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 day 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. REFERNCES 1 Kshetri P and Ningthojam D.S (2016) keratinolytic activities of alkaliphilic Bacillus sp. MBRL 575 from a novel habitat, limestone deposit site in manipur, India. Springer plus DOI 10.1186/s40064-016-2239-9.5:595 2 Lakshmi P.J., Chitturi M.K., and Lakshmi V.V(2013) Efficient degradation of feather by keratinase producing Bacillus sp. Internation jounal of microbiology 608321- 7 3 Richter C.S., Williams C.M., Shih J.C.H., and MacKenzie J.M (1990) isolation, identification, and characterization of a feather-degrading bacterium. Appl. Environ. Microbiol., 56, 1509-1515. Kim J.M., Lim W.J., and Suh H.J.(2001) feather degrading Bacillus sp from poultry waste. Process biochem., 37, 287-291. 5 Galunsky B., Bockle B., and Muller R. (1995) characterization of a keratinolytic serine proteinase from streptomyces sp. Argic.sci.,20,2913-2923. 6 okeke C.N., Dozie I.NS., and Unaeze N.C (1994) A thermostable alkaline active keratinolytic proteinase from crysosporium keratinophylum. Microbiol. Biotechnol., 10,563-567. 7 Manivasagan P., venkatesan J., and Kim S.K. (2013) introduction to marine actinobacteria. Marine microbiology. 8 Saba S and Dhanasekran D. (2010) Iolation and screening of keratinolytic actinobacteria from dumped soil in tiruchirappalli and nammakal, Tamilnadu India. Current research journal of biological sciences 2(2): 124-131. 9 Chitte R.R., Nalawade V.K. and Dey S. (1998) Keratinolytic activity from the broth of a feather-degrading thermophilic streptomyces thermoviolaceus strain SD8. Applied microbiology 28, 131-136. 10 Selvam K., vishnupriya B., and Yamuna M.(2013) Isolation and description of keratinas producing marine actinobacteria from south Indian coastal region. African journal of biotechnology. Vol. 12(1) pp.19-26.