of 0.01 after reaction with
azokeratin for 15 min.

-m

31

DISCUSSION
The combination of the milk-agarose plate assay and the
azokeratin hydrolysis assay was demonstrated to be effective for the purification of the keratinase from the culture
medium of B. licheniformis PWD-1. The milk-agarose plate
assay is a simple method for screening large numbers of
fractions for proteolytic activity. For the identification of
keratinase activity, the azokeratin hydrolysis assay is simple
and specific. The two methods correlated well in that the
active fractions showing proteolytic activity on the milkagarose plate also showed keratinase activity. Therefore,
only one major protease, the keratinase, was present in the
bacterial medium. To further confirm the keratinolytic activity, we also used a third method, measuring the increase in
the production of free amino groups upon hydrolysis of the
native feather keratin. Both the native feather keratin and
synthetic azokeratin have been shown to be more specifically hydrolyzed by keratinase than by other proteases
(Table 2).
The purification of keratinase was effective and efficient.

21.5

1 4.4

A
1 05

phosphorylase b

BSA
ovalbumin

0
keratinase w.

carbonic anhydrase

trypsin inhibitor

lysozyme
4 n4
Iv

0.0

0.2

0.6
0.4
Relative mobility

0.8

1.0

B
FIG. 1. SDS-PAGE of purified keratinase. (A) SDS-polyacrylamide gel. Lanes: 1 and 3, purified keratinase; 2, molecular mass
standards; 4, CM-cellulose-treated preparation; 5, crude enzyme
preparation. (B) Molecular mass standard curve. Standards: phosphorylase b, 97.4 kDa; BSA, 67 kDa; ovalbumin, 43 kDa; carbonic
anhydrase, 31 kDa; soybean trypsin inhibitor, 21.5 kDa; lysozyme,
14.4 kDa.

About 73% of the total activity was retained, while only 1%

of the original total protein remained. However, concentration of the medium by membrane ultrafiltration resulted in a
decrease in the total enzyme units. The total enzyme units
returned to the original level after ion-exchange chromatography (Table 1). This phenomenon was repeatedly observed.
The concentration of the proteins may be such that the

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50C, respectively. Isoelectric focusing determined that the

isoelectric point is 7.25. The keratinase is fairly stable upon
storage at low temperatures. A solution sample (30 ,ug/ml)
was found to lose only 7% of its activity at -20C and 20%
of its activity at 4C after 19 days of storage. However, at
room temperatures (20 to 25C), the half-life of keratinase
activity is 4 to 5 days. The loss of enzyme activity is largely
due to enzymatic autolysis, which was detected by SDSPAGE (data not shown). The UV-visible absorption spectrum of the keratinase was found to be identical to that of
BSA. No absorption peak besides the typical absorption
peaks at 280 and <220 nm for a protein molecule was
detected.
Enzyme specificity. A free amino group assay was used to
compare the substrate specificities of a variety of proteases.
The keratinase was capable of hydrolyzing all the proteins
tested, including BSA, casein, collagen, elastin, and feather
keratin. As demonstrated in Table 2, both the native feather
keratin and synthetic azokeratin were specific substrates for
keratinase. They were degraded less by other proteases.
Sulfhydryl analysis. During the enzymatic hydrolysis of
feather keratin, sulfhydryl groups in the reaction mixture
were monitored (Fig. 2). No increase in the production of
free sulfhydryl groups was detected during the reaction. On
the other hand, the production of free amino groups increased as a result of peptide bond cleavage in a linear
fashion over a 1-h period.

-_

LIN ET AL.

3274

APPL. ENVIRON. MICROBIOL.

TABLE 2. Relative keratinolytic activities of proteases in two different assays
Keratinolytic activity of:

Substrate

Temp (C)

Feather keratin

Azokeratin

Papain

Trypsin

Collagenase

Elastase

Proteinase K

Keratinasea

37
50

0
0

13
16

0
0

17

36

27

51

41
100

37
50

8
7

23
27

2
1

35
47

39
52

64
100

a For the feather keratin substrate, the specific activity of purified keratinase is 118 pmol of leucine equivalents released per h per mg of enzyme protein; for
the azokeratin substrate, the specific activity is 4,700 U, and 1 U is defined as an increase in the A450 of 0.01 after reaction for 15 min under standard assay
conditions.

0.4

0.2

0.3 -[-NH 2]
E 0~~~~~~~~~
E

ID

=10.
2

0.2

0.1

Lo

U)0.1

CD

Time(min)~~~~~~~~~~~m[-HJ
~

0.0
0

*
20

I
40

0.0
60

Time (min)
sulfhydryl

FIG. 2. Analysis of free amino and

enzymatic keratinolysis.

80
groups during

more hydrolytic for elastin and collagen than some elastases

and collagenases (data not shown). Elastin and collagen are
similar to keratin in that they are insoluble, highly structured, and recalcitrant to many proteases. However, exam-

ination of the individual structures of these proteins reveals

quite different assemblies. Collagen, like keratin, is a fibrous
protein and consists of three a chains assembled in a triple
helix conformation, resulting in a rod-like shape. Elastin is
not a fibrous protein but rather is a polymer of globular
proteins assembled in a fibrous arrangement. Both elastin
and collagen are stabilized by covalent intermolecular crosslinkages involving lysine and lysine derivatives (1, 2, 13, 15,
20). These properties provide some degree of resistance to
peptide hydrolysis.
The unique structure of keratin makes it very resistant to
proteolytic digestion. The resistance is due not only to the
supercoiled helical structure of polypeptide chains but also
to the strength of intermolecular disulfide bonds and other
molecular interactions (6, 12). One of the possible mechanisms in breaking down keratin is the reduction of disulfide
bonds. For instance, pretreatment of keratin with a reducing
agent, such as thioglycolate, disrupts the disulfide bonds and
increases digestibility by trypsin (7). However, such a mechanism is not likely in the case of the keratinase. First, it
degrades keratin without a reducing agent or coenzyme, and
second, enzymatic keratinolysis is not coupled with an
increase in the production of detectable sulfhydryls (Fig. 2).
The molecular mechanism of the enzymatic action remains
to be determined.
In summary, a simple colorimetric assay for keratinase
was developed. With this method, a keratinase was purified
from newly discovered B. licheniformis PWD-1. The keratinase was characterized in terms of its biochemical properties
and found to have a broad substrate specificity. The reduction of disulfide bonds to sulfhydryls was not detected in the
hydrolysis process. The use of keratinase as a feed additive
to improve the digestibility of feather meal in chickens has
been demonstrated in our laboratory (10). The purification
and characterization studies of the keratinase have provided
the basis to develop further the production and uses of this
enzyme.
ACKNOWLEDGMENT
We appreciate the grant support from Southeastern Poultry and
Egg Association.

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VOL. 58, 1992

3275

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B. LICHENIFORMIS KERATINASE