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VSRD-TNTJ, Vol. 2 (3), 2011, 128-136

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1
Assistant Professor,
23
UG Student,,
123
Bio-Technology Department, Yeshwant College of IT (Bioinformatic &
Biotechnology), Parbhani, Maharastra, INDIA. *Correspondence : biolichen@gmail.com
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Isolation and Identification of
Feather Degradable Microorganism
1
Tom Sinoy ES*,
2
Chavan Pooja Bhausaheb and
3
Patre Pratiksha Rajendra
ABSTRACT
This study aimed to isolate and identify a new local bacterial strain, which is able to completely degrade keratin-
rich wastes into soluble and useful materials which can be used for many purposes. Bacterial keratinases are of
particular interest because of their action on insoluble keratin substrates and generally on a broad range of
protein substrates. These enzymes have been studied for de-hairing processes in the leather industry and
hydrolysis of feather and keratin. Samples from poultry wastes, soil, water, and feather were collected from
different places in Parbhani. Each sample was plated on feather meal agar plates containing 5 g LG1 feather as
the sole carbon and nitrogen source and the obtained colonies were selected, purified and their growth were
detected on casein agar mediumThe well grown isolates on casein agar mediumwhich producing the largest
clearing zone on casein plate were selected for keratinase assays. Out of 16bacterial isolates, 5 isolates were
selected. The best keratinase producing bacterium kera MS21 was selected and identified based on
morphological, physiological and some biochemical characteristics. It was recorded as a species belonging to
the genus Pseudomonas and identified as Pseudomonas sp. Precipitation and purification of the keratinase
enzyme in addition to factors affecting enzyme activity (pH & temp.) were studied. The enzyme molecular
weight was determined to be of 30 KDa using sodiumdodecyl sulfate polyacrylamide gel electrophoresis
analysis. The optimumtemperature and pH were determined to be 35C and pH 8.0, respectively. The effect of
some proteases inhibitors and activators were also studied.
Keywords : Keratinase, De-Hairing, Protease Inhibitors.
1. INTRODUCTION
Keratin is an insoluble protein macromolecule with very high stability and low degradation rate. Keratin is
mainly present in hair, feather, nails, wool and horns. High protein content of keratin waste can be used as a
good source of protein and amino acids by systemic recycling. Recycling of feather can provides a cheap and
alternative protein feed stuff. Further this can be used for animals feed and for many other purposes. However,
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poor digestibility of keratin is a problemin recycling .
Keratinase is an extracellular enzyme used for the bio degradation of keratin. Keratinase is produced only in the
presence of keratin substrate. Keratinase attacks the disulfide bond of keratin to degrade it. Some microbes have
been reported to produce keratinase in the presence of keratin substrate. Keratinase producing microorganisms
have the ability to degrade chicken feathers, hair, nails, wool etc. Several keratinolytic microorganisms have
been isolated and characterized fromsoil collected nearby feather
Screening:processing units includes Bacteria, Bacillus Sp. FK46, B. licheniformis, B. pumilus sps, Vibrio sps
strain Kr2, Actinobacteria, Streptomyces pactum, S. albus, and Saprophytic and Dermatophlilic fungi,
Aspergillus sps, Rhizomucor sps, Trichophyton mentagrophytes, T. rubrum, T. gallinae, Microsporum canis and
M. gypseum .
In the current study we focused on the isolation and characterization of extracellular keratinase producing
bacteria fromthe soil of poultry shop in Parbhani.
2. MATERIALS AND METHODS
2.1. Isolation Of Feather Degrading Microorganism
One gmsoil was obtained fromLocal poultry farmand added to 9 ml of saline water. This initial dilution was
activated by heat shock at 70
o
C for 15 min. This heat treated sample then serially diluted to reduce initial
number of microorganism(dilution up to 10
-9
in saline water prepared in tubes) and were plated on nutrient agar
mediumand incubated at 35
o
c for 24 hrs. The appeared colonies were checked for spore formers and streaked
on Nutrient agar slants for further study.
2.2. Screening On Casein Agar Plates
Casein agar plates were prepared, and the colonies fromnutrient agar plates was taken by making suspension
and spreaded on casein agar for testing caseinolytic activity of the organism.Bacteria were inoculated on plates
and incubated at 37
o
c for 24 hrs. Colonies showing zone of clearance after addition of tannic acid were selected.
3. CHARACTERIZATION IDENTIFICATION OF ISOLATED FEATHER DEGRADING
BACTERIA
The bacterial isolates were studied for cultural, morphological and Biochemical characters.
3.1. Cultural Characterization
The isolates were observed under the microscope, the colony morphology was noted with respect to color,
shape, size, nature of colony and pigmentation.
3.2. Microscopic Observation
The bacterial isolates were Gram stained and observed under a high power magnifying lens in light microscope.
Tom Sinoy et. al / VSRD Technical & Non-Technical Journal Vol. 2 (3), 2011
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Endospore staining and motility test were performto observe the morphology and motility of the cells.
3.3. Biochemical Characterization
The bacterial isolates were characterized biochemically by indole test, methyl red test, voges proskauer test,
Simmons citrate test, catalase test, oxidase test, urease test, nitrate reduction test, gelatin hydrolysis test, Starch
hydrolysis test, and carbohydrate fermentation test (glucose, sucrose and lactose) etc.
4. KERATINOLYTIC ACTIVITY ASSAY
20 ml 0.1 mol-1 Tris buffer (pH 8) containing 0.1% feather and 40 l of enzyme solution and was incubated for
30 minutes at 55C. The reaction was stop with 500 l 0.1 l mol-1 trichloroacetic acid (TCA) in 0.1 mol-1 Tris
buffer, pH 8. The amino acid liberated were measured as the absorbance at 590 nm against a reagent blank and
the quantity was determined froma standard tyrosine solution (50-500 g ml-1) using a spectrophotometer.
4.1. Effect of pH
Trypticase soy broth (TSB) medium was prepared (pH 3, 4, 5, 6, 7 and 8). The bacterial isolate was inoculated
in to the TSB medium. Inoculated mediums were incubated at 37C for 48 hours. Absorbance of the medium
was measured using spectrophotometer at 590 nmagainst the TSB as blank.
4.2. Effect of Temperature
TSB mediumwas prepared and the bacterial cultures were inoculated into the medium. The inoculated media
were incubated at 4, 25, 30, 35, 40 and 45C. The absorbance of the
5. PRODUCTION AND EXTRACTION OF ENZYME
5.1. Inoculum Development
The selected bacterial colony after its identification and characterization inoculated into feather basal broth
medium. It was incubated in orbital shaker incubator at 35
o
c for 7 days. After that wait for feather degradation.
The PH of mediumshould be 7 i.e. neutral.
5.2. Production of Enzyme
After seven days of incubation, 10 ml of culture mediumwas transferred to1.0 liter medium. The mediumwas
prepared similarly as previously described. All incubations were done at 35
o
c with shaking at 150 rpmin a
controlled environment shaker. This flask is also incubate for 7 days. The purpose of this step is only large scale
(i.e. in sufficient amount for biochemical testing) production of keratinase enzyme.
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6. EXTRACTION OF ENZYME
6.1. Filtration
The culture mediumwas filtered through Whatmann No.1. Filter paper to remove residual undegraded.
The filtrate was then subjected to centrifugation at 10000 rpmfor 10 min to remove bacterial residue. After
centrifugation ammoniumsulphate was added to the supernatant to achieve 30% saturation, which gives the
precipitation of enzyme in suspension, now this Crude enzyme then used for enzyme assay and characterization.
6.2. Assay for Keratinase Activity
Keratinolytic activity of culture filtrates was measured spectro-photometrically; the test described below was
developed in order to simplify analytical work on Keratinase. Azo-keratin hydrolysis provides a colorimetric
assay for enzymatic activity on keratin.
6.3. Synthesis and Enzymatic Hydrolysis of Azo-keratin
Azo-keratin was prepared by a similar method similar to a known procedure for azoalbumin. The use of
azoalbumin as a substrate in the colorimetric determination of peptic and tryptic activity. Ball-milled feather
powder was prepared. A 1 g portion of the feather powder (the keratin source) was placed in a 100-ml round-
bottomed reaction flask with 20 mL of deionized water. The suspension was mixed with a magnetic stirrer. Two
ml of 10% NaHCO (weight per volume) were mixed into the feather suspension (Lin et al., 1992). In a separate
10-ml tube, 174 mg of sulfanilic acid were dissolved in 5 mL of 0.2 N NaOH. Sixty-nine mg of NaNO were
then added to the tube and dissolved. The solution was acidified with 0.4 mL of 5 N HCl, mixed for 2 min and
neutralized by adding in 0.4 mL of 5 N NaOH This solution was added to the feather keratin suspension and
mixed for 10 min. The reaction mixture was filtered and the insoluble azo-keratin was rinsed thoroughly with
deionized water. The azo-keratin was suspended in water and shaken at 50C. for 2 hr and filtered again. This
wash cycle was repeated until the pH of the filtrate reached 6.0-7.0 and the spectrophotometric absorbance of
the washing at 450 nm was less than 0.01. Finally, the wash cycles were repeated at least twice using 50 mM
potassiumphosphate buffer, pH 7.5. The azo-keratin was washed once again with water and dried in vacuum
overnight at 50C. The resulting product is a chromogenic substrate that can be incubated with enzyme solution
to produce and release soluble peptide derivatives that cause an increase in light absorbance of the solution.
6.4. Enzymatic Hydrolysis of Azo-keratin
This procedure tested the keratinolytic activity of keratinase on azo keratin to begin the process, 5 mg of azo-
keratin was added to a 1.5-ml centrifuge tube along with 0.8 mL of 50 mM potassium phosphate buffer, pH 7.5.
This mixture was agitated until the azo-keratin was completely suspended. A 0.2-ml aliquot of supernatant of
crude enzyme was added to the azo-keratin, mixed and incubated for 15 min at 50C with shaking. The reaction
was terminated by adding 0.2 mL of 10% trichloroacetic acid (TCA). The reaction mixture was filtered and
analyzed for activity. The absorbance of the filtrate was measured at 450 nmwith a UV-160 spectrophotometer
.A control sample was prepared by adding the TCA to a reaction mixture before the addition of enzyme solution
A unit of keratinase activity was defined as a 0.01 unit increase in the absorbance at 450 nmas compared to the
Tom Sinoy et. al / VSRD Technical & Non-Technical Journal Vol. 2 (3), 2011
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control after 15 min of reaction.
6.5. Feather Degradation by Crude Enzyme Preparation
0.1 g of autoclaved native whole feather was inoculated in the test tube containing 10ml of crude enzyme
preparations. After incubations for 12 h at 40
0
c , the test tube were inspected for visible degradation of feather
against feather containing distilled water as a blank.
7. RESULT AND DISCUSSION
All the isolates were screened for keratinolytic activity on the Casein agar plates. Keratinolytic activity was
measures in compression with the zone of inhibition produced by on agar surface. The isolate showed the
keratinolytic activity while other isolates didnt show any activity. The keratinolytic activity of H5 isolate is
mentioned in Fig. 1. the isolate produced 26 mmzone of inhibition on Casein agar plates.

Fig. 1 : Keratinolytic Activity of Isolate by making well in Agar Plate

Fig. 2 : Plate shows the Cultural Characteristics of Bacillus species in Starch Casein agar.
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Table 1 : Characterization of Keratinolytic Bacteria
Characterization of Bacteria Result
Cultural
characters
Colony morphology Large, round, irregular, mucoid, fast growing
colonies
Spore staining Spore forming
Gramstaining Grampositive rods
Microscopic
characters
Motility Non motile
Indole Negative
Methyl Red Negative
Voges Proskauer Positive
Citrate utilization Positive
Catalase Positive
Oxidase Negative
Urease Negative
Nitrate reduction Positive
Gelatin liquefaction Positive
Starch hydrolysis Positive
Hydrogen sulphide Negative
Glucose Positive
Sucrose Positive
Biochemical
characters
Lactose Negative
Table 2 : Effect of Different pH on Enzyme Activity.
OD value at 590 nm
Bacterial Isolate
pH 3 pH 4 pH 5 pH 6 pH 7 pH 8
Bacillus sp 0 0.005 0.014 0.075 0.099 0.065
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Table 3 : Effect of Different Temperature on Enzyme Activity
OD value at 590 nm Bacterial Isolate
4C 25C 30C 35C 40C 45C 50C
Bacillus sp. 0 0.194 0.950 0.900 0.845 0.354 0

Fig. 3 : Effect of pH on Enzyme Activity
.
Fig.4 : Effect of Temperature on Enzyme Activity
8. ACKNOWLEDGEMENT
We have great pleasure to present this abstract. There is always a supportive person behind a success so we
would like to thanks to our project guide Mr. TomSinoy E. S. He always inspired us to do work in this project.
Finally we would like to thank s to Yeshwant College of IT (BI & BT) for the financial support and providing
this platform.
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