Study of Keratinolytic Activity of Thermophilic
The purified enzyme was preincubated with different
with feathers (feathers were the sole source of carbon and
inhibitors at 5 mM concentrations for 10 and 60 min
nitrogen) (Adriano Brandelli 2008). Total volume of 50
before the addition of substrate at room temperature
ml in tube with two feathers each weighing 1.8 g. Control
(Adinarayana, K. et al., 2003). The inhibitors used
was maintained with same contents without inoculating
were: phenyl methyl sulfonyl fluoride (PMSF) serine
the organism. The set was incubated at 45°C for 96 h
inhibitor, dithiothreitol (DTT) a reducer of disulfide
and observed for keratinolytic activity at the interval of
bonds, perachloromercuric benzoate (PCMB) thiol group
24 h.
containing amino acid inhibitor and ethylene diamine
tetraacetic acid (EDTA) chelator of divalent metal ions.
7. Degradation of feathers by partially purified protease
The caseinolytic activity was determined at 70°C using
10 ml of partially purified protease of S. viridis SJ-21
glycine NaOH buffer (pH 9.5).
was inoculated in basic salt solution along with feathers
The relative activity was calculated with respect to the
as described above. Control was maintained with same
control without treatment with inhibitors.
contents without inoculating the enzyme. Total volume
of 50 ml in tube with two feathers each weighing 1.8 g.
Table 1. Profile of free amino acids released by S. viridis
The set was incubated at 45°C for 96 h and observed for
from feathers at 45°C. (Analyzed at NDDB, Anand (3.6 g
keratinolytic activity at the interval of 24 h and results
feathers in 50 ml sample)) (HPLC- fluorescence with post
were noted visually and photographs were taken as well.
column derivetization.)
8. Amino acid analysis from degraded feather samples
Aminoacids
μg/ml of sample
μg/g of feather
The samples of above mentioned feather degradation
Aspartic acid
Less than 0.5
Less than 7.0
experiment were sent to National Dairy Development
Threonine
Less than 0.5
Less than 7.0
Board, Anand, Gujarat for amino acid analysis. The
Serine
1.40
19.6
amino acids released due to feather degradation by S.
Glutamic acid
5.45
76.30
viridis SJ-21 were analyzed by HPLC- Fluorescence with
Glycine
4.02
56.28
post column Derivatization.
Alanine
1.15
16.1
cysteine
7.56
105.84
Results and Discussion
Valine
13.45
188.3
Isolation of alkaline protease producing actinomycetes
Methionine
6.37
89.18
from various sources was carried out using alkaline skim
Isoleucine
5.60
78.4
milk agar medium. Growth of the organism is shown in
Leucine
13.35
186.9
Figure 1. On the basis of colony characters, biochemical
Tyrosine
21.65
303.1
activity, spore nature, growth patterns and pigmentation,
Phenyl alanine
19.74
276.36
organism was identified as Saccharonomospora spp.
Lysine
5.74
80.36
(Lacey, J., 1997). And for confirmation, we relied on
Histidine
2.13
29.82
16S r RNA Sequencing. Phylogenetic analysis based
Arginine
6.67
93.38
on 16 S r-RNA sequence of SJ-21 showed that the
sequence exhibited a high level of homology with
6. Degradation of feathers by S. viridis SJ-21.
Saccharomonospora (Figure 2). The sequence was
Chicken feathers (whole feathers) were collected from
submitted to Gene Bank under the NCBI accession
chicken shop. Feathers were first extensively washed in
number JX 262282. Based on all these experimental data,
tap water and finally with double distilled water. Feathers
it was confirmed that isolate represented a novel species
were then steam sterilized and stored at 5°C until used.
of Streptomyces and designated as Saccharomonospora
3 ml fresh culture suspension of SJ-21 of optical density
viridis SJ-21 JX 262282.
0.75 at 680 nm was inoculated in basic salt solution along
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