Purification and characterization of four keratinases ... - ResearchGate

Bioresource Technology 101 (2010) 344–350
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Bioresource Technology
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Purification and characterization of four keratinases produced by Streptomyces sp.
strain 16 in native human foot skin medium
Fuhong Xie a,b,1 , Yapeng Chao a,1 , Xiuqing Yang c , Jing Yang a , Zhiquan Xue a,b , Yuanming Luo d , Shijun Qian a, *
a State Key Laboratories of Transducer Technology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
b Graduate School of the Chinese Academy of Sciences, Beijing 100039, China
c Institute of Biotechnology, Shanxi University, Taiyuan 030006, China
d State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
article
info
abstract
Article history:
Received 13 March 2009
Received in revised form 23 July 2009
Accepted 6 August 2009
Available online 4 September 2009
Keywords:
Keratinase
Native human foot skin
Streptomyces sp. strain 16
Purification
Characterization
Four extracellular keratinases (designated KI, KII, KIII, and KIV) were produced during submerged aerobic
cultivation in a medium containing native human foot skin (NHFS) for enzyme synthesis. The molecular
weights, determined by SDS–PAGE, were 25, 50, 34, and 19 kDa, respectively. Gel filtration of the four
purified enzymes in native conditions indicated that active keratinase KI is a novel homo-octamer, KII
a homo-dimer, and KIII and KIV monomers. All four keratinases exhibited high activities at pH 8.0–
10.0 with an optimal pH of 9.0. The optimal temperature for keratinolytic activity of KI, KII, and KIII
was approximately 50, and 60 °C for KIV. One millimolar of PMSF completely inhibited the keratinolytic
activities of the four enzymes. The N-terminal sequences of KI, KII, and KIII showed that they were different
from previously described enzymes, whereas KIV shared an identical N-terminal sequence with
two other peptidases from Streptomyces.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Keratins are insoluble proteins that are major constituents of
skin, hair, feathers, wool, and nails. Compared to other soluble proteins,
keratins are known for their high stability and extreme resistance
to degradation by proteolytic enzymes such as pepsin, trypsin,
and papain (Gradisar et al., 2005). The mechanical stability of keratin
depends on the tight packaging of proteins in a-helix (a-keratin) or
b-sheet (b-keratin) structures and their high degree of cross-linkages
by disulfide and hydrogen bonds (Yamamura et al., 2002).
Despite the rigid structure, keratin wastes can be efficiently degraded
by a few bacteria and fungi due to the secretion of keratinolytic
peptidases (keratinases) into the culture medium (Onifade
et al., 1998). Keratinases have been reported from several species
of fungi such as Microsporum (Essien et al., 2009), Trichophyton
(Anbu et al., 2008), and from the bacteria Bacillus (Cai and Zheng,
2009; Macedo et al., 2005; Pillai and Archana, 2008), Streptomyces
(Syed et al., 2009; Szabo et al., 2000; Tatineni et al., 2008). Keratinases
have several important uses in biotechnological processes
such as dehairing, catalysis in the leather industry and slow release
* Corresponding author. Address: State Key Laboratories of Transducer Technology,
Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen
Road, Chaoyang District, Beijing 100101, China. Tel./fax: +86 10 64807428.
E-mail address: qiansj@sun.im.ac.cn (S. Qian).
1 These two authors contributed equally to this work.
nitrogen fertilizer application, cosmetics, and preparation of biodegradable
films (Kim et al., 2004).
Streptomyces species are known to secrete multiple peptidases
(Morihara et al., 1967; Sinha et al., 1991). Some of these serine peptidases,
purified from S. griseus (Awad et al., 1972; Johnson and
Smillie, 1974) and S. fradiae (Kitadokoro et al., 1994; Sinha et al.,
1991) have been well characterized structurally and enzymatically.
We are uncertain, however, why most purified keratinases cannot
completely solubilize native keratin (Ignatova et al., 1999).
Keratin-like materials such as scurf often appear in dirty
clothes, but are not effectively hydrolyzed by the peptidases
currently included in detergents. Therefore, enzymes with high
activity on such materials would be useful. In China, large amounts
of foot skin waste are generated by foot care centers and this waste
is usually discarded and is a potential source of environmental
pollution. Native human foot skin waste (NHFS) comes from the
epidermis, in which keratin is the major constituent. Gradisar
et al. used scrapings of Stratum corneum from the human sole, a
NHFS analogue, as the starting materials for keratinase production
(Gradisar et al., 2000). It is possible to use these NHFS for keratinase
production in order to solubilize the scurf on the clothes.
In our previous study (Chao et al., 2007), NHFS was used as the
sole nitrogen source to screen microorganisms with keratindegrading
capabilities. A Streptomyces sp., designated strain 16,
was found to produce a series of keratinases that could efficiently
degrade NHFS. Degradation tests indicated that culture filtrate
0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2009.08.026

F. Xie et al. / Bioresource Technology 101 (2010) 344–350 345
from Streptomyces sp. strain 16 could also efficiently decompose
casein, collagen, and keratin azure.
In this study, we describe the purification and characterization
of four keratinases from Streptomyces sp. strain 16 cultured in
medium containing NHFS.
2. Methods
2.1. Materials
Native human foot skin waste was collected from the foot care
section of the Haidian Hospital in Beijing. In preparation for NHFS
experiments, the human foot skin was washed thoroughly with tap
water, autoclaved, and then dried. The dried NHFS was stored under
dry condition and was used for keratinase production. One part of the
dried NHFS was milled to powders for the keratinase assay. 1-Ethyl-
3-(3-dimethyaminopropyl) carbodiimide (EDAC), p-chloromercuribenzoic
acid (pCMB), phenylmethanesulfonyl fluoride (PMSF), DLdithiothreitol
(DTT), and casein were purchased from Sigma (USA).
Nanosep centrifugal device tubes (molecular size cutoff, 100 or
10 kDa) were purchased from Pall Corporation (USA), while Sephacryl
S-200 resin, Superose 12 10/300 GL column, Hiprep DEAE FF
column (1 ml), and Hiprep Octyl FF column (1 ml) were acquired
from Amersham Biosciences (Sweden). The Folin–Ciocalteu reagent
was purchased from Dingguo Company (China). All reagents used
in this study were analytical grade unless otherwise indicated.
2.2. Microorganism, culture medium, and growth conditions
The microorganism used in this study was Streptomyces sp.
strain 16, which was isolated in our lab for keratinase production
(Chao et al., 2007). The medium for keratinase production was
composed of NHFS, 4 g/l; soluble starch, 5 g/l; yeast extract, 1 g/l;
and K 2 HPO 4 , 0.5 g/l. The pH of the medium was 8.0.
Seed cultures of Streptomyces sp. strain 16 were prepared in a
500 ml Erlenmeyer flask containing 100 ml of culture medium.
After 2 days of incubation, the culture broth was transferred to a
5 L Fernbach culture flask containing 1.5 L of medium for keratinases
production. After 4 days of incubation, the culture was collected
for keratinase purification. All incubations were done
aerobically at 30 °C on an orbital platform shaker at 200 rpm.
2.3. Zymography of the culture filtrate
The zymography of the culture filtrate was based on the method
of Bressollier et al. (1999).The culture broth was centrifuged
(10,000 rpm, 4 °C, 10 min.), and the supernatant was mixed with
2 electrophoresis sample buffer (2 ml 4 stacking gel buffer,
1.6 ml glycerol, 3.2 ml 10% SDS, 0.8 ml 2-mercaptoethanol, and
0.4 ml 1.0% bromophenol blue) (Simpson, 2003) in a 1:1 ratio without
heat denaturation. SDS–PAGE was carried out at 4 °C with a
constant current of 23 mA in a 12% polyacrylamide gel. The gel
was washed with 2.5% Triton X-100 (v/v) in 50 mM Tris–HCl buffer
(pH 7.5) for 30 min. Then washed with 50 mM Tris–HCl buffer (pH
7.5) for 30 min. Casein (1%, w/v) dissolved in 50 mM Tris–HCl buffer
(pH 7.5) was poured onto the gel. After 30 min at 40 °C, the gel
was stained with Coomassie brilliant blue R-250 and then destained
with a water/methanol/acetic acid (50:40:10) solvent. Peptidase
bands appeared as clear zones on a blue background.
2.4. Enzyme assays
2.4.1. Assay for keratinolytic activity
Keratinolytic activity was determined spectrophotometrically
with a modification of the Folin–Ciocalteu method (Ledoux and
Lamy, 1986; Margesin and Schinner, 1991). An appropriate amount
of enzyme was incubated with 1.5 ml of 0.5% sieved (mesh size:
300 lm) NHFS, in 50 mM Tris–HCl buffer (pH 7.5) at 40 °C for
16 h. The reaction was terminated with 3 ml of 10% trichloroacetic
acid (TCA) and placed at room temperature for 30 min. The reaction
mixture was centrifuged, and 5 ml of 0.55 M Na 2 CO 3 was
added to 1 ml of the supernatant, with subsequent addition of
1 ml Folin–Ciocalteu reagent. This mixture was incubated at
40 °C for 15 min. Keratinolytic activity was measured at 680 nm
using a spectrophotometer (721 Visible Spectrometry, China).
One unit (U) of keratinase activity was represented by an increase
in absorbance at 680 nm of 0.01 in 1 h compared with the control
reaction. The control was treated in the same way, except that TCA
was added before incubation.
2.4.2. Assay for proteolytic activity
Protease activity was determined by modifying the Folin–Ciocalteu
method using casein as a substrate. The appropriate amount
of enzyme was incubated with 1 ml 1% casein in 50 mM Tris–HCl
buffer (pH 7.5) at 40 °C for 15 min. The reaction was terminated
by the addition of 3 ml of 10% TCA. Subsequent steps were the
same as those described in the assay of keratinolytic activity.
One unit (U) of protease activity was defined as an increase in
absorbance at 680 nm of 0.01 in 1 min compared with the control
reaction. The control was treated in the same way, except that TCA
was added before incubation.
2.5. Protein determination
Protein concentrations of the enzyme preparations were determined
according to the method described by Bradford (1976),
using bovine serum albumin as the standard.
2.6. Purification procedure
Culture broth was centrifuged (11,366g, 4°C, 10 min) to remove
cells and debris and the supernatant was collected. Solid
ammonium sulfate was added to the supernatant with a saturation
of 80% under gentle stirring, and then kept at 4 °C overnight. The
suspension was centrifuged at 10,000g for 30 min at 4 °C. The
precipitate was then dissolved in 100 ml of 50 mM Tris–HCl buffer
(pH 7.5).
Three milliliter of the dissolved ammonium sulfate precipitate
was loaded onto a Sephacryl S-200 column (3 100 cm) equilibrated
with 50 mM Tris–HCl buffer (pH 7.5) and eluted with the
same buffer at a flow rate of 0.5 ml/min. Fractions of 6.0 ml in each
tube were collected and assayed for keratinolytic activity. The
peaks with keratinolytic activity were named KI, KII, KIII, and KIV
according to the order of elution. Purity was checked via SDS–
PAGE. KIV was found to be homogenous on the SDS–PAGE gel,
while the other three components needed further chromatographic
purification.
2.6.1. Purification of keratinases KI and KII
Following Sephacryl S-200 column chromatography, the KI and
KII fractions were pooled separately, and sequentially applied to a
Hiprep DEAE FF column (1 ml) pre-equilibrated in 50 mM Tris–HCl
buffer (pH 7.5), and a Hiprep Octyl FF column (1 ml) pre-equilibrated
in 50 mM Tris–HCl buffer (pH 7.5) containing 1.5 M
(NH 4 ) 2 SO 4 . The KI fraction was eluted from the Hiprep DEAE FF column
with 20 ml 50 mM Tris–HCl buffer (pH 7.5) containing 0 to
1 M NaCl at a linear flow rate of 2 ml/min. The KII fraction was
eluted using 0.1 M, 0.2 M, 0.5 M, and 1 M NaCl sequentially, in
50 mM Tris–HCl buffer (pH 7.5). The KI fraction was eluted from
the Hiprep Octyl FF column using 20 ml of 50 mM Tris–HCl buffer
(pH 7.5) containing 0 to 1.5 M (NH 4 ) 2 SO 4 at a linear flow rate of

346 F. Xie et al. / Bioresource Technology 101 (2010) 344–350
2 ml/min. The KII fraction was eluted from the Hiprep Octyl FF column
using a reverse step gradient of 50 mM Tris–HCl buffer (pH
7.5) containing 1.0, 0.75, 0.5, 0.3, and 0 M (NH 4 ) 2 SO 4 , sequentially,
at a flow rate of 2 ml/min.
2.6.2. Purification of keratinase KIII
After Sephacryl S-200 column chromatography, fractions of
peak KIII were pooled and adjusted to 1.5 M (NH 4 ) 2 SO 4 . KIII was
eluted by applying 20 ml of 50 mM Tris–HCl buffer (pH 7.5) containing
0 to 1.5 M (NH 4 ) 2 SO 4 at a linear flow rate of 2 ml/min.
3. Results
3.1. Keratinases production
Streptomyces sp. strain 16 was able to grow and produce keratinase
in a medium that contained 4 g/L NHFS. To expedite growth
and increase the biomass of the strain, limited nitrogen sourceyeast
extract was added to the medium in this study. NHFS began
to decrease in the second day of cultivation, and completely disappeared
in the fourth day of cultivation. Keratinase production
reached maximal activity of 74.9 U/ml at four days of cultivation.
2.7. Molecular weight of the purified keratinases
The purity and subunit number of each peptidase was estimated
by SDS–PAGE (12% gel) according to Laemmli (1970). After electrophoresis,
the gel was stained with Coomassie Brilliant Blue R-250
dissolved in a water/methanol/acetic acid (50:40:10) solvent for
1 h, then destained in the same solvent. The molecular weight of
the native peptidases was determined using a Superose 12 10/300
GL column, using thyroglobulin (669,000 Da), ferritin (440,000 Da),
catalase (232,000 Da), lactate dehydrogenase (140,000 Da), and bovine
serum albumin (67,000 Da) (Amersham Biosciences, Sweden)
as standards.
2.8. Effect of pH and temperature on enzyme activity and stability
The optimal pH for enzyme activities was determined at 40 °C
at various pH levels (pH 6.0–12.0). Sodium phosphate was used
for pH 6.0–7.0, Tris–HCl was used for pH 7.5–9.0, and sodium carbonate–bicarbonate
was used for pH 10.0–12.0. For pH stability,
residual activity was measured at pH 7.5 after 1 h of incubation
at 40 °C in the pH buffers being tested.
Enzyme activities were tested at temperatures ranging from
30 °C to80°C in 50 mM Tris–HCl buffer (pH 7.5). Each of the purified
enzymes was incubated for 1 h at the tested temperature, and
residual activity was measured at 40 °C in 50 mM Tris–HCl buffer
(pH 7.5).
3.2. Purification of the keratinases produced by the Streptomyces sp.
strain 16
The zymogram of culture supernatant shown in Fig. 1 indicated
the presence of five bands that showed peptidase activity in the
culture supernatant.
The purification procedures of the four enzymes is outlined in
Fig. 2. When the precipitated enzyme was loaded onto Sephacryl
S-200 column, four major peaks eluted that showed keratinase
activity. The main keratinase in each peak was designated KI, KII,
KIII, and KIV based on their order of elution. However, the fifth
band appeared on the zymogram gel was not detected from the
elution.
After one to two further steps of chromatography, all four keratinases
from Streptomyces sp. strain 16 were ultimately purified
to homogeneity, which was confirmed by SDS–PAGE (Fig. 3).
Molecular weights of subunits of the four enzymes were calculated
as 25 kDa (KI), 50 kDa (KII), 34 kDa (KIII) and 19 kDa (KIV).
The determination of the molecular weight of each of the four
keratinases was performed by gel filtration chromatography on a
2.9. Effects of protease inhibitors, reducing agents, and metal ions on
keratinase activity
To investigate the effects of different inhibitors, reducing
agents, and metal ions on keratinase activity, the residual activity
after allowing the enzyme to stand with the reagent for 30 min
at 30 °C was measured. Phenylmethyl sulphonyl fluoride (PMSF),
para-chloro mercuric benzoate (pCMB), EDAC, and ethylene diamine
tetra acetic acid (EDTA) were used at a concentration of
1 mM as protease inhibitors. Dithiothreitol (DTT) and ascorbic acid
were used at a concentration of 1 mM, and Na 2 SO 3 ,Ca 2+ , and Mg 2+
were used at a concentration of 10 mM.
2.10. N-terminal amino acid sequencing of the purified keratinases
After SDS–PAGE, each purified ketatinase band was transferred
onto a polyvinylidene fluoride (PVDF) membrane (Pall Corporation,
USA). The keratinase band on the PVDF membrane was cut out and
dissolved in 20% (v/v) acetonitrile/water containing 0.001% trifluoroacetic
acid (TFA). The N-terminal sequences of the purified keratinases
were determined by automated Edman degradation using a
pulsed-liquid sequence analyzer (ABI PROCISE TM 492cLC protein
sequencing system, Applied Biosystems, USA).
Fig. 1. Zymogram analysis of keratinases secreted by Streptomyces sp. strain 16.
Arrows indicate the keratinase bands.

F. Xie et al. / Bioresource Technology 101 (2010) 344–350 347
Concentrated culture
Sephacryl S-200
Peak I
DEAE FF
Peak II
DEAE FF
Peak III
Peak IV
Octyl FF
Octyl FF
Octyl FF
KI
KII
KIII
KIV
Fig. 2. Purification strategy for extracting keratinases from Streptomyces sp. strain 16.
kDa
110
M
1 2 3 4
Table 1
Activity determination in different components treated by ultrafiltration.
1 2 3
66
Protein (lg/ml) 244 256 1.5
Keratinolytic activity (U/ml) 37 42 0
Specific activity (U/mg protein) 151.6 164.1 0
45
35
1. The retentate by 100 kDa ultrafiltration.
2. The retentate by 10 kDa ultrafiltration.
3. Filtrate by 10 kDa ultrafiltration.
25
18
14
Fig. 3. SDS–PAGE analysis of the purified keratinases (KI, KII, KIII, and KIV)
produced by Streptomyces sp. strain 16. The molecular marker, KI, KII, KIII, and KIV
are located in lane M, 1, 2, 3, and 4, respectively.
Superose 12 10/300 GL column. The results provided a molecular
weight estimate of 203.2 kDa for KI, 100.8 kDa for KII, 31.8 kDa
for KIII, and 19.2 kDa for KIV. This indicated that KI is a homo-octamer,
KII a homo-dimer, and KIII a monomer and KIV a monomer.
To further verify that KI is an active homo-octomer, ultrafiltration
was conducted. Retentate with a molecular weight of 100 kDa,
retentate with a molecular weight of 10 kDa, and filtrate with a
molecular weight of 10 kDa or less were sequentially collected
and analyzed (Table 1). Most of the enzyme activity was detected
in retentate with a molecular weight of 100 kDa and a molecular
weight of 10 kDa. However, no activity emerged in the filtrate with
a molecular weight of 10 kDa.
3.3. Influence of temperature and pH on the keratinolytic activity of KI,
KII, KIII, and KIV
The four purified keratinases had high activities between pH 8.0
and 10.0, with maximal activity at pH 9.0. The four keratinases
showed low activities at pH 6.0 and 12.0. The effect of pH 7.0 on
each of the four keratinases was different, the keratinase activities
for KI and KIV were more than 50% of their maximal activities, but
KII and KIII activities were less than 30% of their maximal activities.
At pH 11.0, KI showed low keratinase activity, while KII, KIII, and
KIV showed more than 50% of their maximal activities. The four
keratinases showed the same pH stability. More than 85% of the
maximal activities were retained between pH 8.0 and 10.0.
The optimal temperature for keratinases KI, KII, and KIII was
about 50 °C, while the optimal temperature was about 60 °C for
keratinase KIV. KI, KII, and KIII activity dropped fast at temperatures
below 40 °C and above 60 °C. For KIV, the activity at 80 °C
was approximately half of its maximal activity, which was at
60 °C. All four keratinases retained more than 70% of their maximal
activities in the temperature range of 40–60 °C after 1 h incubation.
KIV was more stable than the other three keratinases.
3.4. Effects of chemicals on keratinolytic activities of KI, KII, KIII and
KIV
Protease inhibitors, reducing agents, and metal ions were used
to investigate their effects on the enzyme activities (Table 2). All
four enzymes were significantly inhibited by the serine protease
inhibitor PMSF at 1 mM. This indicates that all of the enzymes belong
to the serine protease family. Moreover, the residual activities
of the four enzymes were evidently not affected by cysteine protease
inhibitor pCMB. However, metalloprotease inhibitor EDTA
strongly inhibited KI, KII, and KIII, although it did not inhibit the
activity of KIV. For all four keratinases, significant increases in keratinolytic
activity were observed by adding reducing agents DTT,
ascorbic acid, and sodium sulfite. Carbonyl group modifier EDAC
had stimulative effects on the activities of KI, KIII, and KIV. The keratinolytic
activities of KI, KIII, and KIV increased by 36%, 100%, and
32%, respectively. The divalent metal ion Ca 2+ showed a stimulative

348 F. Xie et al. / Bioresource Technology 101 (2010) 344–350
Table 2
Effects of the different reagents on the keratinolytic activity of the four keratinases.
Compound Concentration (mM) Residual activities (%)
effect on KI, an inhibitory effect on KIII, and no effect on KII and
KIV. The metal ion Mg 2+ had an inhibitory effect on KII and KIII,
and no effect on KI and KIV.
3.5. Substrate specificities of KI, KII, KIII, and KIV
The ability of the four enzymes to hydrolyze proteinaceous substrates
was examined with casein, NHFS, keratin azure, collagen,
cock feathers, and human hair (Table 3). Results showed that the
four enzymes exhibited high activities toward casein and NHFS,
however, NHFS could not be solubilized by any one of the four keratinases.
All four purified enzymes showed higher activities with
soluble proteinaceous substrates such as casein than with insoluble
proteinaceous substrates such as NHFS. The four keratinases
showed low activities toward keratin azure and collagen. For all
four keratinases, no activity was observed with human hair and
cock feathers as substrates.
3.6. N-terminal sequence
N-terminal amino acid sequencing of the purified keratinases
resulted in unequivocal determination of the first 10 amino acid
residues of the four keratinases (Table 4), with the exception of position
10 in KIII, where no assignment could be made. When compared
to the National Center for Biotechnology Information (NCBI)
protein database by BLAST search, the KI, KII, and KIII sequences
did not show significant homology to known microbial peptidases.
However, the N-terminal sequence of KIV was identical to that of
the 17.6 kDa protease (Sinha et al., 1991) and SFase-2 (Kitadokoro
et al., 1994) from S. fradiae and it showed 70% homology to SGPA
from S. griseus (Johnson and Smillie, 1974).
4. Discussion
KI KII KIII KIV
Control 0 100 100 100 100
pCMB 1 92 86 96 95
EDAC 1 136 107 200 132
PMSF 1 33 0 0 0
EDTA 1 36 7 0 97
DTT 1 195 157 170 256
Ascorbic acid 10 145 107 370 155
Na 2 SO 3 1 177 160 130 180
CaCl 2 10 114 86 20 82
MgCl 2 10 86 28 60 87
In our previous studies, Streptomyces sp. strain 16 was found to
completely degrade NHFS, and keratinases were produced when
Table 3
Substrate specificities of the four keratinases.
Substrate Residual activity * (%)
KI KII KIII KIV
Casein 100 100 100 100
NHFS 93 79.1 89 82
Keratin azure 3.7 0.8 1.0 6.5
Collagen 3.7 1.2 1.0 30
Cock feather 0 0 0 0
Human hair 0 0 0 0
* The proteolytic activity toward casein of each enzyme was used as 100%. Activity
on other substrate was compared with it.
strain 16 was cultured in a medium that contained NHFS. The addition
of yeast extract to the medium was helpful to increase of biomass
and enzyme production. A zymogram of culture filtrates
showed that complete degradation of NHFS was due to the presence
of multiple keratinases. In order to investigate the mechanism
of degradation, we purified the keratinases from the culture broth.
The results showed that four keratinases secreted by Streptomyces
sp. strain 16 were responsible for the degradation of NHFS in the
medium. In the purification procedure, we also tried to purify the
fifth peptidase appeared on the zymogram gel. Unfortunately, we
failed to isolate this enzyme, perhaps due to its low quantity.
Most microbial keratinases are inducible enzymes, as reported
by Cheng et al. (1995). Various keratinous materials such as chicken
feather, feather meal, wool, and bovine hair have been used as
inducers of keratinases (De Toni et al., 2002; Ignatova et al.,
1999; Kumar et al., 2008; Nam et al., 2002). NHFS belongs to soft
keratins from the human sole, which was first used as a keratinase
inducer.
Proteolytic enzymes have dominated the detergent enzyme
market. Nonetheless, there is always a need for new enzymes with
novel properties that can further widen the scope of enzyme-based
detergents. Keratinases could help in the removal of keratinous
soils that are often encountered in the laundry and on which most
normal peptidases fail to act. In this study, the keratinases produced
by Streptomyces sp. strain 16 can efficiently decompose
NHFS, therefore, these keratinases may be candidate enzymes to
be used as additives to the detergents.
Similar to most keratinases reported in the literature, keratinase
KIV is a monomer. However, KII is composed of two subunits in its
native form and the molecular mass of each subunit is approximately
50 kDa. Interestingly, KI is composed of eight subunits in
its native form with a total molecular weight of 203.2 kDa. This
is the first report of a peptidase with eight subunits. A few
keratinases of high molecular weight with multimeric structures
and multiple domains have been reported. Nam et al. isolated a
native-feather-degrading thermophilic anaerobic bacterium identified
as Fervidobacteium islandicum from a geothermal hot stream
(Nam et al., 2002). A homo-multimeric membrane-bound keratinolytic
peptidase was purified from the cell extracts of the anaerobe.
Its molecular weight was estimated to be greater than 200 kDa
with a 97 kDa subunit (Nam et al., 2002). Fervidolysin is a keratinase
(with a molecular weight of 73 kDa) as an immature form from
Fervidobacterium pennivorans (Kluskens et al., 2002). The 1.7 Å resolution
crystal structure of fervidolysin showed that the peptidase
is composed of four domains: a catalytic domain (CD), two b-sandwich
domains (SDs), and the propeptide (PD) domain (Kim et al.,
2004). Keratinases of high molecular weight may consist of multi-domain
structures or oligomeric proteases, which contribute to
their substrate specificities (Kim et al., 2004). Accordingly, the oligomeric
nature of KI and KII is also postulated to contribute to the
substrate specificities.
Several Streptomyces keratinases have been classified as serine
proteases (Bockle and Muller, 1997; Bressollier et al., 1999). Kerotinolytic
activities of studied enzymes were significantly inhibited
by PMSF. Consequently, keratinases KI, KII, KIII, and KIV were classified
as serine proteases. KI was slightly more stable at the same
concentration of PMSF, perhaps due to its homo-multimeric form.
Meanwhile, EDTA also showed similar effects on the keratinolytic
activity of KI, KII, and KIII suggesting that they are metal ion related
enzymes. The activity of KIV was not affected by EDTA. Although
KI, KII, and KIII shared the same optimal temperature, and optimal
pH of the four keratinases were identical, the effects of pH and
temperature on the activities of the four keratinases actually
varied.
According to the difference in molecular mass, characteristics,
and the distinct differences in N-terminal sequences, the four

F. Xie et al. / Bioresource Technology 101 (2010) 344–350 349
Table 4
N-terminal sequences of Streptomyces sp. strain 16 keratinases and Alignment of keratinase KIV, SFase-2, 17.6 kDa protease and SGPA.
Microorganism Keratinase N-terminal residues
1 2 3 4 5 6 7 8 9 10
Streptomyces sp. strain 16 KI a A G N S A S E I R V
KII a A A P G D K D V T A
KIII a A P D I I L A N A
KIV a I A G G E A I Y A A
S. fradiae ATCC 14544 SFase-2 b I A G G E A I Y A A
S. fradiae 17.6 kDa c I A G G E A I Y A A
S. griseus SGPA d I A G G E A I T T G
a
This study.
b (Kitadokoro et al., 1994).
c (Sinha et al., 1991).
d (Johnson and Smillie, 1974).
keratinases of Streptomyces sp. strain 16 are apparently different
peptidase. From the identical molecular weight and N-terminal sequence,
KIV may be the same peptidase as SFase-2 in S. fradiae, and
a homologue of the 17.6 kDa protease in S. fradiae and SGPA in S.
griseus.
Base on the fact that no enzyme among the four keratinases
could solubilize NHFS alone, there must be some cooperation
among them or with some other components in the culture filtrate
in the process of degrading NHFS. Therefore, it will be interesting
to study the cooperation among the keratinases in further
research.
5. Conclusions
In this work, four keratinases were purified from the culture
broth of Streptomyces sp. strain 16 simultaneously, and they differed
from each other in terms of molecular weight, oligomeric
nature, and some physicochemical characteristics. All four keratinases
showed strong keratinolytic activity toward NHFS. The N-terminal
sequences of KI, KII, and KIII showed no similarity to
sequences of known microbial peptidases, whereas keratinase
KIV shared an identical N-terminal sequence with two other peptidases
of Streptomyces. Keratinase KI is a homo-octamer with a
molecular weight of 203.2 kDa. Based on the properties of the four
keratinases, they might become attractive for potential applications
in laundry detergents.
Acknowledgement
This work was financially supported by the National High Technology
Research and Development Program of China (863;
2006AA020204).
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