INTRODUCTION

Actinomycetes are virtually unlimited sources of novel compounds with many therapeutic applications and hold a prominent position due to their diversity and proven ability to produce novel bioactive compounds. There are more than 22,000 known microbial secondary metabolites, 70% of which are produced by actinomycetes, 20% from fungi, 7% from Bacillus spp. and 1-2% by other bacteria. Among the actinomycetes, Streptomyces's group are considered economically important because out of the approximately more than 10,000 known antibiotics, 50-55% are produced by this genus.

A little is known about the actinomycetes diversity of marine sediments, which is an inexhaustible resource that has not been properly exploited. Many reports describe that in India, East coast area is a major source for actinomycetes. Marine microorganisms continue to provide pharmacologically important secondary metabolites which are unique and novel chemical compounds. Marine microbes are continuously explored for drug discovery. Apart from microbes all other marine sources have also provided valuable chemical diversity.

The antimicrobial and antifungal testing techniques including agar well diffusion, agar disc diffusion and tube dilution method. The Minimum Inhibitory Concentration (MIC) test for bacterial cells and fungal spore germination also has to be done.

Marine actinomycetes have been traditionally a rich source for biologically active metabolites. Although heavily studied over the past three decades, actinomycetes continue to prove themselves as reliable sources of novel bioactive compounds. Among the well-characterized pharmaceutically relevant microorganisms, actinomycetes remain major sources of novel, therapeutically relevant natural products.

Bioactive compounds are treated with the cancer cells to check the anticancer property of the compounds.

SUMMARY OF INVENTION

This project work summarise that the actinomycetes is efficiently isolated from the marine soil sample even by changing the composition of the media. Basic level biochemical identification shows the characters of actinomycetes. The isolated actinomycetes was successfully identified by using 16s rRNA sequencing method. Bioactive compounds shows the potent in resemblance and the potent compounds shows the anti microbial and anticancerous activity.

BACKGROUND OF INVENTION

The work in this project reveals that isolation, identification,biochemical and anticancerous activity of actinomycetesand its application of cancer activity. The actinomycetes were isolated from the marine soil sample. The isolation of the actinomycetesis done with the starch casein nitrate agar media and the isolated culture was identified by 16s rRNA technique. Biochemical characters such as starch hydrolysis, casein hydrolysis, Indole test, methyl red were carried out for the biochemical characters. Thus the isolated actinomycetes is confirmed through molecular level characterisation. The media composition was changed slightly from the standard composition even though we got the effective growth of the actinomycetes. Bioactive compounds were subjected to anticancer activity against the cancer cells to check the anticancer property of the compound.

OBJECTIVE OF INVENTION

• Isolation and identification of the actinomycetes by 16s rRNA sequencing.

• Performing the biochemical characters for the potent identification

• Characterization of bioactive compounds upto species level

• Anticanerousactivity of bioactive compounds.

METHODOLOGY

Collection of Soil Sample

The soil sample was obtained from the coastal area, Rameswaram. The collected soil sample was air dried for about 5 days. The soil sample was washed twice with double deionised distilled water. The obtained soil sample was serially diluted for the isolation of actinomycetes. From the serial dilution the dilution rate taken for the actinomycetes was 10"2 and 10"3. The serially diluted soil sample was grown in Starch Casein Nitrate Agar media for its growth and proliferation. STARCH CASEIN NITRATE AGAR COMPOSITION

• Starch - 10g

• Casein - 0.2g

• Dipotassium hydrogen phosphate - 1.5g

• Potassium Nitrate -1.5g

• Sodium Chloride -2g

• Magnesium sulphate - 0.02g

• Calcium Carbonate - 0.05g

• Agar -20g

• Distilled water-1000 ml

Culture preparation

The isolated actinomycetes were grown in starch casein nitrate broth media. A loop full of grown actinomycetes from the conical flask was taken and poured into plate supplemented with cyclohexamide and naldixic acid in the laminar air flow chamber to avoid the contamination. This was incubated at 37°C for 4 days in the incubator. Starch Casein Nitrate agar plates were made and the actinomycetes was streaked to it and kept in incubator at 37°C for 4 days. The growth of the organism was observed. This acts as the primary screening process for nanoparticle production. IDENTIFICATION PORCEDURE DNA preparation method

Growth from mature slant culture of the actinomycetes were inoculated aseptically into 250 ml Erlenmeyer flasks each containing 30 ml of Nutrient broth medium (beef extract 2 g/1, yeast extract 2 g/1, peptone 5 g/1 and sodium chloride 8 g/1, pH 7.1 after sterilization) and incubated in a rotary shaker at 30°C for 2 days at 180 rpm. Cultures were centrifuged at 10,000 rpm for 10 minutes. The 0.1 g of mycelium was transferred in to sterile porcelain dish and crushed with liquid nitrogen. The crushed mycelium was transferred into fresh tube containing 500 ul of TE buffer supplemented with lysozyme (20 mg/ml). The tube was incubated at 37°C for 30 minutes. Biochemical Characterization Starch Hydrolysis Test:

For starch hydrolysis starch agar medium was preferred. This test was used to confirm whether the isolated organism have the ability to produce the amylase hence to determine whether this organisms are Actinomycetes.

Prepare and sterilize starch casein nitrate medium and poured into a petriplates. Streak the plates individually with the culture. Incubate the inoculated plates at 37°C for 24 hrs. Following incubation period, flood iodine solution over the entire starch agar surface. Examine the plates for the presence or absence of the blue-black color surrounding each test organism. Casein Hydrolysis Casein is an exoenzymes. Casein hydrolysis test was carried out inorder determine whether the organism has the ability to produce the protease enzyme. Anti- Cancerous Activity Media preferred for anti-cancerous activity is RPMI 1640 medium. The media is from sigma Aldrich chemicals Co and the media is with glutamine, without sodium bicarbonate. It consist serum of fetal bovine serum and it is used for the growth of cells. Antibiotic of penicillin and streptomycin were also obtained. 3.9.1 Chemicals:

5-diphenyl tetrazolium bromide, 0.25% trypsin EDTA, Ethidium bromide, PBS buffer.

RESULTS AND DISCUSSION

The isolated actinomycetes was identified as Nocardiopsisalkaliphilaby 16s rRNA sequencing.

Starch Hydrolysis Test

Iodine reagent is used to detect the presence of starch in a growth medium. When it is added to starch agar plates, the iodine reagent combines with starch in the medium to produce a blue-brown color. Bacteria producing alpha-amylase, an enzyme destroying starch, exhibit clear halos under their growth indicating that the starch has been digested.

Casein Hydrolysis Test

Protease is an exoenzymes that is produced by some bacteria in order to degrade casein. This test is carried out whether an organism is capable of producing exoenzymes protease.

Anticancerous Activity

Effect of Bioactive compounds on Cell Cytotoxicity

Mitochondrial function-based MTT conversion to formazon crystals is often used as cell viability test in cell culture system. MTT was reduced to purple formazon by mitochondrial succinate dehydrogenase of living cells. In this study, Bioactive compounds exerted cytotoxic effects on MCF-7 Human Breast cancer cell line at the concentration of 2-20ug for a period of 24, 48 and 72 hours. Bioactive compounds inhibited the growth of the MCF-7 Human Breast cancer cell line significantly in a dose and time dependent manner. The cytotoxic activity of this cell line was determined based on the concentration of the compound required to reduce the survival of cells by 50% (IC50). Bioactive compounds were highly cytotoxic to the MCF-7 Human Breast cancer cell lines were at very low concentrations and IC50 dosage differ in time and dose dependent manner. The IC50 values for Bioactive compounds are shown in figure for MCF-7 Human Breast cancer cell line. Since bioactive compoundsare given at a dose 44.5lug were found to be effective for 24 hour treatment it was fixed for further analysis.

Figure: MTT assay- Inhibition of cell viability at different concentration in Various time intervals.

MERITS OF THE INVENTION

• Isolation and Identification of actinomycetes by following the starch casein nitrate agar media shows the good growth on their appearance.

• Marine samples shows the effective results against various steps.

• Marine sediments shows the active growth of actinomycetes due to salt tolerant.

• The bioavailability and the biocompatibility of actinomycetesis more when compared to other microbes.

• Though bioactive compounds are cytotoxic but they have tremendous applications in the field of high sensitivity bimolecular detection and diagnostics, antimicrobials and therapeutics.

DEMERITS OF THE INVENTION

• Maintenance of the actinomycetes is difficult in the way of preventing the contamination.

• Collection of sample is difficult due to availability of resources.

• The chance of contamination of culture during its growth is more.

Claims for the Invention Claim 1:

1. Isolation of actinomycetes from soil derived marine sediments shows the potent and enormous amount of salt tolerant activity

2. Marine sediments shows the subsequent nutritional requirements and also able to discriminate the amount of bioactive compound

3. Actinomycetes shows the capability of collecting the bioactive compounds

Claim 2:

1. Antibiotic production of actinomycetes shows in enormous amount when compare to the other compound

2. Marine sediments shows the comparably large amount of antibiotics because of the salt tolerant

3. Actinomycetes shows the innumerable number of bioactive compounds

Claim 3:

1. Bioactive compound which is isolated through the marine sediments shows the antibiotic production and also the anticancerous activity

2. The Bioactive compound which is isolated and identified shows the anticancer activity

3. Bioactive compound which is treated with the cancer cells shows the potent act of killing the cancer cells

4. When compare to the other compound bioactive compound shows the property of anticancer activity because of it salt tolerant.