Bioactive compound produced from Streptomyces aureofaciens KF532950 and its antimicrobial activity

Bioactive compound produced from Streptomyces aureofaciens KF532950 and its antimicrobial activity

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Original Article

Bioactive compound produced from Streptomyces aureofaciens KF532950 and its antimicrobial activity Sundaram Vennila, Marimuthu Krishnaveni* Department of Biochemistry, Periyar University, Salem 636 011, India

article info

abstract

Article history:

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

Received 29 July 2013

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

Accepted 24 August 2013

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

Available online 1 October 2013

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

Keywords:

substances.

Bioactive substance

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

MKSV_2013

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

Mine soil

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

Sequencing

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

Actinomycetes are of high pharmacological, commercial interest as they are secondary metabolite producers. ActinomyceteseStreptomyces 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-Tyrosine into L-Dopa, dopachrome, which inturn is auto-

oxidized to indol 5,6-quinone to DOPA-melanin.1 Large 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 aureofaciens and its ability to kill pathogens isolated from wound.

* Corresponding author. Tel.: þ91 9894829823 (mobile). E-mail address: [email protected] (M. Krishnaveni). 0976-1209/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijcas.2013.08.007

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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. Subcultures 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-lactamase, slime production.

2.3.

Production and extraction of bioactive compound

Fig. 1 e SDS-PAGE analysis of bioactive compound obtained from Streptomyces aureofaciens.

The isolated strain was grown in yeast extractemalt extract agar medium and kept in an incubator for 4e5 days at 37  C. A loopful 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 obtained was mixed with 25 ml 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.

Materials and methods

2.4. Antimicrobial activity of bioactive compound against wound pathogens

2.1.

Collection of soil sample, isolation

Soil samples were collected from 2 inch depth of the earth surface from mine area, Salem. They were 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 and mixed with 99 ml of sterile distilled water. The soil suspension was 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 bacterial 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.

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 pathogens 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 solubilizing 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 is a readymade one; add 3e5 ml of marker to well.

3.

Results and discussion

3.1.

Isolation of ActinomyceteseStreptomyces

Collection of wound samples and isolation

Purulent materials were collected aseptically with the aid of sterile swab sticks from hospitals located at Namakkal. Culture plates of Nutrient agar e Pseudomonas spp., Mannitol salt agar e Staphylococcus spp. were used. The swab sticks used for

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

Fig. 2 e Phylogenetic tree showing other similar species.

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strain at molecular level. The amplified 16S rDNA PCR product was sequenced using automated sequencer (Synergy scientific, Chennai). The Sequence similarity search was 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 constructed via the neighbour-joining method. Phylogenetic tree reveals that it is very close to Streptomyces subrutilus, misionensis, griseus, aureofaciens (Fig. 2).

4. Plate 1 e Showing zone of inhibition. 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.

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 3.2.

Isolation of bacterial isolates from wound samples All authors have none to declare.

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 isolates such as Staphylococcus spp., Pseudomonas spp. The zone of inhibition was 10e12 mm for Staphylococcus and 13e15 mm for Pseudomonas spp. The results are shown in Plate 1.

3.4.

Molecular mass determination

The produced bioactive substance was subjected to SDSPAGE. 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

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 facilities to carry out the research and Managing Director, Dr. Sankarapandian Selvaraj, Helini Biomolecules, Chennai for helping us in doing bioinformatics work.

references

1. Mencher JR, Heim AH. Melanin biosynthesis by Streptomyces lavendulae. J Gen Microbiol. 1962;28:665e670. 2. Cochrane VW. Physiology of Actinomycetes. Annu Rev Microbiol. 1961;15:1e26. 3. Bauer AW, Kirby WMM, Sherris JC. Antibiotic susceptibility testing by a standard single disk method. Am J Clin Pathol. 1966;45:493e496. 4. Cappuccino James G, Sherman Natalie. Microbiology A Laboratory Manual. 7th ed. Dorling Kindersley (India) Pvt. Ltd; 2009.