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ISOLATION OF KERATINASE PRODUCING MARINE ACTINOBACTERIA
ABSTRACT:
Keratinase is a extracellular proteolytic inducible enzyme with the capability of
degrading insoluble keratin substrates. The aim of this study was to isolate keratinase
producing marine actinobacteria. The sample was collected from marine sediment of
rameshwaram coast, and subjected to isolation of actinobacteria on ISP7 medium. The
obtained two isolates were inoculated on skimmed milk agar plate for the primary
screening of proteolytic activity. Isolates showing proteolytic activity in terms of clear
zone around their colony was studied. These isolates was subjected to the secondary
screening on liquid media modified with feather. The degradation activity of feather by
isolates was observed for a period of 10 to 15 days. The keratinolytic property of
actinobacteria was analysed by using feather as a substrate.
Key words: keratinase, Actinobacteria, ISP7 medium, protrolytic activity, skimmed
milk agar, modified liquid medium.
INTRODUCTION:
Keratinase is a extracellular proteolytic inducible enzyme with the capability of
degrading insoluble keratin substrates. Keratin is found in skin, hair, nail, feather and
wool. Keratins exibits in two forms such as hard keratins, found in addition such as
feather, hair, hoof, and nail and soft keratins found in callus and skin1. The keratin
makes feather recalcitrant to proteases like trypsin, pepsin, papain and so on, thus under
goes degradation process in nature12. Degradation of feather protein (keratinase) by
bacteria found in some species of Bacillus3, parasitic fungi4
, Actinobacteria 5, and
some other microorganisms6
. The actinobacteria degradation of insoluble
macromolecules such as cellulose, lignin, chitin, and keratin depends on the secretion
of extracellular enzymes with the ability to act on substrate surfaces.
Keratinolytic actinobacteria treated with feather as several industrial application such as
cosmetic and pharmaceutical industries, biotechnological industries, medical therapy
and waste management, leather industries, textile, and feed and poultry industry1
.
Thus, the present study describes the isolation, and screening, of keratinase producing
marine actinobacteria sample collected from rameshwaram coast, Tamilnadu, India.
MATERIAL AND METHODS
Sample collection: Marine sediments were collected from rameshwaram coast,
Tamilnadu, India. white chicken feather was collected from chicken processing shop
from surrounding area, cleaned with distilled water several time and kept for shade dry
for period of two days.Medium: International Streptomyces project medium No.7 (ISP 7) was used for the
isolation actinobacteria from marine sediments contained the following 2.3g of ISP7,
1.5ml of glycerol, 1.5g of agar in 50ml of marine water and 50ml of distilled water and
the media was supplemented with potassium di chromate and Nalidixic acid at the
concentration of 50µ/ml.
Preparation of soil suspensions: marine sediment sample was prepared by serial
dilution: 1ml marine sediment sample was mixed in 9ml of sterile distilled water was
mixed and serially diluted till 10-7
. 0.1ml of dilution was taken from 10-4
to 10-7
and
plated. The experiment was perform in duplicates.
Isolation of actinobacteria: ISP7 medium was prepared and sterilized at 1210C
temperature, 15-psi pressure for 15 min in autoclave. Medium was poured in petri plate
once it reached at tolerable temperature i.e 450C Nalidixic acid was added at
concentration of 50µ/ml and allowed to solidify. Spread plate technique was followed
to isolate the actinobacteria. Each plate was inoculated with 0.1ml of inoculums from
10-4
, 10-5
, and 10-6
dilution. The plate was incubated at room temperature for 7 days.
Primary screening of keratinolytic actinobacteria: skimmed milk powder was used
for primary screening of kernatinolytic actinobacteria. 2g of skimmed milk powder was
dissolved in 50ml of distilled water and 2.8g of Nutrient agar was dissolved in 50ml of
marine water and made up to 100ml. The prepared medium was sterilized in autoclave
once the medium reached 450C medium was poured in sterile petri plate. Obtained two
actinobacteria isolates which was maintained in ISP7 medium was inoculated in
skimmed milk plates. The plates were incubated at room temperature and examined the
plates for clear zone formation for 7 days.
Secondary screening of keratinolytic actinobacteria: positive isolates obtained from
the primary screening was subjected to secondary screening in order to isolate the
feather degrading actinobacteria. Modified basal liquid medium was used for secondary
screening with chicken feather as substrate. 0.5% of 1.5g KH2PO4 ,0.05% of 0.15g
MgSO4 and 0.5% of 1.5g Na2Co3 dissolved in 300ml of distilled water, pH was
maintained around 7.8 and used as modified basal liquid medium. The prepared
medium was sterilized in autoclaved. The fresh chicken feather was collected from
chicken processing shop. Feather was wash under tap water followed by distilled water
to remove the blood strains and dust particles and shade dried at room temperature for 2
days. 50ml of sterilized medium is poured in six conical shake followed by sterile
feather was added in concial shake containing medium with the concentration of 0.1%
(0.05g), 0.2% (0.1g), 0.3% (0.15g), 0.4% (0.2g), 0.5% (0.25g) and 1.25g of feather was
added in control. The positive isolates obtained from primary screening in terms of
clear zone was inoculated in feather containing modified basal liquid medium and one
substrate with medium is kept as control to compare the degradation activity. The
inoculated flask was incubated at room temperature in shaker incubator for 150 rpm for
10 to 15 days.DISCUSSION:
Keratinase producing actinobacteria was isolated from Marine sediments collected from
rameshwaram coast, Tamilnadu, India. The sample was inoculated by serial dilution in
ISP7 medium and incubated for 7days at room temperature from which two isolates
were obtained isolate-1, isolate-2. This two isolates was subjected to primary screening
on skimmed milk agar medium among this isolates isolate-2 were found to produce
clear zone. This isolate-2 was inoculated in modified basal liquid medium with feather
as a substrate. Chicken feather was added in different concentration such as 0.1%,
0.2%, 0.3%, 0.4%, 0.5% ,control and incubated for period of 10 to 15 days at room
temperature in shaker incubator at 150rpm. After incubation period the feather was
completely degraded by isolate-2. Complete feather degradation was seen in different
concentration. Thus obtained actinobacteria (isolate-2) shows keratinolytic activity by
degradation of feather.
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