studies on the bio-active compound from streptomyces flavofuscus

R.Sugumaran et al., IJSID, 2012, 2 (6), 551- 558

International Journal of Science Innovations and Discoveries, Volume 2, Issue 6, November-December 2012 551

STUDIES ON THE BIO-ACTIVE COMPOUND FROM STREPTOMYCES FLAVOFUSCUS

R. Sugumaran *· D. Chithradevi · P. Muthukrishnan·1 Department of Microbiology, PSG College of Arts and Science, Coimbatore, Tamil Nadu, India

INTRODUCTION

INTRODUCTION

ISSN:2249-5347IJSID

International Journal of Science Innovations and Discoveries An International peerReview Journal for Science

Research Article Available online through www.ijsidonline.info

Received: 26-11-2012

Accepted: 24-12-2012

*Corresponding Author

Address:

Name:

R. SugumaranPlace:Tamilnadu, IndiaE-mail:[email protected]

ABSTRACT

A recent study was carried out to isolate the psychrophilic actinomycetes fromfarming soil, to determine the antibacterial activity against different human pathogens.Totally, 9 psychrophilic actinomycetes were isolated and 5 were found to be facultativepsychrophiles and other 4 were mesophilic. The isolated psychrophilic strains wereselected to determine antimicrobial activity against human pathogens. Actinomycetesisolates were identified as (Micromonospora sp., Streptomycetes sp., Nocadia sp,). The activeculture filtrate was tested against Escherichia coli, Stapyhlococcus aureus, Serratia

marscescens, Acinetobacter sp., Streptococcus mutants, Bacillus cereus, Shigella flexineri,

Vibrio cholerae, Salmonella typhi, Klebsiella pneumoniae, Pseudomonas auerogenosa, Proteus

vulgaris. All the actinomycetes isolates were able to produce secondary metabolites butMicromonospora sp. (FS1 & FS2) and Streptomyces sp. (FS5) able to show more antibacterialactivity towards the test pathogen. Bioactive compounds were separated by thin layerchromatography and identified using Gas Chromatograpy - Mass Spectrophotometryanalysis (GC-MS). Various compounds were analysed by GC-MS, from that the bioactivecompound Stigmasta-5, 22-dien-3-ol, (3a, 22E) - (CAS) were found to be an active substanceidentified from Onyza floribunda and thus active against Streptococcus pneumoniae,Staphylococcus aureus and Escherischia coli.

Keywords: Streptomyces flavofuscus, antibacterial, human pathogens, GC-MS, Bioactivecompound



INTRODUCTIONActinomycetes are gram-positive bacteria that are wide spread in nature and play a pivotal role in the production ofbioactive metabolites [1]. Among actinomycetes, the member of the genus Streptomyces are considered economicallyimportant because they alone constitute 50% of soil actinomycetes are produced by this genus [2]. They continue to be prolificsources of secondary metabolites with biological activities that ultimately find application as antimicrobial, anticancer agentsor other pharmaceutically useful compounds [3]. Streptomyces spp are soil-dwelling filamentous bacteria with acomplex cycleof morphological differentiation [4]. Members of the genus Streptomyces are potential sources for secondary metabolitespossessing a variety of biological activities, including antibacterial activity, which is used for human and animal treatment. It isestimated this bacteria synthesizes more than 7,000 metabolites [5]. Majority of Streptomycetes and other actinomycetesmembers produce a diverse array of antibiotics including aminoglycosides, glycopeptides, B-lactams, macrolides, nucleosides,peptides, polyenes and tetracyclines.Antibiotic resistant bacteria emerge clinically within months to years following their use [6, 7]. Staphylococcus aureus,a virulent pathogen responsible for a wide range of infections including pimples, pneumonia, osteomyelitis, endocarditis andbacteremia, has developed resistance to most classes of antibiotics; methicillin and vancomycin [8]. Rising numbers ofresistant microbes towards antibiotic has cause for the unresponsive infectious disease in human being [9]. Emerging of novelantibiotic classes has the strategy to control the emerging drug-resistant pathogen. Microbes gaining more resistant towardsthe available antibiotics, so there is in need of screening actinomycetes for antimicrobial compounds [10].Bioactive compounds like macrocyclic lactum, macrolide, quinine and peptide are present in actinomycetes. Thesecompounds have range of activity like antibacterial, antifungal, antitumor etc [11, 12 & 13]. This study aimed to isolateStreptomyces spp from soil and tested for antibacterial activity against bacterial sample. Strain FS4, showing potent activityagainst multiple drug resistant bacteria, was selected for biochemical and molecular characterization.

MATERIALS AND METHODS

Sample collection:Soil samples were collected from different agricultural places in and around south zone of Tamilnadu (Coimbatore) ina sterile glass container and stored at room temperature (30oC).Clinical samples:The test organisms were collected from PSG Institute of Medical Research and Science, Coimbatore and theantibacterial activity were carried against the test organisms. The selective human pathogenic organisms used for antagonisticstudy were Escherichia coli, Stapyhlococcus aureus, Serratia marscescens, Acinetobacter sp., Streptococcus sp., Bacillus subtillis,

Shigella flexineri, Vibrio chlorae, Salmonella typhi, Klebsiella pneumoniae, Pseudomonas aurogenosa, Proteus vulgaris .Isolation of Actinomycetes from soil sample:One gram of soil sample was serially diluted up to 10-8 dilution. The dilutions such as 10-3, 10-4, 10-5 and 10-6 weretaken and inoculated in nutrient agar by spread plate method and the plates were incubated at 30oC for 3 days until thecolonies were developed. Based on the colony nature, the actinomycetes were isolated and inoculated into actinomycetesisolation agar. The plates were incubated at 30oC for a period of 15 days until the growth was observed. The grown colonieswere purified by sub culturing on starch casein nitrate agar supplemented with cyclohexamide (0.01 mg/ml).



Biochemical characteristics:Various biochemical tests were carried out to characterize the type of actinomycetes and to identify the potent strain;Indole, Methyl red, Vogus-proskauer, Citrate utilization, Starch hydrolysis, Catalase and Oxidase tests Sugar utilization testswere performed to determine the ability of the organism to utilize various sugars as a carbon source such as glucose, lactose,mannitol, xylose and sucrose [14].Mass production:The actinomycetes were inoculated into the ISP-4 medium and kept in shaker for 15 days at 200-250 rpm. Theproduction media (ISP-4) consist of soluble starch 10gm, Dipotassium phosphate 1gm, Magnesium phosphate 1gm, Sodiumchloride 1gm, Calcium carbonate 3gm, Ferrous sulphate 1gm, Manganese chloride 1gm, Zinc sulphate 1gm in 1000 ml ofdistilled water with 7.4 pH. The growth of organism was observed at optimal temperature 27-30oC and the optimum pH were7.4.Biological activity:After 15 days, the culture was taken and centrifuged at 3000-5000 rpm for 10 minutes. After centrifugation, the cellfree supernatant was collected and filtered with whatmann no.1 filter paper. Equal volume of ethyl acetate was added tosupernatant, shaked well for 20 minutes and allowed to settle overnight. The aqueous layer was collected in watch glass andkept in hot air oven at 40oC for 4 hours. The dried compound was scrapped, mixed with distilled water and antibacterialactivity was seen against human pathogen by agar well diffusion method. The test pathogenic organisms were swabbed inmuller hinton agar and kept undisturbed for 20 minutes. The extracted bioactive compound mixture was added to each well atdifferent concentration (50 µl, 100 µl & 200 µl). The plates were incubated at 37oC for 24 hours and the diameters of inhibitionzones were measured.

Figure 1: Scheme for the extraction of bioactive compound Fermentation Broth (50 ml)



Extraction, Purification and identification of bioactive compound from the strain:The active compound was separated on silica G 60 grade absorbent by mixture of chloroform and methanol (4:1)solvent. The dried cellular filtrate was mixed with DMSO (Dimethyl sulfoxide) and 10 µl (30mg/ml) of mixture was loaded inTLC plate. The TLC plate was kept in solvent until the solvent run ¾ of the plate. The TLC plates were UV radiated and exposedto iodine vapour for the identification of compounds as spot and the Rf value was calculated [15].The partially purified active compound was analysed by gas liquid chromatography-mass spectrometry (GC-MS). GC-MS system was equipped with a fused silica capillary column (DB 5 - MS Capillary Standard Non - Polar Column) was used toanalyse the compound. The data was processed with GC/MS DSQ II (Thermo GC-MS DSQ II). Column condition wasprogrammed as column oven temperature 70oC rose to 260o C at 6oC /Min [16]. The peaks of compounds in gaschromatography were subjected to mass-spectral analysis. The spectra were analyzed from the available library data, TraceUltra search (version 5.0).RESULT AND DISCUSSIONThe samples were collected from farming land around Coimbatore area. Totally 5 strains were isolated from the soilsample and identified as Micromonospora sp., Streptomycetes sp., and Nocardiopsis sp., based on their morphological natureon the Actinomycetes isolation agar and SCNA plates and it revealed the most of the strains belonging to the white and greycolour and rough nature (Table: 1).Various biochemical tests were proceeded to characterize the isolated strains. Most of the strains were showedpositive reaction to MR and showing the ability to hydrolyze the urea but showing negative reaction for Indole and VP test(Table: 2). Strains FS1 to FS9 utilize glucose, sucrose and xylose as their carbon source (Table: 3). Among the 5 isolates FS1,FS4 and FS5 showed marked antibacterial activity against selected bacterial pathogens (Table: 4).The scheme for the extraction of bioactive compounds from the stain is presented in Figure1. An amount of 0.4 gcrude extract was obtained from 50 ml of fermentation broth after defatted with ketoconozole. Totally 5 strain were isolatedand one strain found to effective towards the test organisms. The effective strain was taken and further studies were done. Thecrude extract of FS4 was analysed by TLC and pure single district band was exhibited.

Table 1: Study of phenotypic characterization of isolated actinomycetesStraincode

Morphology ofhyphae

Myceliumcolour

Type of spore Possible genera GrampositiveFS1 Separate hyphae Pink Monospore Micromonospora sp., +FS2 Rarely branched,separate hyphae Grey to white Monospore Micromonospora sp., +FS3 Spiral, twisted hyphae Grey Small spore Streptomyces sp., +FS4 Long hyphae,branched White Long spore Norcadia sp., +FS5 Long chain spore Sandal Long spore Streptomyces sp., +

The compound were analysed by GC-MS (Fig. 2). The peak obtained at 45.22 shows the presence of compoundStigmasta-5, 22-dien-3-ol, (3a, 22E) - (CAS). The probability of the compound was 60.50 and the molecular weight was 412.This compound was identified from plant onyza Floribunda a resource says this compound has activity against Streptococcus

pneumoniae, Staphylococcus aureus and Escherischia coli (Manguro et al., 2010).



In the present study, the bioactive compound Stigmasta-5, 22-dien-3-ol, (3a, 22E) - (CAS) were purified from the brothculture of the stain and biological activities were studied against gram-positive and gram-negative bacteria. The partiallypurified bioactive compound was analysed by GC-MS. Stigmasta-5, 22-dien-3-ol, (3a, 22E) - (CAS) showed strong antibacterialactivity towards test organisms. The Stigmasta-5, 22-dien-3-ol, (3a, 22E) - (CAS) produced by this are not reported earlierfrom actinomycetes.Table 2: Biochemical properties of isolated actinomycetes

Key: + positive, - negativeTable: 3 Utilization of various carbohydrates by isolated actinomycetes

Key: +++ Strong, ++ moderate, + weakTable 4: Antagonistic effect of actinomycetes against human pathogens

Strain code AFB Indole MR VP Citrate Catalase OxidaseFS1 - - - - + + +FS2 - - - + + + -FS3 - - + - - + +FS4 - - + - - + +FS5 - - + - + + -Strain code Glucose Lactose MannitolFS1 ++ +++ +FS2 +++ + ++FS3 ++ ++ +FS4 +++ + ++FS5 ++ + ++



Figure 1: Scanning electron microscopic image of Streptomyces flavofuscus

Figure 2: Dried secondary metabolites



Figure 4: Iodine exposed TLC plate with bioactive compound

Figure 5: GC-Spectra of an Active fraction: Presence of an Active compound Stigmasta-5, 22-dien-3-ol, (3a, 22E) - (CAS) at theretention time 45.22.



CONCLUSIONBioactive compound produced from Streptomyces flavofuscus were active against the test organism. The activecompound can be used as antibiotic in pharmacological industries. Stigmasterol is one of the groups of plant hormone thatinclude β-sitosterol, campesterol and ergosterol (provitamin D2). Stigmasterol is an unsaturated sterol occurs in plant fats andin number of medicinal herbs. Stigamasterol is used as a precursor in manufacturing semisynthetic progesterone. Researchershave indicated that stigmasterol may be useful in prevention of cancer like ovarian, prostate, breast and colon cancers.ACKNOWLEDGEMENTThe authors would like to thank the Management, Secretary and the Principal of PSG College of Arts and Science forproviding necessary facilities for the completion of this work.

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studies on the bio-active compound from streptomyces flavofuscus

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