Journal of Saudi Chemical Society (2012) 16, 35–44

King Saud University Journal of Saudi Chemical Society

www.ksu.edu.sa www.sciencedirect.com

ORGINAL ARTICLE Biochemical studies on the production of Sparsomycin by Pseudomonas aeurginosa, AZ-SH-B8 using plastic wastes as fermented substrate

Houssam M. Atta *, Hassan G. Radwan

Botany and Microbiology Dept., Faculty of Science (Boys), Al-Azhar University, Cairo, Egypt

Received 3 October 2010; accepted 24 October 2010 Available online 3 November 2010

KEYWORDS Abstract Hundred and twenty microbial isolates could be isolated from different soil samples col- Biochemical studies; lected from different localities in Egypt. One of the bacterial cultures AZ-SH-B8 was found to pro- Pseudomonas aeurginosa; duce a wide spectrum antimicrobial agent when cultivated on plastic wastes. The bacterial culture Parameters; AZ-SH-B8 could be isolated from a soil sample collected from Sharkia governorate, Egypt. From Production of antimicrobial the taxonomic features, the bacterial isolate AZ-SH-B8 matches with Pseudomonas aeurginosa in agent; the morphological, physiological and biochemical characters and confirmation by using API20E Sparsomycin antibiotic system. Thus, it was given the suggested name P. aeurginosa, AZ-SH-B8. The parameters control- ling the biosynthetic process of antimicrobial agent formation includes: innoculum size, different pH values, different temperatures, different incubation period, different carbon and nitrogen

sources and different mineral salts concentrations (KNO3,K2HPO4, MgSO4Æ7H2O and KCl) were fully investigated. The active metabolite was extracted using n-butanol (1:1, v/v) at pH 7.0. The sep- aration of the active ingredient and its purification was performed using both thin layer chromatog- raphy (TLC) and column chromatography (CC) techniques. The physico-chemical characteristics of the purified antibiotic viz. color, melting point, solubility, elemental analysis, spectroscopic charac- teristics and chemical reactions have been investigated. This analysis indicates a suggested empirical

formula of C13H21N3O6S2. The minimum inhibition concentrations ‘‘MICs’’ of the purified antimi-

* Corresponding author. E-mail address: [email protected] (H.M. Atta).

1319-6103 ª 2010 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.

Peer review under responsibility of King Saud University. doi:10.1016/j.jscs.2010.10.019

Production and hosting by Elsevier 36 H.M. Atta, H.G. Radwan

crobial agent were also determined. The purified antimicrobial agent was suggestive of belonging to Sparsomycin antibiotic produced by P. aeurginosa, AZ-SH-B8. ª 2010 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.

1. Introduction tance in Streptomyces lividans, an organism susceptible to the drug. The antibiotic Sparsomycin is a universal translation inhibitor In the present work we describe the isolation of a Bacterial that blocks protein synthesis in all species (Ottenheijm et al., strain from Egyptian soil, which generates an antimicrobial 1986). The broad spectrum of Sparsomycin action indicated agent(s). The identification of this strain is based on the cul- that the drug was targeted to a highly conserved component tural, morphology, physiology and biochemical characteristics. of the translation machinery. The relevance of the target was The bioactive substance was isolated, purified, and spectro- also supported by the fact that mutations inducing high scopic analysis and biological activities were determined. resistance to Sparsomycin have not been reported, and only a moderately resistant strain has been found in Halobacterium 2. Materials and methods salinarium (La´zaro et al., 1996). In fact, it was soon shown that Sparsomycin blocks the pep- 2.1. Microorganism tide bond formation (Jayaraman and Goldberg, 1968). The drug binds and causes important conformational changes in The bacterial AZ-SH-B8 was isolated from soil sample col- the active center. Thus, it was found that lected from Sharkia governorate. It was purified using the soil Sparsomycin can block the binding of substrates at the A-site dilution plate technique described by Williams and Davies (Ravel et al., 1970) but enhances binding to the P-site (Jimenez (1965). et al., 1970). Actually, Sparsomycin has been a very powerful tool in the study of the structure and function of the 2.2. Identification of Bacterial isolate, AZ-SH-B8 (Gale et al., 1981). Recently, the antibiotic was found to inter- act with A2602 in the peptidyl transferase center of 2.2.1. Morphological characteristics the bacterial ribosome (Porse et al., 1999). Morphological characteristics of colonies like color, Gram The drug was initially developed as a potential antitumor reaction, cell shape, spore formation, motility and diffusible agent, although toxicity soon limited its clinical application pigment were investigated. (McFarlane et al., 1966). Nevertheless, the synthesis of a series of Sparsomycin derivatives with higher inhibitory activities 2.2.2. Physiological and biochemical characteristics (van den Broek et al., 1987) has led to a reappraisal of its poten- Lipase (Elwan et al., 1977); protease (Ammar et al., 1991); pec- tial as an anticancer drug (Fiebig et al., 1990; Hofs et al., 1994). tinase (Ammar et al., 1995a); a-amylase (Ammar et al., 1998); Sparsomyin is produced by Streptomyces sparsogenes, Lecithinase was conducted on egg-yolk medium according to which is obviously resistant to the drug. Organisms producing the method of Nitsh and Kutzner (1969) and catalase test toxic compounds, including , use different ap- (Jones, 1949). Aesculin broth has been done according to proaches to defend themselves from their own action (Burdett, Gordon et al. (1974). Nitrate reduction was performed accord- 1996). Not the least frequent approach is to modify the target, ing to the methods of Gordon (1966). Hydrogen sulfide; poly- making it insensitive to the drug. Thus, methylation of specific hydroxyl butyrate accumulation, King A&B, Methyl red, residues within the 16S rRNA makes the of many Voges–Proskauer, indol production, urea test, gelatein lique- aminoglycoside antibiotic producers resistant to their respec- faction, Levan formation, arginine dihydrolase, malonate uti- tive products (Skeggs et al., 1985; Thompson et al., 1985). lization, phenyl alanine deamination, utilization of KCN and The same strategy is used by the producers of macrolides oxidase test were carried out according to Cowan (1974). (Lai et al., 1973), lincosamides (Skinner et al., 1983), pactamy- The utilization of different carbon and nitrogen sources was cin (Ballesta and Cundliffe, 1991), and thiostrepton (Cundliffe carried out according to Shirling and Gottlieb (1966). and Thompson, 1979). The study of these resistance mecha- nisms has provided relevant information on the antibiotic 2.3. Factors effecting on the of the antimicrobial mode of action and on the ribosomal binding site at the molec- agent ular level. It would be important, therefore, to see whether S. sparsogenes, like other antibiotic-producing streptomycetes, has managed to modify the highly conserved Sparsomycin tar- These included inoculum size, incubation period, pH values, get site to make the ribosomes resistant to the drug. Alterna- incubation temperatures; different carbon and nitrogen tively, as in the case of other producers, a different resistance sources, starch, potassium nitrate, K2HPO4, MgSO4Æ7H2O mechanism, such as drug inactivation or permeability barrier and KCl have been determined by the standard methods. alterations, might have evolved (Cundliffe, 1989), perhaps be- cause modification of the target is not possible without seri- 2.4. Fermentation and purification of AZ-SH-B8 antibiotic ously affecting its activity. We have approached the study of Sparsomycin resistance by 2.4.1. Fermentation directly analyzing the producer and by trying to characterize Pseudomonas aeruginosa AZ-SH-B8 was inoculated into Bax- genetic determinants from S. sparsogenes that provide resis- ter bottle containing 5 g dry weight of plastics supplied with Biochemical studies on the production of Sparsomycin antibiotic by Pseudomonas aeurginosa 37

(0.52 · 1013 CFU/disc), after 28 days of incubation at 35 C fil- tration was carried out through Whatman No. 1 and was fol- lowed by centrifugation at 5000 rpm for 15 min. The clear filtrates were tested against the test organism.

2.4.2. Extraction The clear filtrate was adjusted at pH values and extraction pro- cess was carried out using different solvents to be added to the fermentation broth at the level of 1:1 (v/v), respectively. The organic phase was concentrated to dryness under vacuum by using a rotary evaporator.

2.4.3. Precipitation The precipitation process of the antibiotic was carried out Plate 1 A photograph of bacterial isolate AZ-SH-B8 growing using petroleum ether. The compound precipitate was centri- on nutrient agar medium showing Gram negative, short rods fuged at 5000 rpm for 15 min. The antibiotic powder was (·1000). tested for its antibacterial activity by using cup assay method.

40 ml mineral salt solution containing the following ingredi- 2.4.4. Separation ents (g/l): NaNO3, 2.0; K2HPO4, 1.1; MgSO4Æ7H2O, 0.5 KCl, Separation of the antibiotic into its individual components has 0.5 and peptone 2.0. The pH was adjusted at 6.8 before steril- been tried by thin layer chromatography using a solvent sys- ization. Inoculating three discs from bacterial growth tem composed of chloroform and methanol (24:1, v/v).

Table 1 The morphological, physiological and biochemical properties of the bacterial isolate AZ-SH-B8. Characteristic Result Characteristic Result Morphological characteristic Utilization of carbon sources – Color of colonies Yellow L-Arabinose – Gram reaction Negative D-Xylose – Motility + D-Ribose + – Cell shape Rods D-Mannose – Spore former Non-spore former D-Glucose + D-Fructose + Diffusible pigment Blue–green D-Galactose Physiological characteristics – Mannitol + (A) Hydrolysis of – meso-Inositol Protein + – Sucrose Starch – Maltose Lipid + – Lactose Egg-yolk (lecithinase) – Raffinose Oxidase test + – Trehalose Catalase test + – Starch (B) Pigment production Utilization of nitrogen source Pyocyanin pigment + L-Glycine Carotenoid pigmt + L-Alanine Fluorescent pigment + L- + Biochemical characteristics L-Leucine + – Degradation of Esculine + L-Valine – Gelatin liquefaction + L-Lysine +

–H2S production + L-Proline + – Nitrate reduction + L-Tyrosine + – Urea test L-Arginine + – Indole production Growth in presence of different NaCl concentrations (%) – Levan formation from sucrose – Arginine dihydrolase + 1+ – Poly b-hydroxy butyrate accumulation 3+ – Utilization of KCN + 5+ 7 – Phenyl alanine deamination + Growth at different temperature (C) – Voges–Proskauer test 20–43 + – Methyl red test 45 + = positive and = negative. 38 H.M. Atta, H.G. Radwan

2.4.5. Purification Table 2 Identification of bacterial isolate AZ-H-B8 by The purification of the antibiotic was carried out by using sil- API20E system. ica gel column chromatography. A column of 2.5 · 50 cm was Test Result used for this purpose. Chloroform and methanol 10:1 (v/v), was used as an eluting solvent. The column was left for over NO3 = nitrate reduction + night until the silica gel (BDH – 60–120 mesh) was completely TRP = indole production settled. One milliliter crude extract to be fractionated was GLU = acidification ADH = arginine dihydrolase + added on the silica column surface and the extract was ad- URE = urease sorbed on top of silica gel. Fifty fractions were collected (each ESC = esculin hydrolysis of 5 ml). Antibiotic activities were performed for each separate GEL = gelatin hydrolysis + fraction. PNPG = p-nitrophenyl-b-D-galactopyranoside (b-galactosidase) 3. Physico-chemical properties of AZ-SH-B8 antibiotic [GLU] = glucose assimilation + [ARA] = arabinose assimilation 3.1. Elemental analysis [MEN] = mannose assimilation [MAN] = mannitol assimilation + [NAG] = N-acetylglucosamine assimilation + The elemental analysis C, H, O, N, and S was carried out by [MAL] = maltose assimilation the microanalytical center of Cairo University, Egypt. [GNT] = gluconate assimilation + [CAP] = caprate assimilation + 3.2. Spectroscopic analysis [ADI] = adipate assimilation + [MLT] = malate assimilation + [CIT] = citrate assimilation + The IR, UV, mass spectrum and HPLC were determined at the [PAC] = phenyl-acetate assimilation micro analytical center of Cairo University, Egypt. (+) = positive result and () = negative result. 3.3. Reaction of the antibiotic with certain chemical test

For this purpose the following reactions were carried out: Mol- and phenyl alanine deamination are positive, whereas utiliza- ish’s reaction, Fehling reaction, Sakaguchi reaction, Ninhydrin tion of urea, levan formation from sucrose, production of in- reaction, Ehrlish reaction, nitroprusside reaction, ferric chlo- dole, methyl red and Voges–Proskauer, and poly b-hydroxy ride reaction, and Mayer reaction. butyrate accumulation are negative. It utilizes, D-ribose, D-glucose, D-fructose and mannitol and 3.4. Biological activity no growth on L-arabinose, D-xylose, D-mannose, D-galactose, meso-inositol, lactose, raffinose, sucrose, maltose, trehalose The minimum inhibitory concentration (MIC) has been deter- and starch was detected. Good growth was detected on mined by cup method assay (Kavanagh, 1972; National L-serine, L-leucine, L-lysine, L-proline, L-tyrosine, and L-argi- Committee for Clinical Laboratory Standards, 2003). nine. No growth on glycine, L-alanine and L-valine was de- tected. Good growth in the presence of NaCl up to 5% was 3.5. Characterization of the purified antibiotic detected. Good growth was detected within temperature range from 20 to 43 C. The antibiotic produced by P. aeruginosa, AZ-SH-B8 was tried to be identified according to the recommended international 4.2. Identification of bacterial isolate, AZ-SH-B8 by API20E system references of (Umezawa, 1967, 1977; Berdy, 1974, 1980a–c) and British pharmacopoeia 2000. The most potent bacterial isolate, viz. AZ-SH-B8, gives positive results with nitrate reduction, arginine dihydrolase, gelatin 4. Results hydrolysis, glucose assimilation and assimilation of mannitol, N-acetylglucosamine, gluconate, caprate, adipate, malate and 4.1. Characterization of the bacterial isolate, AZ-SH-B8 citrate (Table 2).

Identification of these strains to the species as well as their specific 4.3. Identification of the bacterial isolate, AZ-SH-B8 characteristics will be given here after for each individual strain. This isolate (Plate 1 and Table 1) was isolated from El- This was performed basically according to the recommended Sharkia governorate, thus symbolized AZ-SH-B8. Color of international Key’s viz. (Buchanan and Gibson, 1974; Krieg, colonies is yellow, Gram negative, motile, short rods, non- 1984; Hensyl, 1994). On the basis of the previously collected spore former, aerobic. It produces blue green water soluble data and in view of the comparative study of the recorded pigment, hydrolyses protein, and lipid, oxidase and catalase properties of bacterial isolate, AZ-SH-B8 Table 1 in relation (+), but do not hydrolyse starch and lecithin. The fluorescent, to reference strain, viz. P. aeruginosa, and confirmed by pyocyanin and carotenoid pigments can be produced. Degra- API20E strip system (Table 2) it could be stated that bacterial dation of esculine is positive; production of H2S, gelatine isolate, AZ-SH-B8 is suggestive of being related to P. aerugin- liquefaction, KCN, nitrate reduction, arginine dihydrolase osa, AZ-SH-B8 (Table 3). Biochemical studies on the production of Sparsomycin antibiotic by Pseudomonas aeurginosa 39

concentration of K HPO , MgSO Æ7H O and KCl is 1.1 g/l; Table 3 A comparative study of the characteristics of the 2 4 4 2 0.5 g/l and 0.5 g/l, respectively. bacterial isolate, AZ –SH-B8 in relation to Reference strain, P. aeruginosa. 4.6. Fermentation, separation, precipitation and purification Characteristics AZ-SH-B8 Pseudomonas antimicrobial agent(s) aeruginosa Morphological characteristic The fermentation process was carried out for 28 days at 35 C. Gram reaction Negative Negative Cell form Rod shape Rod shape Filtration has been determined through Whatman No. 1 and Motility Motile Motile followed by centrifugation at 5000 rpm for 15 min. Only clear filtrates (supernatant) were tested for their Physiological and biochemical characteristic antimicrobial activity. The clear filtrate (15 l) was adjusted Diffusible pigment production Blue-green Blue-green at pH 7.0 then the extraction process was carried out. Hydrolysis of Butanol was added to the fermentation broth at the level Protein + + Starch of 1:1 (v/v). The organic phase was collected, evaporated Lipid + + under reduced pressure using a rotary evaporator. The Lecithin (egg-yolk) + + residual syrup was dissolved in least amount of DMSO Levan formation + + and filtered. The filtrates were tested for their antimicrobial Arginine dihydrolase activity. Oxidase reaction + + Only one fraction was obtained with petroleum ether (b.p. Catalase test + + 40–60 C) by centrifugation at 5000 rpm for 15 min. Crude Phenyl alanine deamination + + deep green was tested for their antimicrobial activities by using Poly-b-hydroxybutyrate accumulation + + cup diffusion method. Growth at 41 C++ Separation of the antibiotic into individual components has Utilization of carbon sources been carried out by thin layer chromatography (TLC). L-Arabinose The obtained results revealed that one band at Rf 0.7 exhib- D-Xylose ited obvious inhibitory effects against the growth of both D-Ribose + + Gram positive and Gram negative bacteria and fungi (unicellu- D-Mannose lar and filamentous fungi). D-Glucose + + The purification of the antibiotic was carried out by using D-Fructose + + D-Galactose silica gel column chromatography. The active fractions were Mannitol + + concentrated. The maximum activity was recorded at fraction meso-Inositol No. 28. A summary of the amplified scheme for the extraction Sucrose was recorded and purification of the antibiotic was biosynthe- Trehalose sized by P. aeruginosa, AZ-SH-B8. Utilization of nitrogen sources L-Glycine d 4.7. Physicochemical characteristics of the purified antibiotic L-Alanine + L-Leucine + + The physical characteristics such as melting point 360 C and L-Valine d soluble in water, chloroform, n-butanol, methanol, ethanol, L-Lysine + + DMSO, carbon tetra chloride, but insoluble in petroleum L-Proline + + ether, hexane and benzene were investigated. L-Arginine + + L-Tyrosine + + 4.7.1. Elemental analysis + = positive, = negative, d = doubtful. A study of the elemental analysis of the antibiotic showed the following C = 41.13; H = 5.58; N = 11.07, O = 25.30 and S = 16.90: leads to an emperical formula of: C13H21N3O6S2. 4.4. Screening for the antimicrobial activities 4.7.2. Spectroscopic characteristics The antimicrobial agent produced by the bacterial isolate The spectroscopic characteristics of the antibiotic revealed the exhibited various degrees of activities against Gram positive presence of the maximum absorption peak in infra-red absorp- and Gram negative bacteria and fungi (unicellular and filamen- tion spectrum represented by 19 peaks (Fig. 1); UV at 270 and tous fungi) (Table 4). 300 nm (Fig. 2). Mass-spectrum showed that the molecular weight is 379 (Fig. 3) and NMR-spectrum was also determined 4.5. Factors effecting on the biosynthesis of the antimicrobial (Fig. 4). agent 4.7.3. Biochemical reaction of antibiotic Maximum antimicrobial activity biosynthesis could be re- The reactions revealed the detection of certain groups in the corded with different inoculum sizes for three discs; incubation molecule investigated. The antibiotic showed positive results period for 28 days; pH 6.0, temperature 35 C; glucose best with nitroprusside, ferric chloride and Mayer reactions (Table carbon source; peptone best nitrogen source as well as, the best 5). 40 H.M. Atta, H.G. Radwan

Table 4 Antimicrobial potentialities of the active microbial isolates isolated from various localities under solid state by fermentation using (plastic). Organism Mean values of inhibition zone (in mm) against number Bacteria Fungi G +ve Cocci G +ve Bacilli G ve strain Unicellular Multicellular Micrococcus Staph. B.subtilis, B.pumilus, E. coli, Klebsiella P. aeurginosa, Candida Asp. Penicillum lutues, aureus, NCTC NCTC NCTC pneumonia, ATCC 10145 albicans, flavus chresogenum ATCC 9341 NCTC 7447 10400 8214 10416 NCIMB 9111 IMRU 3669 AZ-SH-B8 27.0 26.0 27.5 27.0 26.0 26.0 24.0 22.0 21.0 20.0

Figure 1 IR spectrum of antimicrobial agent produced by Pseudomonas aeurginosa, AZ-SH-B8.

Figure 2 Ultraviolet absorbance of antimicrobial agent produced by Pseudomonas aeurginosa, AZ-SH-B8.

4.7.4. Characterization of the antimicrobial agent the antibiotic is suggestive of belonging to Sparsomycin antibi- On the basis of the recommended keys (Umezawa, 1977; otic (Table 6). Berdy, 1974, 1980a–c; British pharmacopoeia, 2000) for the identification of antibiotics produced by P. aeruginosa, 4.7.5. Biological activities of the purified antibiotic AZ-SH-B8 and in view of the comparative study of the Data of the antimicrobial spectrum of antibiotic indicated that recorded properties of the antibiotics, it could be stated that the antibiotic is fairly active against both Gram positive and Biochemical studies on the production of Sparsomycin antibiotic by Pseudomonas aeurginosa 41

Figure 3 Mass spectrum of antimicrobial agent produced by Pseudomonas aeurginosa, AZ-SH-B8.

Figure 4 NMR spectrum of antimicrobial agent produced by Pseudomonas aeurginosa, AZ-SH-B8.

Identification process has been carried out according to the Table 5 Summarizes the response of the antimicrobial agent Key’s giving in Bergey’s Manual of Determinative Bacteriol- produced by Pseudomonas aeurginosa, AZ-SH-B8 to certain ogy, 8th edition (Buchanan and Gibbson, 1974), Bergey’s biochemical reactions. Manual of Determinative Bacteriology, vol. I (Krieg, 1984) and Bergey’s Manual of Determinative Bacteriology, 9th edi- Chemical test Result Remark tion (Hensyl, 1994). The morphological characteristics and Molish’s reaction Absence of sugar moiety microscopic examination of the isolate were compared with Fehling test Absence of free aldehyde the identified bacteria. As the isolate Gram negative reaction, or keto sugar rod shape, motile, aerobic, oxidase test is positive it was iden- Ninhydrin test Absence of free-NH2 group tified as Pseudomonas (Hensyl, 1994). Sakaguchi reaction Arginin is Absent In view of the recorded data, the identification of bacterial Nitroprusside reaction + Presence of Sulfur Ferric chloride reaction + Presence of di-ketons group isolate AZ-SH-B8 was suggestive of being belonging to P. Ehrlish reaction Absence of indolic acid aeruginosa, AZ-SH-B8. Mayer reaction + Presence of nitro group In view of all the previously recorded data, the identifica- tion of bacterial isolate AZ-SH-B8 was suggestive of being belonging to P. aeruginosa, AZ-SH-B8, a bacterial with a wide spectrum antibiotic productivity (Radwan, 2008). Gram negative bacteria and unicellular and filamentous fungi Maximum antimicrobial activity biosynthesis could be re- (Table 7). corded withdifferent inoculum sizes for three discs; incubation period for 28 days (Adinarayana et al., 2002); pH 6.0 5. Discussion (El-Henawy, 2006); temperature 35 C(Kunnari et al., 1997; Atta, 1999); glucose best carbon source (Yasutaka et al., The P. aeruginosa, AZ-SH-B8 was isolated from Sharkia gov- 2004); peptone best nitrogen source (Hosokawa et al., 2001) ernorate. The isolate was found to produce a wide spectrum as well as, the best concentration of K2HPO4, MgSO4Æ7H2O antimicrobial agent against Gram-positive and Gram-negative and KCl is 1.1 g/l; 0.5 g/l and 0.5 g/l, respectively (Khalifa, bacteria and was slightly active against unicellular and filamen- 2008; Radwan, 2008). tous fungi when cultivated on plastic wastes (Ammar et al., The active metabolites were extracted by n-butanol at pH 2001; Scheller and Conrad, 2005). 7.0 (Atta et al., 2009a,b; Atta, 2010). 42 H.M. Atta, H.G. Radwan

Table 6 A comparative study of the characteristic properties of the antimicrobial agent produced by P. aeurginosa, AZ-SH-B8 in relation to reference antibiotic (Sparsomycin). Characteristic Purified antimicrobial agent Sparsomycin (1) Melting point 360 C 361 C (2) Molecular weight 379 379.45 (3) Chemical analysis C 41.13 41.13 H 5.58 5.58 N 11.07 11.07 O 23.30 23.30 S 16.90 16.90 Ultra violet 270 and 300 270 and 302

Formula C13H21N3O6S2 C13H21N3O6S2 Active against Active against Gram positive and Active against Gram positive Gram negative bacteria and and Gram negative bacteria unicellular and filamentous fungi and unicellular and filamentous fungi ND = no data and = negative results.

Table 7 The antimicrobial spectrum of the antibiotic by using chloroform, n-butanol, methanol, ethanol, DMSO, carbon tet- paper disc diffusion method (Kavanagh, 1972). ra chloride, but insoluble in petroleum ether, hexane and ben- zene. Similar results were recorded by (Lotfi et al., 2003; El- Test organisms MIC (lg/ml) concentration Tayeb et al., 2004; Atta, 2010). of antibiotic produced by P. aeurginosa, AZ-SH-B8 A study of the elemental analysis of the antibiotic showed the following C = 41.13; H = 5.58; N = 11.07, O = 25.30 (A) Bacteria and S = 16.90: lead to an imperical formula of: (a) Gram positive cocci C H N O S . Staph. aureus, NCTC 7447 3.9 13 21 3 6 2 Micrococcus luteus, ATCC 9341 2.6 The spectroscopic characteristics of the antibiotic revealed (b) Gram positive bacilli the presence of the maximum absorption peak in UV at 270 Bacillus subtilis, NCTC 10400 2.6 and 300 nm, infra-red absorption spectrum represented by 19 (c) Gram negative bacteria peaks. Mass-spectrum showed that the molecular weight is Escherichia coli, NCTC 10416 5.85 379 and NMR-spectrum was also determined. The biochemi- Klebsiella pneumonia, NCIMB 9111 7.8 cal tests of the antibiotic gave positive reaction with Nitroprus- (B) Fungi side, Ferric chloride and Mayer reactions (Table 7). Similar (a) Unicellular fungi studies were conducted by (Bastian et al., 2001; Arya, 2005; Candida albican, IMRU 3669 31.25 Imnagaki et al., 2006; Sekiguchi et al., 2007; Radwan, 2008). (b) Filamentous fungi The MIC of the antibiotic under study exhibited to be fairly Asp. flavus, IMI 111023 46.87 active against both Gram positive and Gram negative bacteria Penicillium chrysogenium 62.5 and unicellular and filamentous fungi. Similar investigations and results were attained by Matsuoka and Saski (2004), Imnagaki et al. (2006) and Sekiguchi et al. (2007). Identification of the antibiotic according to recommended international keys indicated that the antibiotic is suggestive Separation of antibiotic into individual components has of belonging to Sparsomycin antibiotic (Umezawa, 1977; been tried by thin-layer chromatography using a solvent sys- Berdy, 1974, 1980a,b,c; Fredrick and Noller, 2003; Atta tem composed of chloroform and methanol (24:1, v/v) as the et al., 2009a,b). developing solvent (Zhang et al., 2007; Atta et al., 2009a,b). The band with an Rf value at 0.7 which indicated the presence of one compound. For the purpose of purification process, the References antibiotic was allowed to pass through a column chromatogra- phy packed with silica gel and the eluting solvent was com- Adinarayana, K., Ellaiah, P., Srinivasulu, B., Bhavani, R., Adinara- posed of chloroform and methanol (9:1, v/v), 50 fractions yana, G., 2002. Response surface methodological approach to were collected and tested for their activities. The maximum optimize the nutritional parameters for neomycin production by activity was recorded at fraction No. 28. Similarly, many Streptomyces marinensis under solid-state fermentation. Process workers used a column chromatography packed with silica Biochem. 38, 1565–1572. Ammar, M.S., Haroun, B.M., El-Sehrawi, M.H., Atta, H.M., 2001. gel and an eluting solvent composed of various ratios of chlo- Fermentation, isolation, characterization and biological activities roform and methanol similarly (Howells et al., 2002; Kharel of foromacidin A, B & C antibiotics produced by Pseudomonas et al., 2004; Radwan, 2008). gladioli under solid state fermentation (S.S.F.) condition. In: 4th The physico-chemical characteristics of the purified antibi- International Conference (Science and development) Cairo, 27–29 otic revealed that, melting point is 360 C, soluble in water, March 2001. Biochemical studies on the production of Sparsomycin antibiotic by Pseudomonas aeurginosa 43

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