Journal of Environmental Sciences 19(2007) 1125–1128
Screening for a new Streptomyces strain capable of eﬃcient
CHAO Ya-peng, XIE Fu-hong, YANG Jing, LU Jing-hua, QIAN Shi-jun ∗
State Key Laboratories of Transducer Technology, Institute of Microbiology, Chinese Academy of Sciences,
Beijing 100080, China. E-mail: firstname.lastname@example.org
Received 18 October 2006; revised 12 December 2006; accepted 25 December 2006
Keratinous wastes could be degraded by some microorganisms in nature. Native human foot skin (NHFS) was used as sole nitrogen
source to screen microorganisms with keratin-degrading capability. From approximately 200 strains, a strain of Streptomyces sp. strain
No.16 was found to possess the strongest keratinolytic activity, and the total activity in the culture was 110 KU/ml with speciﬁc activity
of 2870 KU/mg protein (KU: keratinase unit). Substrate speciﬁcity test indicated that the crude keratinase could degrade keratin azure,
human hair, cock feathers and collagen. The optimal pH of the crude keratinase ranged from 7.5 to 10 and the temperature ranged from
40°C to 55°C. Metal chelating agent ethylenediamine tetraacetic acid obviously stimulated the keratinolytic activity but suppressed
the proteolytic activity. To our knowledge, this is the ﬁrst report on speciﬁc induction of keratinases by NHFS from an actinomycete.
Moreover, excellent characteristics of its crude keratinase may lead to the potential application in waste treatment and recovery, poultry
and leather industry, medicine, and cosmetic development.
Key words: keratinolytic capability; Streptomyces sp.; native human foot skin (NHFS); keratinase; substrate speciﬁcity
Introduction impala in health care and cosmetic, speed up healing
process, help anti-fungi medicine to attain the target in
Keratin is an insoluble structural protein of skin, both tetters, remove the scar and regenerate the epithelia. It is
keratin and its derivatives (e.g. feather, hair, wool, and also the special medicine of trauma, and so on.
horn) are known for their high stability. The keratin chain The aim of this study was to screen a microorganism
is tightly packed in the α-helix (α-keratin) or β-sheet (β- with high ability to degrade the keratinous wastes, espe-
keratin) and then turns into a supercoiled polypeptide chain cially native human foot skin (NHFS) and feathers, and to
(Parry and North, 1998), resulting in mechanical stability exploit for the application of keratinase in waste resources.
and resistance to common proteolytic enzymes, such as
pepsin, trypsin, and papain. In addition, cross-linking of 1 Materials and methods
the protein chains by cysteine bridges confers high me-
chanical stability and resistance to proteolytic degradation 1.1 Materials
of the keratins. It is becoming a part of solid waste since
it is tough to degrade due to the highly rigid structure. Keratin azure, casein and azocasein were purchased
Hence, there is a demand for developing biotechnological from Sigma Co. (USA). The fresh human foot keratin was
alternatives for recycling of such wastes. collected from feet care section of Haidian Hospital, Bei-
There are only a few reports on enzymatic keratin degra- jing. The ultraﬁltration membrane (molecular size cutoﬀ,
dation. These enzymes reported are produced by some 10 kDa) was purchased from Pall Company (USA), Folin-
species of Bacillus, Streptomyces, and fungi (Yamamura Ciocalteu reagent was from Dingguo Company (China).
et al., 2002; Williams et al., 1990; Takami et al., 1999; Other reagents were either analytical or biological grade.
Beockle et al., 1995; Nickerson et al., 1963; Gradisar 1.2 Isolation and screening of keratin-degrading strains
et al., 2000; Mignon et al., 1998; Tawﬁk and Rawa,
2001). They have potential applications in biotechnology, Collection and pretreatment of human foot keratin
for example, the conversion of keratin to peptides and The fresh NHFS was pre-autoclaved and then dried at
rare amino acids. Moreover, these proteinases deserve 100°C. One part of treated samples was deposited in dry
attention because of their role in skin physiology, including condition for liquid cultivation and the remaings were
formation and degradation. It can dissolve the scurf and milled into powder for “clear circle” demonstration and
*Corresponding author. E-mail: email@example.com.
1126 CHAO Ya-peng et al. Vol. 19
Culture medium and Schinner, 1991) using azocasein as a substrate. The
Liquid medium 2 (L−1 ): 5 g glycerol, 0.5 g K2 HPO4 , 1 g enzyme solution (1 ml) was incubated with 1 ml of 1% azo-
yeast extract, 4 g human keratin, distilled water was added casein in 50 mmol/L Tris/HCl buﬀer (pH 7.5) at 40°C for
to 1 L, pH 8.0. Solid medium 1 (L−1 ): 5 g glycerol, 0.5 g 15 min. The reaction was terminated by addition of 3 ml of
K2 HPO4 , 1 g yeast extract, 4 g human keratin, 15 g agar, 6% TCA. After 10 min, reaction mixture was ﬁltered with
distilled water was added to 1 L, pH 8.0. Liquid medium absorbent cotton. Treatment of supernatant and subsequent
2 (L−1 ): 5 g glycerol, 0.5 g K2 HPO4 , 4 g human keratin, assay procedure were the same as described above.
distilled water was added to 1 L, pH 8.0. Solid medium 2
1.3 Keratin degradation by culture supernatant
(L−1 ): 5 g glycerol, 0.5 g K2 HPO4 , 4 g human keratin, 15
g agar, distilled water was added to 1 L, pH 8.0. Preparation of crude keratinase
Strain collection After cultivating the strain in a 5-L Fermenter for 4 d,
Plates with solid medium 1 were used to collect micro- the supernatant was collected by centrifugation at 11366×g
bial strains from diﬀerent sources. A total of 82 strains for 10 min and concentrated by ultraﬁltration (molecular
were obtained from a soil sample collected near a chicken- size cutoﬀ, 10 kDa). Ammonium sulfate was added to
processing stall in market, 17 strains were isolated from eighty percent of saturation. The precipitate was recovered,
a sample of the cloaca, and 32 strains were obtained dissolved in 50 mmol/L Tris/HCl buﬀer (pH 7.5), dialyzed
from an earth sample of a deer-breeding zoo in China. and freeze-dried.
Other strains used in screening were either collected from Substrate speciﬁcities of the crude keratinase
China General Microbiological Culture Collection Center Keratin azure, human hair, cock feather, collagen, and
or obtained from our lab collection. NHFS were used as substrate to determine the keratinolytic
Screening methods activities. Proteinase K and trypsin with same proteolytic
The isolates were inoculated in 250 ml ﬂasks with 50 ml activities were also determined under the same conditions.
liquid medium and incubated on a shaker at 30°C for 6 The procedure to determine the activity is described in
d, then the supernatants were separated by centrifugation “Assay for keratinolytic activity”.
at 11366×g for 10 min. The keratinolytic strains were Eﬀects of pH and temperature
screened with three diﬀerent ways, determination of pro- The optimal temperature for the determination of the
teinase activity using azo-casein as substrate, measurement crude keratinase activity using azocasein and keratin
of transparent circle size on solid agar plate including ranged from 30 to 70°C. The optimal pH was determined at
NHFS, and observation of the disintegration of NHFS by 40°C with 50 mmol/L using sodium phosphate (pH 6–6.5),
the supernatants. Tris-HCl (pH 7–9), and boric acid buﬀer (pH 9–12).Three
Medium optimization parallels were conducted for each test.
The optimal parameters for carbon source (0.5% glyc- Eﬀects of chelating agents
erol or 0.5% soluble starch), nitrogen source (0.4% human One milliliter of crude keratinase was mixed with 1 ml
keratin, 0.4% polypeptone or 0.4% gelatin respectively) 20% EDTA (pH 8.0) and incubated overnight. The activity
were determined. Initial pH of the medium (6.5, 7.0, 7.5, was analyzed with azocasein and NHFS as substrate.
8.0, 8.5, 9.0, 9.5), cultivation temperature and time (3,
4, 5, 6, 7 d) were also optimized. The testing strain was 2 Results
grown only for 4 d when fermentation was conducted in a 2.1 Screening for microorganisms degrading NHFS
Assay for keratinolytic activity More than 200 microorganisms obtained from diﬀerent
The keratinolytic activity was determined spectropho- sources (including Aspergillus, Streptomyces, Bacillus,
tometrically using a modiﬁed Folin-Ciocalteu method Vibrio) were screened. Five strains were found to have
(Ledoux and Lamy, 1986; Margesin and Schinner, 1991). good proteolytic activities when inoculated on solid agar
The enzyme solution (0.5 ml) incubated with 0.5% sieved plate by observing the diameter of clear zones. Strain
(mesh size: 300 µm) NHFS (1.5 ml), in 50 mmol/L No.16 exhibited the most potent activity with the observa-
Tris/HCl buﬀer (pH 7.5) at 40°C for up to 16 h. The tion of the largest clear zone (Fig.1). The same results were
reaction was terminated with 3 ml of 10% trichloroacetic also observed for proteolytic and keratinolytic activities.
acid (TCA) and then the reactants were allowed to stand for This strain was obtained from China General Microbiolog-
30 min. After ﬁltration with absorbent cotton, 5 ml of 0.55 ical Culture Collection Center, belonging to Streptomyces
mol/L Na2 CO3 was added to 1 ml of the supernatant with sp. strain No.16. Fermentation broth of strain No.16 also
subsequent addition of 1 ml of Folin-Ciocalteu reagent, degraded keratin azure as indicated by the blue color of the
and then incubated at 40°C for 15 min. The keratinolytic solution (Fig.2), the supernatant also completely degraded
activity was measured at 680 nm with a 721 visible the NHFS after incubation for 24 h, indicating its strong
spectrometer (China) and expressed in keratinase units degrading activity for NHFS (Fig.3). In a conclusion, the
(KU). One KU is deﬁned as an increase of 0.01 OD at 680 strain No.16 possessed potent keratinolytic activity.
nm in 1 h. 2.2 Optimization of cultivation conditions
Assay for proteolytic activity A series of experiments were conducted to optimize
Proteinase activity was detected by modiﬁed Folin- keratinase production. The results showed that soluble
Ciocalteu method (Ledoux and Lamy, 1986; Margesin starch was more suitable as carbon source than glycerol,
No. 9 Screening for a new Streptomyces strain capable of eﬃcient keratin degradation 1127
Table 1 Activities of the crude keratinase on diﬀerent rigid proteins
Enzyme Activities (KU/ml)
NHFS Keratin Hair Feather Collagen
Crude keratinase 1.94 1.69 1.63 1.56 1.56
Proteinase K 0.75 0.06 0 0.05 0
Trypsin 0.81 0.08 0.05 0.08 0.02
most potent activity for NHFS. It also degraded the other
four tested rigid proteins (keratin azure, human hair, cock
feather, and collagen), eﬃciently indicating its activity
on a broad range of substrates. Proteinase K and trypsin
Fig. 1 Clear zone on the agar plate containing NHFS.
exhibited weaker activity on diﬀerent types of keratins
except for NHFS. The keratinase was obviously more
active on NHFS than proteinase K and trypsin. Therefore,
the keratinase produced by strain No.16 could be applied
for keratinous waste treatment, medicine, and cosmetic
2.4 Inﬂuence of pH and temperature on enzyme activity
Casein, keratin azure and NHFS were used as substrates
for pH and temperature optimization. The results (Fig.4)
indicated that optimal pH of the keratinase ranged from 7.5
to 10 with NHFS as the substrate, from 7 to 11 with casein
as the substrate, and from 7.5 to 8.5 with keratin azure as
the substrate, respectively. Correspondingly, the optimal
temperatures ranged from 40°C to 55°C with NHFS as
Fig. 2 Degradation of keratin azure by culture supernatant. Left: boiled the substrate, 40°C to 65°C with casein as the substrate,
culture supernatant; right: native culture supernatant. and 50°C to 65°C with keratin azure as the substrate,
respectively. It was surprised that both the optimal pH
and temperature for keratinolytic activity on NHFS were
higher in comparing with those for casein and keratin
azure, implying that the keratinase could be used under
2.5 Inﬂuence of EDTA on the keratinolytic and prote-
olytic activity of the crude keratinase
The inﬂuence of EDTA on enzymatic activity is listed in
Table 2. The keratinolytic activity of the crude keratinase
was obviously stimulated by EDTA. The result implys
that activity of the keratinase isolated in this test was
independent of metal ions. While the proteolytic activity
Fig. 3 Complete degradation of NHFS by culture supernatant. (1) control
of the crude keratinase was slightly inhibited by EDTA,
(boiled culture supernatant); (2), (3) and (4) are complete degradation by
culture supernatant. indicating that the mechanism for keratinolytic activity of
the crude keratinase was diﬀerent from that for proteolytic
the optimal initial pH was 8.0–8.5, and the duration of activity.
cultivation was 4–5 d. Weak keratinolytic activity was
observed when polypeptone was used as sole nitrogen 3 Disccussion
source, indicating that the production of keratinase by
strain No.16 was specially induced by keratins. The en- Keratinases play an important role in fungal invasion
zymatic activity in the ﬁnal culture was 110 KU/ml with of skin and skin formations. The keratinases from the
speciﬁc activity of 2870 KU/mg protein under the optimal dermatophytes Trichophyton spp., Microsporum spp., and
Table 2 Eﬀects of chelation on the enzyme activity (unit: KU/ml)
2.3 Substrate speciﬁcities of the crude keratinase
Substrate Crude keratinase
Keratinolytic activities of the crude enzyme from strain Control EDTA
No.16 were determined using diﬀerent types of keratins
NHFS 100 144.8
as substrates, and the results are summarized in Table Casein 100 86.7
1. Results showed that the crude keratinase exhibited the
1128 CHAO Ya-peng et al. Vol. 19
Streptomyces sp. strain No.16 was found to exhibit re-
markable kerainolytic activity and its keratinase could act
eﬃciently on diﬀerent keratins without strict limitations
of pH, temperature and ions. It is very important to apply
the microorganism in feather waste treatment and resource
recovery, other keratinous waste removal, processing of
the leather industry, medicine, and cosmetic development.
Further work should be focused on analysis of eﬀective
components in the crude keratinase and on improvement
of degradative activity of strain No.16.
The authors thank P&G Technology (Beijing) Co., Ltd
for the ﬁnancial support. We also thank Dr. Liu Tiehan for
his helpful discussion.
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