2 , 4-Di-tert-butylphenol , the bioactive compound produced by Streptomyces sp . KB 1

Article history: Received on: 20/08/2015 Revised on: 23/09/2015 Accepted on: 10/10/2015 Available online: 12/11/2015 Streptomyces sp. KB1(TISTR 2304), identified by 16S rDNA gene, was collected from the air sample at Aonang, Krabi province, Thailand and deposited in the GeneBank database (accession number KF939581.1). It could produce bioactive compounds and excrete into liquid culture medium within 4 days of incubation period at 30 °C, 200 rounds per minute in shaking incubator. Bioactive compounds in culture broth supernatant were extracted with 3-times of ethyl acetate (EA), evaporated by rotary evaporator at 45 °C under reduced pressure, purified by silica gel column chromatography and eluted by gradient solvent system of ethyl acetate: hexane at ratio of 1:9, 3:7, 4:6, 6:4 and 10:0, v/v. Eight pooled fractions were obtained, name PF1 PF8, from basis of their TLC profile. Only PF4 displayed as purified active compound which showed anti-methicillin resistant Staphylococcus aureus (MRSA) activity. The PF4 was predicted as 2, 4-Di-tert-butylphenol (C14H22O) with molecular weight 206.2.


INTRODUCTION
Actinomycetes are gram positive, aerobic, branching and unicellular microorganisms having high Guanine-Cytosine percent (about 69-75%) in their DNA and ubiquitous in nature (Ventura et al., 2007).They have significant capability for degradation of complex organic compounds and they are also invaluable bacteria as they are well known for production of wide range of active secondary metabolites (Solanki et al., 2008).From 22,500 of total bioactive compounds that have been reported.It was found that about 45% ( 10,125) have been produced by actinomycetes, 38% ( 8,550) by fungi, and 17% ( 3,825) by unicellular bacteria (Berdy, 2005).Among actinomycetes, Streptomyces species alone produce .around 8,100 of bioactive compounds and contributing majorly towards the reservoir of important bioactive compound with less contribution from other genera (Renu et al., 2015).They were well known that bioactive compounds produced by Streptomyces were used to develop as many important substrates in medical field such as antibiotics, immunomodulators, anticancer drugs, antiviral drugs, herbicides, and insecticides (Watve et al., 2001;Rahman et al., 2010).Today around 11,900 antibiotics had been discovered by 1994 of which around 7,140 (60%) were produced by these bacteria (Procopio, 2012;Safey et al., 2013).From the problem of old antibiotics for infectious disease treatment is failure leads to infectious diseases are increase (Garcia, 2009;Oancea and Stoia, 2010).Therefore, novel antibiotics had been continuously invented and developed (Raja and Prabakarana, 2011).The interesting specie of Streptomyces used in the present study was collected from the air sample at Aonang, Krabi province, Thailand, identified by means of partial 16S rDNA gene sequence analysis at National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathum Thani province, Thailand, submitted to GenBank (accession number KF939581.1)and finally named as Streptomyces sp.KB1.An unknown species, Streptomyces sp.KB1, is one of an important bioactive compound-producing strain.In our previous studies, it showed broad spectrum of antimicrobial activity against representative of Gram-positive bacteria (Staphylococcus aureus TISTR 517, Micrococcus luteus TISTR 884 and Bacillus cereus TISTR 11778), Gram-negative bacteria (Escherichia coli TISTR 887, Salmonella typhimurium TISTR 292 and Pseudomonas aeruginosa TISTR 1467) and yeasts (clinical isolate of Candida albicans and Cryptococcus neoformans).Therefore, the aim of this present study was to extract, separate, purify the bioactive compounds produced by Streptomyces sp.KB1 and elucidate the chemical structure after obtain the pure active compound.

Microorganisms, Media and Cultural conditions
Producing strain: Streptomyces sp.KB1 was collected from air sample at Ao-nang, Krabi province, Thailand, by using Biosampler, Microflow 90 (Aquaria, Italy), at flow rate 100 l/min for 30 min according to the manufacture's instruction and isolated as pure culture on half-formula of Luria Bertani (LB/2; Himedia, India) agar medium (5 g/l Tryptone, 2.5 g/l Yeast extract, 5 g/l NaCl and 15 g/l Agar powder).The isolate was identified by means of 16S rDNA gene sequence analysis at BIOTEC, Pathum Thani province, Thailand, and published in GenBank database (NCBI, USA).Firstly, it was cultured on LB/2 agar medium at 30 °C in static incubator (WTB binder, Germany) for 5 days.Later, isolated colony was inoculated into 10 ml of LB/2 broth medium in 25  150 mm of screw cap test tube, incubated at 30 °C, 200 rpm in shaking incubator (Thermo scientific, USA) for 2 days and the cells were stored in 15% glycerol at -80 °C until use.

Indicator strains
Staphylococcus aureus TISTR 517 and clinical isolate of methicillin-resistant Staphylococcus aureus 142 (MRSA 142) were streaked on Muller Hinton (MH) agar medium (Himedia, India), incubated at 37 °C in static incubator for 24 h.Single colony was inoculated into 10 ml of LB broth medium in 25  150 mm of screw cap test tube, incubated at 37 °C, 200 rpm in shaking incubator for 24 h.The cells were stored in 15% glycerol at -80 °C until use.

Starter preparation
1  1 cm 2 of initial streak of 5-days culture of Streptomyces sp.KB1 was inoculated to 10 ml of LB/2 broth medium in 25  150 mm of screw cap test tube.Inoculum was incubated at 30 °C, 200 rpm in shaking incubator for 2 days and assigned as starter.

Fermentation
12 l of supernatant was prepared by 6-times of cultivation of Streptomyces sp.KB1.Briefly, 2 l of LB/2 broth medium was prepared in a large container and divided as 200 ml to ten flasks of 1,000 ml baffled flasks.All flasks were autoclaved at 121 °C for 15 min.Then, 1% starter of Streptomyces sp.KB1 was inoculated to all flasks, incubated at 30 °C, 200 rpm in shaking incubator for 4 days.After incubation, all of the culture broth was centrifuged to separate the cell sediment at 4 °C, 10,000 rpm for 30 min and harvested the supernatant.12 l of supernatant was filtered through Whatman No. 1 filter paper by using vacuum pump (Gast, U.S.A).The clear supernatant was tested for the activity against S. aureus TISTR 517.

Extraction of bioactive compounds from clear supernatant
The clear supernatant (12 l) was extracted 3-times with ethyl acetate (EA) at ratio 1:1 (v/v), harvested and pooled the solvent layer.The pooled EA extracts were evaporated by using rotary evaporator at 45 °C under reduced pressure.EA extract slag was weighed, dissolved in the least amount of 100% acetone and named as crude EA extract.

Separation of the bioactive compounds
Separation of the bioactive compounds into theirs components was conducted by thin layer chromatography (TLC).The dissolved crude EA extract was spotted on 1  5 cm of silica TLC plate and developed in the gradient solvent system of EA: hexane in ratio of 1: 9 -10: 0 (v/v).The separated compound(s) were visualized under UV at 254 nm (absorbance) and 365 nm (fluorescence) or detected by spraying with sulfuric acid and heating at 150 °C for 20 sec.

Purification of the bioactive compound
The bioactive compounds were purified by using silica gel column (4.5  60 cm) chromatography.EA : hexane (1:9, v/v) was used as an eluting solvent.The column was left for overnight until the silica gel 60 -120 mesh size of chromatography grade (Merck, Germany) was completely settled.10 ml of dissolved crude EA extract was intermixed with 5 g of silica gel, poured into the silica gel column surface and gradually added a suitable solvent systems.Fractions (each of 10 ml) were collected as regular intervals at flow rate 2.5 ml/min, loaded on silica TLC plate, developed with same solvent system and observed the pattern of spot(s).The fractions that exhibited same Rf values were pooled.The pooled fractions (PF) were evaporated to remove the solvents at appropriate condition.The compounds sediment was weighed, dissolved with 100% acetone for final concentration of 10 mg/ml and investigated the anti-MRSA activity by applying from bioautography method.

Anti-MRSA activity-guided isolation
Bioautography is a method to localize antimicrobial activity on a chromatogram (Patil et al., 2013).In present study, it was applied to investigate the anti-MRSA 142 activity.Briefly, 2, 4, 6 and 8 µl of 10 mg/ml of each dissolved compounds were spotted on 1  5 cm of silica TLC strip.The silica TLC strip was air dried to remove the residue acetone in fume hood at room temperature for 20 min and then placed on Muller Hinton agar (MHA) plate.Finally, the MHA plate were poured with semi-solid MHA medium (0.7% agar) which had 1.5  10 8 CFU/ml of MRSA 142, placed at room temperature for 15 min and incubated in static incubator at 37 °C for 24 h.The plates were observed the clearance formed around the spot on silica TLC strip.

Spectroscopy of bioactive compounds Proton and carbon nuclear magnetic resonance (NMR) spectra
1H and 13C NMR, HMBC, HMQC, DEPT 90, DEPT 135, and COSY spectra were obtained from FT-NMR spectrometer (Bruker, model AVANCE DPX-300, Germany) at center for scientific and technological equipments, Walailak University, Thailand.In this process was operated at 300 MHz for proton and 75 MHz for carbon.Chloroform (CDCl 3 ) was used as deuterated solvents in NMR experiments.Referent signals were the signals of residual undeuterated solvents at δ 7.24( 1 H) and 77.0 ( 13 C) ppm for CDCl 3 .

Fourier transforms infrared (FT-IR) absorption spectra
FT-IR absorption spectrum of bioactive compound was obtained from FT-IR spectrophotometer (Bruker, model TENSOR 27, Germany) at center for scientific and technological equipments, Walailak University, Thailand.The compounds were examined as dry film on NaCl cell.

Ultraviolet (UV) absorption spectra
UV absorption spectrum of bioactive compound was obtained from UV/VIS spectrophotometer (JASCO, model V-630, Japan) at center for scientific and technological equipments, Walailak University, Thailand.

Mass Spectra
Mass spectrum of bioactive compound was obtained from Mass spectrometer (Thermo, model MAT 95 XL, Germany) at scientific equipment center, Prince of Songkla University, Thailand.

Anti-MRSA activity of bioactive compounds
Anti-MRSA efficacy of bioactive compounds was determined by agar well diffusion method (Kekuda et al., 2012).Single colony of 24 hours-culture of MRSA 142 was inoculated to 5 ml of LB broth medium in 15  150 mm of screw cap test tube and then incubated at 37 °C, 200 rpm in shaking incubator for 24 hours.Cell suspension was approximately adjusted to a 0.5 McFarland standard turbidity by 0.85% NaCl and swabbed on sterilized MHA plates using sterile cotton swab and drilled well by a 5 mm diameter of sterile cork borer.50 µl of bioactive compounds (1mg/ml of 100% DMSO), Oxacillin (200 µg/ml), Vancomycin (200 µg/ml) and DMSO (100%) were transferred into labeled wells.The plates were incubated at 37 °C in static incubator for 24 hours.The plates were observed the zone of inhibition formed around the each well.

Cultivation of producing strain
Medium and incubation period plays a pivotal role on bioactive compounds production and theirs antimicrobial activity (Reddy et al., 2011).This study, Streptomyces sp.KB1 was cultivated in LB/2 broth medium for 4 days.The cultivation in decrease of nutritional value from LB to LB/2 did not impact on bioactive compounds production (data not shown).This phenomenon was supported by research which reported that the best antimicrobial activity of Streptomyces lydicus A2 was seen by cultivating it in LB/2 broth medium (Lertcanawanichakul et al., 2015).

Extraction
EA is solvent that was used to extract the most of bioactive compounds or antibiotic because maximum bioactive compounds or antibiotic yield was observed in residue extracted by using EA (Parthasarathi et al., 2012).In this study, 2.82 g of crude EA extract was extracted from 12 l of clear supernatant.Crude EA extract, prepared as 50 mg dry weight/ml with 100% DMSO, showed inhibition zone of 20.67  0.58 mm against MRSA142.

Separation
TLC experiment was performed to investigate the suitable solvent system for using as eluted solvent system in purification process.After TLC plates were developed with ten gradients elution of EA: hexane, sprayed with sulfuric acid and heated at 150 °C for 20 sec.Different spots of compounds on each TLC plate were showed.From this process, solvent systems of EA: hexane in ratio of 1:9, 3:7, 4:6, 6:4 and 10:0 were selected to use for separating the each compound.

Anti-MRSA activity-guided isolation
After 8 PFs were investigated anti-MRSA activity.It was found that PF3, PF4 and PF5 showed activity against MRSA 142.They were selected, investigated the purity from spot feature on silica gel TLC plate when developed with many solvent systems including 2:8 and 4:6 (v/v) of dichloromethane: hexane, 0.5:9.5 (v/v) of EA: hexane and 4:6 (v/v) of EA: dichloromethane.From spot feature on silica gel TLC plate, only PF4 was selected to investigate the purity of compound in the later time.

Spectroscopy
After PF4 in CDCl 3 was investigated the purity by screening 1 H NMR, the result showed that this compound has high purity (Figure 1).The 1 H NMR spectra of this compound showed the presence of a total 21 protons, of which 18 protons were present as six tert-methyls at  1.34 (9H,s, 3 × CH 3 ) and 1.46 (9H, s, 3 × CH 3 ).The remaining three protons were present in the downfield aromatic region at 6.63 (1H, d, J = 8.2 Hz), 7.11 (1H, dd, J = 8.2, 2.4 Hz), and 7.34 (1H, d, J = 2.4 Hz), suggesting that this compound is atrisubstituted aromatic compound.This suggestion was supported by 13 C NMR spectra which showed the presence of 10 carbon signals, of which six downfield carbon signals between 115.1 and 151.8 ppm were in the aromatic region, whereas the remaining four carbon signals were in the upfield region between 29.7 and 34.7 ppm.Of six aromatic carbon signals, three were quaternary carbons (135.2, 143.0, and 151.8 ppm), suggesting that the aromatic substitutions were at these carbons.The most downfield quaternary carbon at 151.8 ppm indicated the presence of a phenolic OH group at this carbon in the molecule.Among the four upfield carbon signals, the two at 34.3 and 34.7 ppm were tertiary carbons and the remaining two at 29.7 and 31.6 ppm were methyl signals.This clearly suggested the presence of two tertbutyl substitutions on the two remaining quaternary carbons at 135.2 and 143.0 ppm in the aromatic ring (Figure 2).Finally, on the basis of the splitting patterns and coupling constants of the three aromatic protons, the chemical structure of bioactive compound or PF4 was deduced as 2, 4-Di-tertbutylphenol.The Mass spectrum revealed that molecular weight and formula were 206.2 g/mol and C 14 H 22 O, respectively (Figure 5).Baseed on the various spectral data, it can be predicted that the molecule structure of bioactive compound or 2, 4-Di-tertbutylphenol as Figure 6 and its anti-MRSA 142 efficacy was showed in Figure 7.

CONCLUSION
Bioactive compounds produced from Streptomyces sp.KB1 (TISTR 2304) showed anti-MRSA 142 activity.After purification step, based on the spectral data, the bioactive compound was deduced as 2, 4-Di-tert-butylphenol which had molecular weight, molecular formula and UV visible maximum absorbance were 206.2 g/mol, C 14 H 22 O and 276 and 282 nm, respectively.It still showed the anti-MRSA activity.

Fig. 3 :
Fig. 3: FTIR spectrum of pure active compound.The FT-IR spectrum (Figure3) of bioactive compound showed various peaks following at 3532 cm -1 indicates the presence of O-H, at 2960 cm -1 indicates the presence of C-H stretching in -CH 3 or -CH 2 , at 1506 -1606 cm -1 indicates the presence of CC stretching in aromatic compounds, at 1363 cm -1 indicates the presence of C-H bending in tert-butyl group and at

Table 1 :
Illustrate various data of each PF.