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Original Article

Bioactive compound produced from Streptomycesaureofaciens KF532950 and its antimicrobial activity

Sundaram Vennila, Marimuthu Krishnaveni*

Department of Biochemistry, Periyar University, Salem 636 011, India

a r t i c l e i n f o

Article history:

Received 29 July 2013

Accepted 24 August 2013

Available online 1 October 2013

Keywords:

Bioactive substance

MKSV_2013

Mine soil

Sequencing

* Corresponding author. Tel.: þ91 9894829823E-mail address: Krishnavenim2011@gma

0976-1209/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.ijcas.2013.08.007

a b s t r a c t

Aim: Actinomycetes are gram-positive bacteria, common in nature, play a significant role

in biotechnology, as it produces vitamins, enzymes, anti-tumour agents, immune-

modifying agents, mainly antibiotic compounds. Various bioactive substances are pro-

duced by a wide variety of microorganisms including several species of bacteria and fungi.

Hence, it was decided to isolate soil bacteria that is able to produce biologically active

substances.

Methods: The bioactive compound producing strain was isolated from soil and the same

was sequenced, submitted to gene bank for accession number. Related species was known

by tree construction. The bacterial isolates from wound were tested for its susceptibility

against bioactive compound isolated from Streptomyces aureofaciens by disc diffusion assay.

The produced compound was subjected to SDS-PAGE analysis for molecular weight

determination.

Results: The isolated strain was obtained accession number KF532950, it contains high GC

content of 63.6%. The bioactive compound produced from Streptomyces aureofaciens showed

positive result against Staphylococcus spp. and Pseudomonas spp. and its molecular weight

ranged from 14.3 to 97.4 Kd.

Conclusion: From the results obtained, it is concluded that, our strain produces bioactive

compound which may be further characterized for its properties.

Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights

reserved.

1. Introduction oxidized to indol 5,6-quinone to DOPA-melanin.1 Large

Actinomycetes are of high pharmacological, commercial

interest as they are secondary metabolite producers. Acti-

nomyceteseStreptomyces are able to produce dark brown

coloured pigment in culture media. Melanin biosynthesis

takes place in a protein or tyrosine containing medium.

Melanin synthesis involves tyrosinase, converting L-Tyro-

sine into L-Dopa, dopachrome, which inturn is auto-

(mobile).il.com (M. Krishnaveni).2013, JPR Solutions; Publi

scale production of these pose a problem as it grows slowly

in media and are able to grow in solid to liquid media. Soil

actinomycetes receive special attention as they produce

antibiotics and its indicator properties of some of their

pigments. Hence, an attempt has been initiated to study

the bioactive substance produced from Streptomyces aur-

eofaciens and its ability to kill pathogens isolated from

wound.

shed by Reed Elsevier India Pvt. Ltd. All rights reserved.

Fig. 1 e SDS-PAGE analysis of bioactive compound

obtained from Streptomyces aureofaciens.

i n t e rn a t i o n a l j o u rn a l o f c h em i c a l a n d an a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 1 5 3e1 5 5154

2. Materials and methods

2.1. Collection of soil sample, isolation

Soil samples were collected from 2 inch depth of the earth

surface frommine area, Salem. Theywere collected in a sterile

bottles, air dried for one week prior to isolation. This helps in

decreasing the population of gram-negative bacteria. 1 g of the

soil sample was taken andmixed with 99ml of sterile distilled

water. The soil suspensionwas shaken vigorously under room

temperature (25� 2 �C) on an orbital shaker at 200 rpm for 1 h.

One ml soil suspension form concentration 106 was pipetted

and lawn on to starch casein agar at pH 7.2 Fungal and bac-

terial contaminants were prevented by adding 100 mg/l

Cycloheximide and 20 mg/l Nalidixic acid to the medium just

before use and incubated at 30 �C for a period of one month.

Strains of actinomycetes were selected and further purified by

repeated streaking on yeast extractemalt extract agar-ISP2

and were preserved in slants at 4 � 2 �C.

2.2. Collection of wound samples and isolation

Purulent materials were collected aseptically with the aid of

sterile swab sticks from hospitals located at Namakkal. Cul-

ture plates of Nutrient agar e Pseudomonas spp., Mannitol salt

agar e Staphylococcus spp. were used. The swab sticks used for

Fig. 2 e Phylogenetic tree show

the sample collection were streaked directly on the labelled

agar plates and incubated at 37 �C for 24 h. After incubation,

the cultures were examined for significant growth. Sub-

cultures were then made into plates of nutrient agar and

incubated for another 24 h. The standard procedures were

adopted for identification. The isolates were tested for b-lac-

tamase, slime production.

2.3. Production and extraction of bioactive compound

The isolated strain was grown in yeast extractemalt extract

agar medium and kept in an incubator for 4e5 days at 37 �C. Aloopful of spores were scraped from the plate and inoculated

into yeast extractemalt extract broth. It was kept in rotatory

shaker at 150 rpm for a period of 14 days. The fermented

biomass obtainedwasmixedwith 25ml ethyl acetate by using

mortar and pestle. The crude pigment was collected,

concentrated by evaporation. After evaporation of the solvent,

the weight of crude pigment was measured and stored in a

sterile vial.

2.4. Antimicrobial activity of bioactive compoundagainst wound pathogens

The crude culture filtrate was screened for biological activity

against Staphylococcus spp., Pseudomonas spp. by well diffusion

method.3 The 18 h old broth cultures of test bacterial patho-

gens were inoculated by making a lawn on nutrient agar by

using sterile cotton swab. 100 ml culture filtrate was added on

Mueller Hinton agar plate.4 The diameter of inhibition zone

was measured after 24 h of incubation at 37 �C.

2.5. SDS-PAGE analysis

The sample was prepared by diluting culture filtrate in solubi-

lizing buffer at ratio of 1:1, which were placed for 10 min in a

boiling water bath. After cooling to room temperature, the

samples were spinned for 1 min. This step does not applicable

to the protein marker (medium range protein marker, Genei,

Bangalore)as it isa readymadeone;add3e5ml ofmarker towell.

3. Results and discussion

3.1. Isolation of ActinomyceteseStreptomyces

Streptomyces strains were isolated from soil sample collected

at mine area, Salem. Number of colonies was found from each

starch casein agar plate. Colonies selected from each plate

ing other similar species.

Plate 1 e Showing zone of inhibition.

i n t e r n a t i o n a l j o u r n a l o f c h em i c a l a n d a n a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 1 5 3e1 5 5 155

were 2e3 based on colony appearance. Colonies observed

after 5 days were taken because actinomycetes are considered

as slow grower. The isolates were inoculated in ISP2 agar

media and stored at 4 �C for further investigation.

3.2. Isolation of bacterial isolates from wound samples

Staphylococcus spp., and Pseudomonas spp. are the two isolates

obtained from wound. These isolates showed positive result

for b-lactamase, slime production.

3.3. Bulk production of bioactive compound from S.aureofaciens and its antibacterial activity

Bulk production was carried out in ISP2 broth by incubating

the culture for 5 days at 37 �C. The antibacterial activity of

pigment produced at 100 ml was assessed against wound iso-

lates such as Staphylococcus spp., Pseudomonas spp. The zone of

inhibitionwas 10e12mm for Staphylococcus and 13e15mm for

Pseudomonas spp. The results are shown in Plate 1.

3.4. Molecular mass determination

The produced bioactive substance was subjected to SDS-

PAGE. After destaining the gel, clear band was observed on

the white light illuminator. The band was ranged from 14.3 to

97.4 Kd. The protein band was compared with medium range

protein marker. The result is depicted in Fig. 1.

3.5. 16S rDNA sequencing

The DNA isolated was amplified using 16S rDNA universal

primers and sequenced for the identification of bacterial

strain at molecular level. The amplified 16S rDNA PCR product

was sequenced using automated sequencer (Synergy scienti-

fic, Chennai). The Sequence similarity searchwas done for the

16S rDNA sequence using online search tool called BLAST. The

sequence obtained for the organism was submitted to gene

bank for accession number. Phylogenetic analysis was con-

structed via the neighbour-joining method. Phylogenetic tree

reveals that it is very close to Streptomyces subrutilus, mis-

ionensis, griseus, aureofaciens (Fig. 2).

4. Conclusion

The strain isolated from mine soil sample was S. aureofaciens,

have GC content of 63.6% and able to produce melanin like

bioactive compound that are active against Staphylococcus

spp., Pseudomonas spp.

Conflicts of interest

All authors have none to declare.

Acknowledgement

The author thank Honourable Vice-Chancellor, Dr. K.

Muthuchelian Avl, Registrar, Dr. K. Angamuthu Avl, Periyar

University, Salem for their administrative support. The author

also thank Managing Director, Mr. D. Jagadeesh Kumar,

Chrompark Research Centre, Namakkal for providing lab fa-

cilities to carry out the research and Managing Director, Dr.

Sankarapandian Selvaraj, Helini Biomolecules, Chennai for

helping us in doing bioinformatics work.

r e f e r e n c e s

1. Mencher JR, Heim AH. Melanin biosynthesis by Streptomyceslavendulae. J Gen Microbiol. 1962;28:665e670.

2. Cochrane VW. Physiology of Actinomycetes. Annu RevMicrobiol. 1961;15:1e26.

3. Bauer AW, Kirby WMM, Sherris JC. Antibiotic susceptibilitytesting by a standard single disk method. Am J Clin Pathol.1966;45:493e496.

4. Cappuccino James G, Sherman Natalie. Microbiology ALaboratory Manual. 7th ed. Dorling Kindersley (India) Pvt. Ltd;2009.