Biotechnology Reports 1–2 (2014) 2–7 Contents lists available at ScienceDirect Biotechnology Reports j ournal homepage: www.elsevier.com/locate/btre Isolation and partial purification of erythromycin from alkaliphilic Streptomyces werraensis isolated from Rajkot, India a b b b c Gaurav V. Sanghvi , Dipak Ghevariya , Subhash Gosai , Riddhi Langa , Niketa Dhaduk , c a b,∗ Prashant D. Kunjadia , Devendra J. Vaishnav , Gaurav S. Dave a Department of Pharmaceutical Sciences, Saurashtra University, Rajkot, India b Department of Biochemistry, Saurashtra University, Rajkot, India c B. N. Patel Institute of Paramedical Sciences, Bhalej Road, Anand, India a r t i c l e i n f o a b s t r a c t Article history: An alkaliphilic actinomycete, BCI-1, was isolated from soil samples collected from Saurashtra University Available online 17 May 2014 campus, Gujarat. Isolated strain was identified as Streptomyces werraensis based on morphological, bio- chemical and phylogenetic analysis. Maximum antibiotic production was obtained in media containing Keywords: ◦ sucrose 2%, Yeast extract 1.5%, and NaCl 2.5% at pH 9.0 for 7 days at 30 C. Maximum inhibitory compound Actinomycetes ◦ was produced at pH 9 and at 30 C. FTIR revealed imine, amine, alkane (C C) of aromatic ring and p-di sub- Streptomyces stituted benzene, whereas HPLC analysis of partially purified compound and library search confirmed 95% Erythromycin peaks matches with erythromycin. Chloroform extracted isolated compound showed MIC values 1 ␮g/ml Fermentation against Bacillus subtilis, ≤0.5 ␮g/ml against Staphylococcus aureus, ≤0.5 ␮g/ml against Escherichia coli and 2.0 ␮g/ml against Serretia GSD2 sp., which is more effective in comparison to ehtylacetate and methanol extracted compounds. The study holds significance as only few alkaliphilic actinomycetes have been explored for their antimicrobial potential. © 2014 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). 1. Introduction useful antibiotics and 60% of antibiotics used in agriculture [10]. Major types of antibiotics produced by Streptomyces are amino- Among the antibiotic producing microbes, the class Actinobac- glycosides, anthracyclins, glycopeptides, ␤-lactams, macrolides, teria represents a broad range of valuable and prominent sources of nucleosides, peptides, polyenes, polyethers, and tetracyclines [11]. pharmaceutically active metabolites. Actinomycetes are responsi- In spite of the availability of new antimicrobial products, the ble for the production of about half of the discovered secondary development of new antimicrobial agents, preferably naturally metabolites [1], notably antibiotics [2], antitumour agents [3], occurring with novel mechanisms of action, is an urgent thera- immunosuppressive agents [4] and enzymes [5]. Each actino- peutic need with increase in drug resistant pathogens, and the mycetes strain has probably genetic potential for producing 10–20 magnitude at which these pathogens are transmitted among peo- secondary metabolites [6]. Terrestrial actinomycetes are one of the ple. Even though much work on the terrestrial actinomycetes is abundant sources of secondary metabolites and the vast majority done but still especially soil remains the richest versatile source for of these compounds are derived from the single genus Strepto- new and clinically important antibiotics [12]. myces. Streptomyces are distributed widely in terrestrial and marine In view of the above, in the present study, we have described habitats [7] and are of commercial interest due to their unique the morphological, biochemical and phylogenetic characteristics capacity to produce novel metabolites. The genus Streptomyces of isolated alkaliphilic strain Streptomyces werraensis. Strain was was classified under the family Streptomycetaceae, which includes further explored for production of antimicrobial compounds. Gram-positive aerobic members of the order Actinomycetales and suborder Streptomycineae within the new class Actinobacteria [8,9]. They produce approximately 75% of commercially and medically 2. Methods 2.1. Sample collection and isolation of microorganism ∗ Corresponding author at: Department of Biochemistry, Saurashtra University, Soil sample was collected from the Saurashtra University cam- Rajkot, Gujarat, India. Tel.: +91 281 2578507x489; fax: +91 281 2585083. E-mail addresses: [email protected], [email protected] (G.S. Dave). pus, Rajkot, Gujarat, India. 1 g soil was suspended in 9 ml of sterile http://dx.doi.org/10.1016/j.btre.2014.05.003 2215-017X/© 2014 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). G.V. Sanghvi et al. / Biotechnology Reports 1–2 (2014) 2–7 3 −2 −3 double distilled water. Diluted aliquots (0.1 ml) of 10 , 10 , of chloroform or ethyl acetate or methanol in a separating funnel. −4 −5 10 and 10 were spread on the isolation plates containing The filtered supernatant was extracted by chloroform in ratio of starch caseinagar, oatmeal agar and actinomycetes isolation agar 1:1 (v/v). The yellow colored residual crude active compound was (Himedia, Mumbai) containing combination of penicillin and chlor- purified by thin layer chromatography (TLC) in a running solvent ◦ amphenicol. Plates were incubated at 28 C for 7–14 days. Stock system of methanol and chloroform. Two fractions with different Rf ◦ culture of isolated strain was preserved in 15% glycerol (v/v) at 4 C. values recovered from TLC plates were dissolved in 10% Dimethyl- sulfoxide (DMSO) and bioassayed against the test microorganisms. 2.2. Physiological, biochemical and cultural characteristics Purification of this crude compound was carried out in column chromatography technique on silica gel (MerckLtd. India) using Morphological, biochemical and cultural characteristics of the chloroform-methanol (Rankem Ltd. India) gradient (11:3) as run- isolated strain was studied as described in Bergey’s manual. Carbo- ning solvent system. Extract were collected and characterized by hydrate utilization was determined by growth on carbon utilization FTIR and HPLC analysis. ◦ medium supplemented with 1% carbon sources at 30 C. Temper- ature range for growth was determined on actinomycete isolation 2.7. Antimicrobial testing activity agar by growing at different temperatures (10, 15, 20, 30, 37, 42 ◦ and 50 C). Hydrolysis of starch was evaluated on starch agar media. Antimicrobial activity of isolated active metabolite was studied Reduction of nitrate was determined and all cultural characteristics by agar well diffusion method. Freshly grown colonies of bacterial were recorded after six days of incubation. strains were inoculated into 25 ml of nutrient broth (NB, Hi-media) in a shaking water bath for 4–6 h until turbidity reached to 0.5O.D. 8 −1 × 2.3. In vitro screening of isolates for antimicrobial activity (660 nm). Final inoculum was adjusted to 5 10 CFUml to each ◦ agar plate. The plates were incubated at 37 C and the zones of inhi- Pure isolates were spot inoculated on actinomycetes isolation bition were measured after 24 h. Pure solvent served as a control. agar medium (Hi-Media, Mumbai) and plates were incubated at ◦ 30 C for six days followed by inversion for 40 min over chloroform 2.8. HPLC and FTIR analysis of antibiotic sample in fumehood. Colonies were then covered with a 0.6% agar layer of nutrient agar medium (for bacteria), previously seeded with two Extracted purified antibiotic fractions were characterized Gram positive (Bacillus subtilis and Staphylococcus aureus) and two by HPLC (High Performance Liquid chromatography) and FTIR Gram negative strains (Escherichia coli and Serretia sp.) to evaluate (Fourier Transform infrared resonance) chromatography. HPLC antimicrobial activity. of bioactive metabolite was determined at 215 nm with mobile phase of Acetonitrile-Methanol-0.2 M Ammonium acetate-Water 2.4. Phylogenetic analysis (45:10:10:35) in C18 column. FTIR spectra of the purified antibi- otic fractions were analyzed after homogenization of the sample The 16SrRNA gene was amplified with primers with KBR. The FTIR spectra were recorded on SHIMADZU AUX 220 −1 forward (5 -GAGTTTGATCC TGGCTCA-3 ) and reverse (5 - spectrometer in the range of 4000–400 cm . ACGGCTACCTTGTTACGACTT-3 ). Amplified PCR product was sequenced and nucleotide sequence was matched using BLAST 3. Results and discussion program. Phylogenetic tree was constructed using neighbor- joining method [13]. Sequence of the isolate was submitted to Present study focuses on isolation of potent antibiotic producing GenBank (Accession ID: JQ964039). alkaliphilic actinomycetes. Fifty actinomycetes strains were iso- lated from ten soil samples collected from the different places of 2.5. Submerged fermentation Saurashtra University, Rajkot, Gujarat, India. Among the isolated pure strains, only one actinomycetes culture, BCI-1 was found to Seed culture media for submerged fermentation with following produce wide spectrum of antimicrobial activities (Gram-positive composition (g/l) was used: soybean meal 30, glucose 10, glyc- and Gram-negative bacteria). BCI-I was characterized by 16srRNA erol 10, (NH4)2HPO4 1, (NH4)2SO4 3.5, CaCO3 5.10% of inoculum sequencing and identified as S. werraensis. In general, Streptomyces was added in 100 ml production media with composition: (g/l): are primarily saprophytic and are best known microorganism from sucrose 35, yeast extract 15.0, NaCl 4, KH2PO4 3, K2HPO4 2 and soils where