www.sospublication.co.

in Journal of Advanced Laboratory Research in Biology

We- together to save yourself society e-ISSN 0976-7614

Volume 3, Issue 3, July 2012 Research Article

Optimization of Extracellular Keratinase Production by Aspergillus terreus Isolated from

Chicken's Litter

Mostafa Koutb1,2*, Fatthy Mohamed Morsy1, Magdy Mohamed Khalil Bagy1 and Elhagag Ahmed Hassan1
1*
Botany Department, Faculty of Science, Assiut University, Assiut-71516, Egypt.
2
Umm Al-Qura University, Faculty of Applied Science, Biology Department, Mecca, Saudi Arabia.

Abstract: In this current study 45 fungal isolates were isolated from chicken's litter on Feather Agar Medium
(FAM) were screened for determining the potent keratinase producing isolates. Out of these fungal isolates, twelve
species and one species variety exhibited various degrees of keratinolytic activities from which A. terreus showed
the highest keratinase production (12.6U/ml). The optimum temperature and initial pH for keratinase production by
A. terreus were 40°C and 8, respectively. The highest keratinase production was observed for a period 25 days. The
optimum ionic strength for the enzyme production was 80mM NaCl. Deprivation of K +, Fe2+, Mg2+, Ca2+ or Zn2+
from the culture medium drastically reduced the keratinase production by A. terreus. In contrast, sulfur deprivation
did not significantly affect the keratinase production. The Km and Vmax values for A. terreus keratinase were 8.64mg
keratin and 56.7U/mg proteins, respectively. The optimum temperature, pH and ionic strength for keratinase activity
were 35°C, 7.8 and 80-100mM NaCl, respectively.

Keywords: Aspergillus terreus, Chicken's litter, Fungi, Keratinase production.

1. Introduction [16]. The objective of this study was to select highly

keratinase producing non-dermatophytic fungi isolated
Keratinases (E.C. No. 3.4.99.11), a group of from chicken’s litter and further characterization of
proteinases, are important for hydrolyzing feather, hair, keratinase with optimizing its production process by the
wool, collagen and casein to clean obstructions in the most potent keratinase producing fungal isolate.
sewage system during wastewater treatment [1]. These
enzymes are also used or could be applied in the food 2. Materials and Methods
industry, textiles, medicine, cosmetics, leather and
poultry processing industry [2, 3]. 2.1 Isolation of keratinolytic fungi from chicken's
Keratinases are produced only in the presence of litter
keratin-containing substrates. Microbial keratinases are The dilution-plate method (Johnson et al., 1959)
predominantly extracellular; however, a few cells- was used to isolate the keratinolytic fungi on Feather
bound and intracellular keratinases have also been Agar Medium (FAM). The composition of (FAM)
reported [4-8]. Among non-dermatophytic fungi, medium (g/l) is: Agar, 15.0; Feather powder (defatted
keratinases showing attractive biochemical properties with chloroform-methanol 1:1 (V/V) over 24 h, ground
are Aspergillus [9, 10], Trichoderma [11], Doratomyces and sifted through a 0.2-mm screen.), 10.0; K2HPO4,
[12], Myrothecium [13], Paecilomyces [14], 1.5; MgSO4.7H2O, 0.025; CaCl2, 0.025; FeSO4.7H2O,
Scopulariopsis [15] and also Acremonium, Alternaria, 0.015; ZnSO4.7H2O, 0.005; Actidione, 0.5;
Beauveria, Curvularia, and Penicillium [16]. Besides Chloramphenicol, 0.25;Volume was adjusted to 1000ml
the biotechnological interest, these investigations may with distilled water and pH was adjusted to 6.5. The
help in understanding the role of fungi in the growing colonies were isolated by aseptic transfer to
degradation of complex keratinous substrates in nature Sabouraud dextrose agar medium (SDA) and incubated
*Corresponding author:
E-mail: [email protected]; Phone: +9660597029270.
Optimization of Extracellular Keratinase by A. terreus Isolated from Chicken's Litter Koutb et al

at 30°C. Fungal growth was identified based on the determined after two weeks of inoculation. A. terreus
macroscopic aspect of colonies and micro- was grown in BSM medium as control and the same
morphological examinations. medium deprived of Mg2+, K+, Ca2+, Zn2+or Fe2+ at both
optimum temperature and initial pH. After two weeks
2.2 Screening for extracellular keratinase the yield of keratinase produced was determined.
production
Extracellular keratinase production was tested in 2.6 Kinetics of A. terreus keratinase
45 fungal isolates on basal salt medium (BSM) The Km value of keratinase for keratin substrate
containing (g/l): K2HPO4, 1.5; MgSO4.7H2O, 0.025; was determined using Lineweaver–Burk plots of
CaCl2, 0.025; FeSO4.7H2O, 0.015 and ZnSO4.7H2O, various keratin concentrations against keratinase
0.005. Defatted white chicken's feather powder (50mg) activity.
was added to each flask at initial pH 7.5. Media were
inoculated with a spore suspension obtained from 10 2.7 The ionic strength, temperature and pH-
days old cultures of each fungal isolate under dependency of the keratinase activity
investigation and incubated for 15 days at 30°C. The The pH-dependency of the keratinase activity was
filtrate containing extracellular keratinase was examined in a pH range from 2.3 to 10. Different buffer
centrifuged at 10000 x g for 15 min at 4ºC. The systems were used in accordance with the respective pH
supernatant containing keratinase was analyzed. The ranges: 50mM Glycine–HCl buffer for the pH range
screened fungal isolates were ordered into 4 groups from 2.3 to 5, 50mM citrate buffer for the pH range
according to keratinase production. The fungal isolates from 6 to 6.6, 50mM Tris–HCl buffer for the pH range
producing (more than 10U/ml) designed as high (H) from 7 to 9 and 50mM Glycine-NaOH buffer for the pH
producers, (6-10U/ml) as moderate (M) producers, range from 9 to 10. The Ionic strength dependency of
(1-6U/ml) as low (L) producers and (less than 1U/ml) the keratinase activity was examined at different NaCl
as non producing). Extracellular keratinase production concentrations of 0 to 2M NaCl in the reaction mixture.
was expressed in a number of enzyme units per ml. The optimum temperature for the keratinase activity
was determined in a temperature range of 20–50°C
2.3 Assay for keratinase activity using the buffer system 50mM Tris–HCl (pH 7.8 at
Keratinase activity was measured as described by ambient temperature).
Yu et al., [17] with some modifications. The reaction
mixture contained 50mM Tris–HCl buffer (pH 7.8), 2.7 Scanning electron microscopy (SEM)
20mg keratin powder and 1ml of fungal filtrate To determine the degree of degradation of feather,
containing crude extracellular keratinase in a total samples from 7 days culture were freeze-dried,
volume of 4ml. The reaction was left for 4 h at 37°C incubated in 5% cold buffered glutaraldehyde for two
with shaking and subsequently, the released amino days at room temperature. The samples were washed
acids from keratin proteolysis were measured by the with sodium cacodylate buffer for three times (30
ninhydrin method as described by Muting and Kaiser minutes each) and postfixed in 1% osmium tetroxide
[18]. One unit of activity was defined as the amount of for two hours. Then, the samples were washed in the
enzyme that catalyzes the release of 1μg amino acids same buffer for three times (30 minutes each) and
per hour. Keratinase specific activity was defined as the dehydration by using an ascending grade of ethanol 30,
number of keratinase units per mg extracellular protein. 50, 70, 90% for two hours and 100% for two days and
The total extracellular protein produced by the in amyl acetate for two days. After that, the samples
keratinase producing fungi was measured in were dried in the critical point drainer using liquid
extracellular crude enzyme as described by Bradford carbon dioxide, and each sample stickled on a metallic
[19]. block by using silver paint. In Gold Sputter Apparatus,
the samples were evenly gold coated in thickness of
2.4 Optimization of keratinase production 15nm. By JEOL JSM 5300 Lv scanning electron
A. terreus was selected for further studies on microscope, the samples then were examined at 15 kV
optimization (incubation temperature, period, initial and photographed.
pH) of keratinase production and kinetics of keratinase
activity. 3. Results and Discussion

2.5 Ionic strength and metal ions dependency 3.1 Isolation and screening of keratinase producing
The Ionic strength dependency of the keratinase fungi
production was examined at different salt Results in the keratinase production of fungi in
concentrations ranging from 0.0 to 2M NaCl in the submerged culture are shown in Table (1).
BSM culture medium. The keratinase yield was

J. Adv. Lab. Res. Biol. 211

Optimization of Extracellular Keratinase by A. terreus Isolated from Chicken's Litter Koutb et al

Table 1. Keratinase production and specific activity of fungal isolates from chicken litter.

Test Visual Extracellular keratinase Extracellular Specific activity

Fungal species Growth production U/ml protein mg/ml U/mg protein
Acremonium strictum + 5.38L 0.19 27.08
Aspergillus flavus + 7.35M 0.18 41.13
A. flavus var. columnaris + 6.48M 0.18 36.52
A. fumigatus + 7.12M 0.26 27.05
A. niger + 7.81M 0.19 41.21
A. terreus + 12.60 H 0.30 42.27
Chrysosporium keratinophilum + 7.88M 0.12 63.90
C. tropicum + 11.63H 0.14 80.68
P. funiculosum + 5.99L 0.23 26.37
Scopulariopsis brevicaulis + 10.35H 0.21 47.82
S. brumptii + 5.82L 0.18 31.78
Trichoderma harzianum + 4.90L 0.17 29.22
T. viridi + 5.91L 0.22 26.95
The keratinase production was measured after two weeks of inoculation of fungi; H= High production of keratinase (more than 10U/ml);
M= Moderate production of keratinase (6-10U/ml); L= Low production of keratinase (less than 6U/ml).

Twelve species and one species variety out of 45 3.2 Optimization of keratinase production
fungal isolates exhibited various degrees of Keratinase production by A. terreus was optimized.
keratinolytic activities when cultivated onto chicken Aspergillus terreus showed an optimum pH range from
feather liquid medium and 32 fungal isolates showed no 7-8 for keratinase production (Fig. 1A). The maximum
keratinolytic activity (data not shown). Three out of the enzyme production was recorded at 40○C, whereas
13 keratinase producing fungal isolates were considered below or above this temperature the keratinase
as highly keratinase productive. (First group) The production declined (Fig. 1B). The effect of different
highest keratinase activity was reached with A. terreus incubation periods of keratinase production was
followed by Chrysosporium tropicum and investigated (Fig. 2A).
Scopulariopsis brevicaulis. The second group contains
moderate producers for keratinases such as Aspergillus
flavus, A. flavus var. columnaris, A. niger and
Chrysosporium keratinophilum. The remaining fungal
isolates have low productivity of keratinase as shown in
Table (1). Aspergillus terreus showed the highest
keratinase productivity (12.6U/ml) with a specific
activity (42.27U/mg proteins) and thus, it was selected
for further optimization and kinetics experiments.
It can be concluded that keratinolytic activity is
relatively widespread among common fungi. Many
fungi excrete enzymes into the environment and can be
used as producers of keratinolytic enzymes. A. terreus
showed the highest keratinase productivity (12.6U/ml). Fig. 1(a). Effect of pH on keratinase production by Aspergillus
Keratinases of non-dermatophytes are of particular terreus. The experiments were repeated three times and mean
interest because of safety in fungal handling and values and standard errors are shown.
application. In this study, five potent keratinase
producing non-dermatophytic fungi were isolated. In
spite of its wide applications in the breakdown of
keratinous wastes, keratinases of dermatophytes are
dangerous to use in its crude form due to contamination
of the crude enzyme with spores of such dangerous
fungi. In contrast to dermatophytes, the crude extracts
of keratinases isolated from non-dermatophytic fungi
are completely safe to use without the expensive and
laborious purification processes. It has been suggested
that screening for non-pathogenic microorganisms with
keratinolytic activity may prevent the need for isolation
and purification of the enzymes [20]. It is worth to
mention that all fungi isolated in this study from Fig. 1(b). Incubation temperature on keratinase production by
chicken litter are non-dermatophytes. Aspergillus terreus. The experiments were repeated three times
and mean values and standard errors are shown.

J. Adv. Lab. Res. Biol. 212

Optimization of Extracellular Keratinase by A. terreus Isolated from Chicken's Litter Koutb et al

Ca2+ and Mg2+ have been reported to cause a 3-fold

increase in the enzymatic activity [23]. These metal
ions might be associated with the stabilization of the
tertiary structure conformation of metalloproteases and
protect these enzymes against autoproteolysis [24].

Fig. 2(a). Effect of incubation period on keratinase production by

Aspergillus terreus. The experiments were repeated three times
and mean values and standard errors are shown.

Keratinase production was detected after 2 days of

incubation. The maximum productivity was detected
after 25 days after which the production declined. The Fig. 2(b). Effect of Ionic strength on keratinase production by
optimum Ionic strength for keratinase production was Aspergillus terreus. The experiments were repeated three times
recorded at 80mM NaCl, and then sharply declined and mean values and standard errors are shown.
with increasing NaCl concentration in the culture
medium (Fig. 2B). The effect of the basal salt medium
constituents on keratinase production was studied by
depriving the medium from each one of constituents
separately. Deprivation of Ca, Mg, Fe, K+ or Zn2+
strongly affected the keratinase production, indicating
that these elements are essential for the keratinase
production. Sulfur deprivation did not significantly
affect keratinase production (Fig. 2C). In the present
study, the optimum production of keratinase enzyme(s)
from A. terreus was recorded within incubation period
25 days. The production of keratinase was
proportionally increased with the incubation time up to
25 days, after that the production of keratinase Fig. 2(c). Effect of metal ions deprivation on keratinase production
decreased. The optimum production of keratinase by Aspergillus terreus. The experiments were repeated three times
enzyme(s) was recorded within an incubation and mean values and standard errors are shown.
temperature of 35○C. The optimum pH ranges from 7-8
for keratinase production, indicating that the fungus 3.3 Kinetics of A. terreus extracellular keratinase
productivity is not affected by the increase in pH value The Km and Vmax values for keratinase of A. terreus
of the medium due to ammonium release as reported by keratinase were determined using Lineweaver–Burk
Gupta & Ramnani [21]. The optimum concentration of plots. The Km and Vmax values for A. terreus keratinase
NaCl for keratinase production by A. terreus is 80 to were 8.64mg keratin and 56.7U/mg proteins,
100mM NaCl and then declined with increasing NaCl respectively (Fig. 3). The optimum pH for keratinase
concentration in the culture medium. Keratin activity was 7.8 (Fig. 4A). A. terreus keratinase showed
degradation processes a large amount of sulfur- an optimum temperature at 35-40○C (Fig. 4B). The
containing amino acids [22]. Keratin proteolysis optimum Ionic strength for A. terreus keratinase
releases sulfur-containing amino acids and sulfhydryl activity was 80-100mM NaCl. The keratinase activity
groups [21]. Sulfur deprivation did not significantly sharply decreased in Ionic strength higher than 100Mm
affect the keratinase productivity by A. terreus, possibly NaCl (Fig. 4C).
because of the release of sulfur-containing amino acids Although, increasing the enzyme specific activity
and sulfhydryl groups from keratin degradation which by purification to homogeneity, is very useful for
might provide the fungus with its sulfur requirements laboratory studies. There is no need for such
for growth and keratinase production. Zn 2+, k+ Ca2+, purification when the target is to use the enzyme in
Mg2+ or Fe2+ deprivation strongly reduced keratinase commercial applications. That is because the high
productivity. These results indicate that these cations specific activity of the expensive purified enzyme will
play an important role in the keratinase productivity and be lost again when mixing the enzyme with the crude
possible regulation of enzyme active conformation. keratin wastes in commercial applications. In addition,
a large amount of the enzyme is always lost with

J. Adv. Lab. Res. Biol. 213

Optimization of Extracellular Keratinase by A. terreus Isolated from Chicken's Litter Koutb et al

various degrees depending on the purification optimum pH for A. terreus keratinase activity was (7.8).
protocols. In contrast keratinase of Trichophyton schoenleinii and
Trichophyton mentagrophytes var. erinacei showed an
optimum pH 5.5 [25].

Fig. 3. Lineweaver–Burk plots of various keratin concentrations

against Aspergillus terreus keratinase activity.
Fig. 4(c). Effect of Ionic strength on keratinase activity by
Aspergillus terreus. The experiments were repeated three times
and mean values and standard errors are shown.

In agreement with our results, Moallae et al., [26]

reported that keratinase activity from Trichophyton
vanbruseghemii was optimum at pH 8. Similarly, the
optimum pH for A. oryzae was 8 [10]. The optimum
temperature for A. terreus keratinase were 35-40ºC.
Keratinases of Scopulariopsis brevicaulis and
Trichophyton sp. HA-2 showed similar optimum
temperature 35-40ºC [27, 28]. In contrast, the optimum
temperature for A. oryzae was 50ºC [10]. The optimum
Fig. 4(a). Effect of pH on keratinase activity by Aspergillus terreus. Ionic strength for keratinase activity was 80-100mM
The experiments were repeated three times and mean values and NaCl. Higher NaCl concentrations sharply reduced the
standard errors are shown. keratinase activity possibly by inhibiting its interaction
with keratin substrate. Accordingly, the NaCl
concentration higher than 100mM sharply reduced both
growth and keratinase production by A. terreus.

Fig. 4(b). Effect of temperature on keratinase activity by Aspergillus

terreus. The experiments were repeated three times and mean
values and standard errors are shown.

The Km and Vmax values for A. terreus crude

extracellular keratinase were evaluated from a
Lineweaver-Burk plot and found to be 8.64mg keratin
and 56.7U/mg protein, respectively, indicating a high
purity of the enzyme. The Km and Vmax values of the
pure enzyme of A. oryzae were found to be 8.47mg/ml Fig. 5(a). Scanning electron micrograph of feather degradation by
and 71.43U/ml, respectively [10]. The properties of A. terreus. colonization of fungal mycelia on feather surface.
microbial degrading enzymes appear to differ according
to the producing species of microorganism. The

J. Adv. Lab. Res. Biol. 214

Optimization of Extracellular Keratinase by A. terreus Isolated from Chicken's Litter Koutb et al

3.4 Scanning electron microscope References

Scanning electron micrograph of feather
degradation by A. terreus showed that most of the [1]. Godfrey, T. (1996). Protease in wastes treatment.
feathers were degraded after 7 days. The shaft of In: Industrial Enzymology, ed. Godfrey, T. and
feathers with barbs (Fig. 5A) could be clearly observed West, S. pp. 315–316. London: Macmillan
in SEM micrographs of uninoculated feathers. A Press Ltd.
considerable degradation of feather shaft and barbs was [2]. Birch, G.G., Parler, K.J. & Worgan, J.T. (1976).
observed and fungal mycelia aggregate with an Food from waste. In: Enzyme and food
extracellular matrix to degrade the surface (Fig. 5B, C). processing. London: Applied Science, pp. 19–65.
[3]. Chessen, A. (1990). Improving the nutritional
value of feeds for pigs and poultry with enzyme
supplement-current benefits and future prospects.
In: Enzymes in der Tierernahrung. pp. 25–37.
Zurich: Institut fur Nutzlierwissenchaften.
[4]. Friedrich, A.B. & Antranikian, G. (1996). Keratin
degradation by Fervidobacterium pennavorans, a
novel thermophilic anaerobic species of the order
thermotogales. Appl. Environ. Microbiol., 62:
2875–2882.
[5]. El-Naghy, M.A., El-Ktatny, M.S., Fadl-Allah,
E.M. & Nazeer, W.W. (1998). Degradation of
chicken feathers by Chrysosporium georgiae.
Mycopath., 143: 77–84.
[6]. Onifade, A.A., Al-Sane, N.A., Al-Musallam, A.A.
& Al-Zarban, S. (1998). A review: potentials for
biotechnological applications of keratin-degrading
microorganisms and their enzymes for nutritional
Fig. 5(b). Scanning electron micrograph of uninoculated chicken improvement of feathers and other keratins as
feather colonization of fungal mycelia on feather surface. livestock feed resources. Bioresour. Technol., 66:
1–11.
[7]. Riessen, S. & Antranikian, G. (2001). Isolation of
Thermoanaerobacter keratinophilus sp. nov., a
novel thermophilic, anaerobic bacterium with
keratinolytic activity. Extremoph., 5: 399–408.
[8]. Nam, G.W., Lee, D.W., Lee, H.S., Lee, N.J., Kim,
B.C., Choe, E.A., Hwang, J.K., Suhartono, M.T.
& Pyun, Y.R. (2002). Native feather degradation
by Fervidobacterium islandicum AW-1, a newly
isolated keratinase producing thermophilic
anaerobe. Arch. Microbiol., 178: 538–547.
[9]. Santos, R.M.D.B., Firmino, A.A.P., de Sa, C.M.
& Felix, C.R. (1996). Keratinolytic activity of
Aspergillus fumigatus Fresenius. Curt. Microbiol.,
33: 364-370.
[10]. Farag, A.M. & Hassan, M.A. (2004). Purification,
characterization and immobilization of a
Fig. 5(c). Scanning electron micrograph of degradation of chicken keratinase from Aspergillus oryzae. Enz. Microb.
feathers after 7 days and colonization of fungal mycelia on feather Technol., 34: 85–93.
surface. [11]. Cao, L., Tan, H., Liu, Y., Xue, X. & Zhou, S.
(2008). Characterization of a new keratinolytic
4. Conclusion Trichoderma atroviride strain F6 that completely
degrades native chicken feather. Lett. Appl.
Aspergillus terreus was the best keratinase Microbiol., 46: 389–394.
producing fungus in our screening. The optimization of [12]. Gradisar, H., Kern, S. & Friedrich, J. (2000).
keratinase production and its characterization showed Keratinase of Doratomyces microsporus. Appl.
potential characteristics for its possible application in Microbiol. Biotechnol., 53: 196–200.
commercial keratin degradation. [13]. Moreira-Gasparin, F.G., de Souza, C.G., Costa,
A.M., Alexandrino, A.M., Bracht, C.K., Boer,

J. Adv. Lab. Res. Biol. 215

Optimization of Extracellular Keratinase by A. terreus Isolated from Chicken's Litter Koutb et al

C.G., Peralta, R.M. (2009). Purification and recycling poultry feathers as a feed ingredient.
characterization of an efficient poultry feather Biores. Technol., 96: 1703-1708.
degrading-protease from Myrothecium verrucaria. [21]. Gupta, R. & Ramnani, P. (2006). Microbial
Biodegrad., 20: 727–736. keratinases and their prospective applications: an
[14]. Gradisar, H., Friedrich, J., Krizaj, I. & Jerala, R. Overview. Appl. Microb. Biotechnol., 70: 21–33.
(2005). Similarities and specificities of fungal [22]. Veselá, M. & Friedrich, J. (2009). Amino Acid
keratinolytic proteases: comparison of keratinases and Soluble Protein Cocktail from Waste Keratin
of Paecilomyces marquandii and Doratomyces Hydrolysed by a Fungal Keratinase of
microsporus to some known proteases. Appl. Paecilomyces marquandii. Biotechnol. Bioproc.
Environ. Microbiol., 71: 3420–3426. Engin., 14: 84- 90.
[15]. Anbu, P., Gopinath, S.C.B., Hilda, A., Lakshmi, [23]. Venter, H., Osthoff, G. & Litthauer, D. (1999).
P.T. & Annadurai, G. (2005). Purification of Purification and characterization of a
keratinase from poultry farm isolate- metalloprotease from Chryseobacterium
Scopulariopsis brevicaulis and statistical indologenes Ix9a and determination of the amino
optimization of enzyme activity. Enz. Microbiol. acid specificity with electrospray mass
Technol., 36: 639-647. spectrometry. Protein Expr. Purif., 15: 282–295.
[16]. Marcondes, N.R., Taira, C.L., Vandresen, D.C., [24]. Kumar, C.G. & Takagi, H. (1999). Microbial
Svidzinski, T.I., Kadowaki, M.K. & Peralta, R.M. alkaline proteases: From a bioindustrial
(2008). New feather-degrading filamentous fungi. viewpoint. Biotechnol. Adv., 17: 561–594.
Microb. Ecol., 56: 13-17. [25]. Qin, L.M., Dekio, S., Jidoi, J. (1992). Some
[17]. Yu, R.J., Harmon, S.R. & Blank, F. (1968). biochemical characteristics of a partially purified
Isolation and purification of an extracellular extracellular keratinase from Trichophyton
keratinase of Trichophyton mentagrophytes. J. schoenleinii. Zentralbl Bakteriol., 277:236–244.
Bacteriol., 96: 1435-1436. [26]. Moallaei, H., Zaini, F., Larcher, G. Beucher, B.
[18]. Muting, D. & Kaiser, E. (1963). & Bouchara, J.P. (2006). Partial purification and
Spectrophotometric method of determining of α- characterization of a 37 kDa extracellular
amino-N in biological materials by means of the proteinase from Trichophyton vanbreuseghemii.
ninhydrin reaction. Hoppe-Seyler's Zeitschrift für Mycopath., 161: 369-375.
Physiologische Chemie, 332:276–289. [27]. Malviya, H.K., Rajak, R.C. & Hasija, S.K. (1992).
[19]. Bradford, M.M. (1976). A rapid and sensitive Purification and partial characterization of two
method for the quantitation of microgram extracellular keratinases of Scopulariopsis
quantities of protein utilizing the principle of brevicaulis. Mycopath., 119: 161–165.
protein-dye binding. Analyt. Biochem., 72: 248- [28]. Anbu, P., Hilda, A., Sur, H., Hur, B. & Jayanthi,
254. S. (2008). Extracellular keratinase from
[20]. Bertsch, A. & Coello, N. (2005). A Trichophyton sp. HA-2 isolated from feather
biotechnological process for treatment and dumping soil. Intern. Biodeterior. Biodegrad., 62:
287-292.

J. Adv. Lab. Res. Biol. 216