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Keratinolytic activity of Bacillus megaterium F7-1,
a feather-degrading mesophilic bacterium
Geun-Tae Parka, Hong-Joo Sonb,c,Ã

a
  Research and University-Industry Cooperation, Pusan National University, Busan 609-735, Republic of Korea
b
  Major of Life Science & Environmental Biochemistry, School of Applied Life Science, Pusan National University,
Miryang, Gyeongnam 627-706, Republic of Korea
c
 Joint Research Center of Pusan National University-Fraunhofer IGB, Pusan National University, Miryang, Gyeongnam
627-706, Republic of Korea

Received 23 October 2006; received in revised form 1 February 2007; accepted 4 February 2007




     KEYWORDS                            Summary
     Bacillus megater-
                                         The aim of this study was to investigate environmental conditions affecting chicken
     ium;
                                         feather degradation and keratinolytic enzyme production by Bacillus megaterium
     Feather degrada-
                                         F7-1, a feather-degrading mesophilic bacterium. B. megaterium F7-1 degraded
     tion;
                                         whole chicken feather completely within 7 days. The bacterium grew with an
     Keratinolytic en-
                                         optimum at pH 7.0–11.0 and 25–40 1C, where maximum keratinolytic activity was
     zyme;
                                         also observed. The production of keratinolytic enzyme by B. megaterium F7-1 was
     Poultry waste
                                         inducible with feather. Keratinolytic enzyme production by B. megaterium F7-1 at
                                         0.6% (w/v) skim milk was 468 U/ml, which was about 9.4-fold higher than that
                                         without skim milk. The amount of keratinolytic enzyme production depended on
                                         feather concentrations. The degradation rate of autoclaved chicken feathers by cell-
                                         free culture supernatant was 26% after 24 h of incubation, but the degradation of
                                         untreated chicken feathers was unsuccessful. B. megaterium F7-1 effectively
                                         degraded feather meal, duck feather and human nail, whereas human hair and sheep
                                         wool showed relatively low degradation rates. B. megaterium F7-1 presented high
                                         keratinolytic activity and was very effective in feather degradation, providing
                                         potential use for biotechnological processes of keratin hydrolysis.
                                         & 2007 Elsevier GmbH. All rights reserved.




                                                                       Introduction
    ÃCorresponding author. Major of Life Science & Environmental
                                                                          Environmental wastes are found in large quan-
Biochemistry, School of Applied Life Science, Pusan National
University, Miryang, Gyeongnam 627-706, Republic of Korea.
                                                                       tities in many countries. Although some of them
Tel./fax: +82 55 350 5544.                                             contain a considerable amount of protein and
    E-mail address: shjoo@pusan.ac.kr (H.-J. Son).                     various carbon compounds, little attention is given

0944-5013/$ - see front matter & 2007 Elsevier GmbH. All rights reserved.
doi:10.1016/j.micres.2007.02.004

    Please cite this article as: Park G-T, Son H-J. Keratinolytic activity of Bacillus megaterium F7-1, a feather-degrading mesophilic
    bacterium. Microbiol Res 2006; (2007), doi:10.1016/j.micres.2007.02.004
                                                    ARTICLE IN PRESS
2                                                                                                            G.-T. Park, H.-J. Son

to utilizing or recycling these wastes in a techno-                    skim milk hydrolyzing protease (Son et al., 2004a).
logical way. Additionally, the accumulation of some                    Following this, we attempted to improve the ability
of these wastes in nature is considered to be a                        of this strain to produce protease using casein as a
serious source of pollution and health hazards.                        enzyme substrate (Son et al., 2004b; Son, 2005).
Therefore, their proper disposal may be considered                     Here, for chicken feather recycling, we report
as a means of avoiding environmental pollution.                        some substances that lead to a high keratinolytic
   Recently, we have focused on the utilization of                     enzyme production, using soluble keratin as a
some polymeric wastes, mainly feather waste.                           testing enzyme substrate, and describe the optimal
Feathers are generated in large amounts as a waste                     condition for chicken feather degradation by
byproduct at commercial poultry-processing plants,                     B. megaterium F7-1.
reaching millions of tons per year worldwide
(Manczinger et al., 2003). Since feathers are almost
pure keratin protein, feather wastes represent a                       Materials and methods
potential alternative to more expensive dietary
ingredients for animal feedstuffs. Generally, they                     Microorganism and culture conditions
become feather meal used as animal feed after
undergoing physical and chemical treatments.                              B. megaterium F7-1 used in this study was
These processes require significant energy and also                     recently isolated from a poultry waste in Korea
destroy certain amino acids (Papadoulos and                            (Son et al., 2004a). In order to examine the feather
Ketelaars, 1986). Therefore, biodegradation of                         degradation and keratinolytic enzyme production,
feather keratin by microorganisms represents an                        the cells were cultivated in the basal salts medium
alternative method to improve the nutritional                          containing 0.1% (w/v) chicken feathers unless
value of feather waste and to prevent environment                      otherwise indicated. For shaken culture in flasks,
contamination.                                                         50 ml of the medium was dispensed into each of
   Because of a high degree of cross-linking by                        250-ml Erlenmeyer flasks followed by inoculation
cysteine disulfide bonds, hydrogen bonding, and                         with 1 ml of B. megaterium F7-1 culture
hydrophobic interactions, keratin is insoluble and                     (2 Â 107 cells/ml) grown in nutrient broth (Difco)
not degradable by proteases such as trypsin,                           at 30 1C for 24 h. Cultivations were performed at
pepsin, and papain (Williams et al., 1990). Never-                     30 1C and 200 rpm for 5 days in a rotary shaker
theless, feathers do not accumulate in nature,                         unless otherwise stated. The cultures were cen-
since feather keratin can be degraded by keratino-                     trifuged at 12,000 rpm for 5 min, and the super-
lytic enzyme of some microorganisms (Onifade                           natant was used as a crude enzyme preparation.
et al., 1998). In this regard, keratinolytic enzymes                      The basal salts medium used contained the
may have important uses in biotechnological                            following (g/l): 0.5 NH4Cl, 0.5 NaCl, 0.1 K2HPO4,
processes involving keratin-containing wastes from                     0.2 KH2PO4, 0.1 MgCl2 Á 6H2O, and 0.1 yeast extract
poultry and leather industries through the devel-                      (Williams et al., 1990). The pH was adjusted to 7.5
opment of non-pollution processes. So far, only                        prior sterilization. Chicken feathers were obtained
some species of saprophytic and parasitic fungi,                       from a poultry-processing plant. They were washed
thermophilic actinomycetes, and Bacillus strains                       extensively with tap water and dried at 60 1C for
have been reported to be able to degrade feather                       72 h, and then kept at 4 1C until used.
keratin (Onifade et al., 1998). Most of these strains
have been isolated from poultry waste using                            Dimethyl sulfoxide soluble feather keratin
nutrient-rich medium and have been shown to                            preparation
degrade feathers at 50–60 1C. More recently, it was
reported that some bacterial strains, such as                             Soluble keratin was prepared from chicken
Bacillus sp. strain kr16 (Werlang and Brandelli,                       feathers by the method of Wawrzkiewicz et al.
2005), Chryseobacterium sp. strain kr6 (Riffel                         (1987). Native chicken feathers (10 g) suspended in
et al., 2003) and Vibrio sp. strain kr2 (Sangali and                   500 ml of dimethyl sulfoxide were solubilized by
Brandelli, 2000), degrade feathers at 30–37 1C.                        heat treatment in a reflux condenser at 100 1C for
However, there are few reports on the nutritional                      1 h. Soluble keratin was then precipitated by
conditions (carbon and nitrogen source require-                        addition of cold acetone (1 l) at À70 1C for 2 h
ments) that improve the feather degradation and                        followed by centrifugation at 12,000 rpm for
keratinolytic enzymes production by mesophilic                         10 min. The precipitate was washed twice with
feather-degrading microorganisms.                                      distilled water, and then suspended in 0.1 M
   Recently, we have reported on the isolation of                      phosphate buffer (pH 7.0) up to obtain a keratin
Bacillus megaterium F7-1, which is able to produce                     suspension 0.06% (w/v) of protein concentration.

    Please cite this article as: Park G-T, Son H-J. Keratinolytic activity of Bacillus megaterium F7-1, a feather-degrading mesophilic
    bacterium. Microbiol Res 2006; (2007), doi:10.1016/j.micres.2007.02.004
                                                 ARTICLE IN PRESS
Keratinolytic activity of Bacillus megaterium                                                                                         3

This keratin suspension was used as a substrate for
keratinoytic activity determination.

Keratinolytic activity assay and protein
determination

   Keratinolytic activity was assayed with soluble
keratin as a substrate, according to the method of
Wawrzkiewicz et al. (1987). An enzyme solution
(2 ml) was mixed with 3 ml of 0.06% (w/v) soluble
keratin in 0.1 M phosphate buffer (pH 7.5) and
incubated for 3 h at 30 1C. The reaction was
terminated by adding 1 ml of 10% (w/v) trichlor-
oacetic acid and centrifuged at 13,000 rpm for
10 min at 4 1C. The absorbance of the supernatant                   Figure 1. Feather degradation by B. megaterium F7-1
                                                                    after: (A) 0, (B) 2, (C) 3, and (D) 4 days.
was measured at 280 nm. One unit (U) of keratino-
lytic activity was defined as the amount of enzyme
that resulted in an increase in absorbance at
280 nm of 0.01 under the above conditions. The                      pointed that feather-degrading activity of mesophilic
protein concentration was determined by the                         organisms may be an interesting property for
Bradford (1976) method with bovine serum albumin                    biotechnological use because these microorganisms
as the standard.                                                    will be less energy consuming than the thermophilic
                                                                    ones. In this respect, B. megaterium F7-1 is another
                                                                    bacterial strain that can be used for the biodegrada-
Determination of bacterial growth and                               tion of poultry and abattoir wastes.
feather degradation

  Bacterial growth was determined by total plate
count on nutrient agar. Feather in cultures was                     Time course of feather degradation and
harvested by filtration with Whatman number 3                        keratinolytic activity in B. megaterium
filter paper, washed twice with distilled water and                  F7-1 culture medium
dried at 105 1C to constant weight. The percentage
of feather degradation was calculated from the                        The time course of feather degradation and
differences in residual feather dry weight between                  keratinolytic activity by B. megaterium F7-1
a control (feather without bacterial inoculation)                   culture grown in a basal salts medium containing
and treated sample.                                                 0.1% (w/v) chicken feather is shown in Fig. 2. The
                                                                    maximum keratinolytic activity of B. megaterium
                                                                    F7-1 was about 58 U/ml after 5 days of cultivation.
Results and discussion                                              Maximum enzyme activity was observed in the late
                                                                    logarithmic growth phase. Feather degradation by
   The degradation of whole chicken feather by B.                   B. megaterium F7-1 was accompanied by alkalini-
megaterium F7-1 is shown in Fig. 1. Partial degrada-                zation of the culture medium and by a pH elevation
tion was observed after incubation at 30 1C for 2 days.             from 7.5 to 8.8. This tendency to alkalinize the
Most of the feather was degraded after 4 days except                medium results from the production of ammonia by
the feather shaft, which was completely degraded                    means of the deamination of peptides and amino
after 7 days of cultivation. Williams et al. (1990)                 acids originating from keratin degradation. It has
demonstrated that Bacillus licheniformis PWD-1                      been stated that keratinolytic fungi often alkalinize
degraded chicken feather completely at 50 1C in 10                  culture media (Kaul and Sumbali, 1999).
        ¨
days. Bockle et al. (1995) reported that Streptomyces
pactum DSM40530 partially degraded native chicken                   Effect of culture temperature on
feather at 50 1C. On the other hand, it was reported                keratinolytic enzyme production
that some keratinolytic bacteria, such as Vibrio strain
kr2 (Sangali and Brandelli, 2000), Chryseobacterium                   To determine the optimum temperature for
strain kr6 (Riffel et al., 2003) and Bacillus strain kr16           keratinolytic enzyme production, cultures of B.
(Werlang and Brandelli, 2005), present maximum                      megaterium F7-1 in shake flask were carried out at
feather-degrading activity at 30–37 1C. These authors               different temperatures (10–45 1C). As shown in

 Please cite this article as: Park G-T, Son H-J. Keratinolytic activity of Bacillus megaterium F7-1, a feather-degrading mesophilic
 bacterium. Microbiol Res 2006; (2007), doi:10.1016/j.micres.2007.02.004
                                                                                                             ARTICLE IN PRESS
4                                                                                                                                                                                         G.-T. Park, H.-J. Son




                                                                                                                                                                                   Feather degradation (%) & Growth (CFU/ml, x 108)
                                                                                  100                                                                                100




                              Keratinolytic activity (U/ml) & Final pH (x 10-1)
                                                                                   80                                                                                80



                                                                                   60                                                                                60



                                                                                   40                                                                                40



                                                                                   20                                                                                20



                                                                                       0                                                                             0
                                                                                           0        1        2            3         4         5        6        7
                                                                                                                      Culture time (days)

Figure 2. Time course of feather degradation and keratinolytic enzyme production and B. megaterium F7-1 growth.
(K), keratinolytic activity; (’), feather degradation; (m), final pH; (J), growth.


                                                                                  70                                                                                 10

                                                                                  60
                                                                                                                                                                     8
                          Keratinolytic activity (U/ml)




                                                                                                                                                                           Growth (CFU/ml, x 109)
                                                                                  50

                                                                                                                                                                     6
                                                                                  40


                                                                                  30
                                                                                                                                                                     4

                                                                                  20
                                                                                                                                                                     2
                                                                                  10


                                                                                   0                                                                                 0
                                                                                       5       10       15       20      25      30      35       40       45   50
                                                                                                                      Temperature (°C)

Figure 3. Effect of temperature on keratinolytic enzyme production and B. megaterium F7-1 growth: (K),
keratinolytic activity; (’), growth.


Fig. 3, the keratinolytic enzyme production in-                                                                                  2000; Riffel et al., 2003). Contrarily, keratinolytic
creased gradually up to 30 1C. The highest kerati-                                                                               enzyme production by B. megaterium F7-1 occurred
nolytic enzyme production was obtainable between                                                                                 at a broader temperature range of 15–45 1C than
25 and 35 1C, at which keratinolytic activities were                                                                             Vibrio strain kr2 and Chryseobacterium strain kr6.
60 and 57 U/ml, respectively. No keratinolytic
enzyme production was observed at 10 and 45 1C
because of an absence of bacterial cell growth at                                                                                Effect of medium pH on keratinolytic
such temperatures. This strain had the ability to                                                                                enzyme production
grow from 15 to 40 1C and the optimum growth
temperature was determined to be 25–40 1C.                                                                                         The optimum pH for keratinolytic enzyme pro-
  Keratinase production of Vibrio strain kr2 and                                                                                 duction was determined by growing B. megaterium
Chryseobacterium strain kr6 was observed during                                                                                  F7-1 at pH 4.0–11.0 and 30 1C. As shown in Fig. 4,
cultivation in the range 25–37 1C, with an optimum                                                                               the keratinolytic enzyme production in B. mega-
at 30 and 25 1C, respectively (Sangali and Brandelli,                                                                            terium F7-1 was obtained at a broad initial pH of

    Please cite this article as: Park G-T, Son H-J. Keratinolytic activity of Bacillus megaterium F7-1, a feather-degrading mesophilic
    bacterium. Microbiol Res 2006; (2007), doi:10.1016/j.micres.2007.02.004
                                                                        ARTICLE IN PRESS
Keratinolytic activity of Bacillus megaterium                                                                                                    5

4.0–11.0, with an optimum at pH 6.5–11.0. The                                         slightly increased the enzyme production in the
enzyme activity was considerably smaller in the                                       feather medium. The addition of glycerol and
lower range of pH 4.0–5.5. B. megaterium F7-1 was                                     mannitol interfered with the enzyme induction by
able to grow over a wide range of pH 5.0–11.0.                                        feather. The concentration of glucose in the
Especially, cell growth at pH 7.0–11.0 was signifi-                                    medium did not appear to matter, since similar
cantly higher compared to pH 5.0 and 5.5. How-                                        results were obtained when the glucose concentra-
ever, poor growth was observed below pH 4.5.                                          tion was increased to either 0.25% or 0.5% (w/v)
   B. licheniformis PWD-1 exhibited best keratinase                                   (data not shown).
activity under neutral conditions (Wang and Shih,                                        Previous studies have shown that the synthesis of
1999), whereas Bacillus pumilis tended to do best                                     extracellular keratinases is constitutive or partially
in a weakly acidic environment (Kim et al., 2001).                                    inducible (Kanekar et al., 2002). Our studies on
Keratinase production of Vibrio strain kr2 and                                        keratinolytic enzyme production by B. megaterium
Chryseobacterium strain kr6 was observed at pH                                        F7-1 indicated that the major regulatory mechan-
5.0–8.0 (Sangali and Brandelli, 2000; Riffel et al.,                                  ism is feather substrate induction. Santos et al.
2003). Our results indicated that B. megaterium F7-                                   (1996) reported that the presence of glucose in the
1 is a novel alkali-tolerant bacterium, which can be                                  medium decreased the keratinolytic activity of
more efficiently applied to the alkaline condition.                                    Aspergillus fumigatus cultures. It has been also
                                                                                      reported that the presence of glucose decreases
                                                                                      the keratinase production of Streptomyces fradiae
Effect of culture medium composition on                                               (Shama and Berwick, 1991).
feather degradation and keratinolytic                                                    The effect of nitrogen sources on keratinolytic
enzyme production                                                                     enzyme production is also shown in Table 1. B.
                                                                                      megaterium F7-1 produced appreciable level of
   The influence of the addition of various carbon                                     keratinolytic enzyme when cultivated in a medium
sources to the basal salts medium with or without                                     containing feather as the sole nitrogen source.
feather supplement is shown in Table 1. In the                                        Additionally, beef extract, casein, gelatin, skim
absence of feather, B. megaterium F7-1 hardly                                         milk, tryptone, and yeast extract had a positive
produced kerainolytic enzyme in media containing                                      influence on enzyme production, resulting in 17%,
all carbon sources tested. However, feather greatly                                   26%, 12%, 36%, 29%, and 9% increases over the
increased the production of keratinolytic enzyme                                      control. On the other hand, urea and inorganic
compared to those obtained in the media without                                       nitrogens reduced enzyme production considerably,
feather supplements. This result suggests that                                        and corn steep liquor and polypeptone had no
chicken feather as the sole carbon and energy                                         effect. Such results were in accordance with
source supports keratinolytic enzyme production.                                      the previous findings of Malviya et al. (1992)
Additionally, fructose, galactose, and glucose                                         for Chrysosporium queenslandicum. However, our

                                                       70                                                           9

                                                                                                                    8
                                                       60
                                                                                                                    7
                       Keratinolytic activity (U/ml)




                                                                                                                        Growth (CFU/ml, x 109)




                                                       50
                                                                                                                    6

                                                       40                                                           5

                                                       30                                                           4

                                                                                                                    3
                                                       20
                                                                                                                    2
                                                       10
                                                                                                                    1

                                                        0                                                           0
                                                            3   4   5    6   7        8     9     10     11    12
                                                                                 pH

Figure 4. Effect of initial pH on keratinolytic enzyme production and B. megaterium F7-1 growth: (K), keratinolytic
activity; (’), growth.

 Please cite this article as: Park G-T, Son H-J. Keratinolytic activity of Bacillus megaterium F7-1, a feather-degrading mesophilic
 bacterium. Microbiol Res 2006; (2007), doi:10.1016/j.micres.2007.02.004
                                                    ARTICLE IN PRESS
6                                                                                                            G.-T. Park, H.-J. Son

Table 1. Effect of carbon and nitrogen sources on feather degradation and keratinolytic enzyme production by B.
megaterium F7-1

Carbon source        + Feather (0.1%, w/v)                  ÀFeather            Nitrogen           + Feather (0.1%, w/v)
(0.1%, w/v)                                                                     source
                     Keratinolytic       Feather            Keratinolytic       (0.02%, w/v)       Keratinolytic       Feather
                     activity            degradation        activity                               activity            degradation
                     (U/ml)              (%)                (U/ml)                                 (U/ml)              (%)

None                 62.8                70                 0.0                 None               54.8                69
Fructose             66.2                71                 2.3                 Beef extract       64.3                81
Galactose            66.4                72                 1.9                 Casamino acid      48.9                61
Glucose              67.4                74                 2.4                 Casein             68.9                74
Glycerol             45.5                35                 1.8                 Corn steep         54.2                72
                                                                                liquor
Lactose              63.7                67                 1.9                 Gelatin            61.3                78
Maltose              63.1                65                 1.8                 Malt extract       39.4                48
Mannitol             53.4                61                 1.7                 Polypeptone        55.9                65
Rhamnose             64.6                69                 1.5                 Skim milk          74.5                87
Sorbitol             65.3                72                 1.7                 Tryptone           70.6                83
Sucrose              64.0                67                 1.8                 Yeast extract      59.8                70
                                                                                Urea               33.6                61
                                                                                (NH4)2SO4          28.2                28
                                                                                NH4Cl              25.4                30
                                                                                NH4NO3             30.7                38
                                                                                KNO3               27.6                41
                                                                                NaNO2               2.1                 7
                                                                                NaNO3              23.1                28




results contrasted with that reported for                              applicable in the preparation of feed additives to
B. licheniformis PWD-1, in which NH4Cl encouraged                      improve the digestibility of feather meal in the
growth and keratinase production (Williams et al.,                     poultry farm, than in the treatment of feathers as
1990).                                                                 environmental pollutants.
   Figure 5 shows that keratinolytic enzyme produc-                       The effect of different chicken feather concen-
tion increased with skim concentrations from 0% to                     trations on keratinolytic enzyme production is
0.6% (w/v), and then stayed at about the same                          shown in Fig. 6. The amount of keratinolytic
level. In a medium containing 0.6% (w/v) skim milk,                    enzyme production depended on feather concen-
enzyme production by B. megaterium F7-1 was                            trations. Keratinolytic enzyme production in-
468 U/ml, which was about 9.4 times higher than                        creased as the amount of feather increased
that without skim milk (50 U/ml). Thus, the 0.6%                       (0.1–1.6%, w/v); but when the concentration was
(w/v) was the concentration selected for the                           raised to 1.8% (w/v), enzyme production slightly
following experiments.                                                 decreased. The highest enzyme production was
   The degradation of autoclaved and untreated                         obtained at 1.6% (w/v) feather. Cheng et al. (1995)
chicken feathers by cell-free culture supernatants                     reported that 1% (w/v) feather powder gave the
was studied. A 20 ml of cell-free culture super-                       highest keratinase activity for B. licheniformis
natant, containing 460 U/ml of keratinolytic activ-                    PWD-1. Similar result was also reported for Bacillus
ity, was incubated with 40 ml of 0.1% (w/v) chicken                    sp. FK46 (Suntornsuk and Suntornsuk, 2003). The
feathers in 0.1 M phosphate buffer (pH 7.5). The                       latter authors reported that higher concentrations
mixture was then shaken for 6, 12, 18, 24, 30 and                      (3% and 5%, w/v) may cause substrate inhibition or
36 h at 30 1C and 200 rpm. The degradation of                          repression of keratinase production. On the other
autoclaved chicken feathers by the culture super-                      hand, high feather concentration increased med-
natant was 26% after 24 h of incubation (data not                      ium viscosity which possibly resulting in oxygen
shown). Even incubation over 24 h, no further                          limitation for bacterial growth (data not shown).
degradation was achieved. The degradation of                              The keratin degradation capability of B. megater-
untreated chicken feathers was unsuccessful.                           ium F7-1 was also investigated using feather meal,
These results indicated that the cell-free culture                     duck feather, human hair, human nail and sheep wool
supernatant from B. megaterium F7-1 may be more                        as substrate. All keratin substrates except feather

    Please cite this article as: Park G-T, Son H-J. Keratinolytic activity of Bacillus megaterium F7-1, a feather-degrading mesophilic
    bacterium. Microbiol Res 2006; (2007), doi:10.1016/j.micres.2007.02.004
                                                                                        ARTICLE IN PRESS
Keratinolytic activity of Bacillus megaterium                                                                                                                                    7

                                                             500                                                                            100



                                                             400                                                                            80




                             Keratinolytic activity (U/ml)




                                                                                                                                                       Feather degradation (%)
                                                             300                                                                            60



                                                             200                                                                            40



                                                             100                                                                            20



                                                               0                                                                            0
                                                                   0.0    0.1     0.2      0.3     0.4    0.5     0.6     0.7     0.8    0.9
                                                                                          Skim millk concentration (%)

Figure 5. Effect of skim milk concentration on feather degradation and keratinolytic enzyme production by B.
megaterium F7-1: (K), keratinolytic activity; (’), feather degradation.


                                                             700                                                                           100

                                                             600
                                                                                                                                           80
                       Keratinolytic activity (U/ml)




                                                                                                                                                  Feather degradation (%)
                                                             500

                                                                                                                                           60
                                                             400

                                                             300
                                                                                                                                           40

                                                             200
                                                                                                                                           20
                                                             100

                                                               0                                                                            0
                                                                   0.0   0.2    0.4     0.6     0.8 1.0 1.2 1.4 1.6         1.8    2.0   2.2
                                                                                              Feather concentration (%)

Figure 6. Effect of feather concentration on feather degradation and keratinolytic enzyme production by B.
megaterium F7-1: (K), keratinolytic activity; (’), feather degradation.


meal were cut into fragments (0.5 cm long) and                                                            (Bressollier et al., 1999). Our strain is also compar-
washed with distilled water. Each flasks contained                                                         able to S. albidoflavus which could degrade only 10%
50 ml medium and were inoculated with 1 ml of B.                                                          of hair keratin (Bressollier et al., 1999), and
megaterium F7-1 culture (2 Â 107 cells/ml) to which                                                       Thermoactinomyces candidus which could degrade
0.1% (w/v) of a selected keratin was added. The                                                           11% of sheep wool (Ignatova et al., 1999). On the
keratin substrates revealed different degradation                                                         other hand, Atalo and Gashe (1993) reported that
rates by B. megaterium F7-1 (data not shown). Of                                                          thermophilic Bacillus sp. PA-001A hydrolyzed 90%,
the five keratin substrates used, feather meal was                                                         60% and 50% of the sheep skin, feather and horn used
completely degraded (100%) after 10 days followed                                                         in the medium, and hair was supported poor growth.
by duck feather (86%) and human nail (28%). Human                                                            Since the keratinolytic enzyme assay methods
hair (18%) and sheep wool (19%) showed relatively                                                         and growth media are different, it is difficult to
low degradation rate. The degradation rate of                                                             compare the enzyme productivity of our result with
feather meal by B. megaterium F7-1 was higher                                                             those in the literature directly from the activity
than that by Streptomyces albidoflavus (67%)                                                               level in fermentation broth. However, the results

 Please cite this article as: Park G-T, Son H-J. Keratinolytic activity of Bacillus megaterium F7-1, a feather-degrading mesophilic
 bacterium. Microbiol Res 2006; (2007), doi:10.1016/j.micres.2007.02.004
                                                    ARTICLE IN PRESS
8                                                                                                            G.-T. Park, H.-J. Son

presented here clearly indicate the strong kerati-                     Kim JM, Lim WJ, Suh HJ. Feather-degrading Bacillus
nolytic activity of B. megaterium F7-1 upon chicken                       species from poultry waste. Process Biochem
feather as the keratin source. B. megaterium F7-1                         2001;37:287–91.
shows potential for biotechnological use because it                    Malviya HK, Rajak RC, Hasija SK. Synthesis and regulation
completely degrades chicken feather after 7 days                          of extracellular keratinase in three fungi isolated from
                                                                          the grounds of a gelatin factory, Jabalpur, India.
of cultivation. Further studies on the purification,
                                                                          Mycophathologia 1992;120:1–4.
biochemical characterization, and molecular biol-                      Manczinger L, Rozs M, Vagvolgyi Cs, Kevei F. Isolation and
ogy studies of the genes encoding keratinolytic                           characterization of a new keratinolytic Bacillus
enzyme(s) will be needed to eventually improve                            licheniformis strain. World J Microbiol Biotechnol
the keratin-degrading capacity of B. megaterium                           2003;19:35–9.
F7-1. We are now investigating the purification and                     Onifade AA, Al-Sane NA, Al-Musallam AA, Al-Zarban S.
properties of keratinolytic enzyme from B. mega-                          Potentials for biotechnological applications of keratin-
terium F7-1.                                                              degrading microorganisms and their enzymes for
                                                                          nutritional improvement of feathers and other kera-
                                                                          tins as livestock feed resources. Biores Technol
                                                                          1998;66:1–11.
Acknowledgments                                                        Papadoulos MC, Ketelaars EH. Effects of processing time
                                                                          and moisture content on amino acid composition and
                                                                          nitrogen characteristics of feather meal. Anim Feed
  This work was supported by 2006 Joint Research
                                                                          Sci Technol 1986;14:279–90.
Center of PNU-Fraunhofer IGB Grant of Pusan                            Riffel A, Lucas F, Heeb P, Brandelli A. Characterization of
National University.                                                      a new keratinolytic bacterium that completely de-
                                                                          grades native feather keratin. Arch Microbiol
                                                                          2003;179:258–65.
                                                                       Sangali S, Brandelli A. Feather keratin hydrolysis by a
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    Please cite this article as: Park G-T, Son H-J. Keratinolytic activity of Bacillus megaterium F7-1, a feather-degrading mesophilic
    bacterium. Microbiol Res 2006; (2007), doi:10.1016/j.micres.2007.02.004

				
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Mary Bernadette Vallesfin Egloso Mary Bernadette Vallesfin Egloso English Teacher http://adelaide17madette.multiply.com/
About My friends call me Addie. I want to become a doctor someday and serve my countrymen after studying medicine in the Philippines. I also want to become a sophisticated investor and business owner someday. I truly believe in what Robert Kiyosaki said in his books. It is very important to keep on improving oneself, as we live in this dynamic and competitive world. I love swimming and singing.