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					                                          Journal of Environmental Sciences 19(2007) 1125–1128




                  Screening for a new Streptomyces strain capable of efficient
                                     keratin degradation
                   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: chaoyp@sun.im.ac.cn

                               Received 18 October 2006; revised 12 December 2006; accepted 25 December 2006



Abstract
   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
                                                                            activity determination.
*Corresponding author. E-mail: qiansj@sun.im.ac.cn.
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
fermenter.
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
                                                                                                            azure

                                                                             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
                                                                             development.
                                                                             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
                                                                             broad conditions.
                                                                             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
cultivation conditions.
                                                                               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

                                                                           4 Conclusions
                                                                              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.
                                                                           Acknowledgements
                                                                              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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