studies on the production of actinomycin by nocardioides luteus,...

1282Current Trends in Biotechnology and PharmacyVol. 5 (3) 1273 -1281 July 2011, ISSN 0973-8916 (Print), 2230-7303 (Online)

AbstractEfforts have been devoted to explore the

antimicrobial activity of local egyptianactinomycetes isolates. 127 pure actinomycetesisolates were recovered from the stones at thelocation of the tomb of Tell Basta (the site of theruins of the old temple of Bast) which is south-east of the Nile Delta at the township of Zagazigin Egypt. The most competent isolate showedpromising activity against some pathogenicyeasts, fungi and bacteria. It was identified asNocardioides luteus. Using 2 ml of 2 days oldinoculum of Nocardioides luteus in fermentationmedium consisting of (g/l): starch, 10; yeastextract, 4; NaCl, 10; K

2HPO

4, 2; MgSO

4.7H

2O,

0.05; CaCO3, 0.02 and FeSO

4.7H

2O, 0.01,

adjusted before autoclaving at pH 7 andincubated for 7 days with shaking rate 200 rpmresulted in maximum antimicrobial activityagainst Candida albicans ATCC 10231. Theminimum inhibitory concentration (MIC) of theculture was determined to be 0.4 µg/ml. Trialshave been conducted to separate and identify theactive compound(s) responsible for theanticandidal activity. Based on the spectroscopicdata from UV, IR, 1H-NMR and 13C-NMRspectra, the isolated compounds were identifiedas derivatives of actinomycins class. To ourknowledge, this is the first time not only to studysystematically the bioactivities of Nocardioidesluteus; but also to report actinomycin production

by a Nocardioides spp. Cytotoxicity of both theculture filtrate extract and the impure compoundwere tested against different tumor cell lineswhere they showed remarkable cytotoxicityagainst all challenged cell lines.

Keywords: actinomycetes, antimicrobial,actinomycin, cytotoxicity.

Introduction Actinomycetes are a group of bacteria,

whose members are famous for producingantibiotics for medicinal use. They provid us witha large database of compounds with differentstructures; including aminoglycosides,anthracyclines, glycopeptides, â-lactams,macrolides, nucleosides, peptides, polyenes,polyketides, quinones, actinomycins,tetracyclines and even alkaloids. Only a veryminute quantity of terrestrial and marineactinomycetes were actually isolated and testedfor antibacterial activity. In the time intervalbetween 1981-2006, 19 out of 24 naturallyoriginating compounds acting as the startingmaterial of marketed drugs, were isolated fromsoil actinomycetes (1,2,3).

Within the actinomycetes, Streptomyces isthe most abundant genus that accounts for 70-80% of secondary metabolites, with smallercontributions from genera such asSaccharopolyspora, Amycolatopsis,

Studies on the Production of Actinomycin byNocardioides luteus, a Novel Source

El-Refai, H.A.1*, AbdElRahman1, H.Y., Abdulla, H.2, Hanna, Atef G.1,Hashem, A.H.3, El-Refai A.H.1 and Ahmed, E.M.1

1Chemistry of Natural and Microbial Products Dept., National Research Center, Dokki, Cairo, Egypt2 Dept. of Botany, Faculty of Science, Suez Canal University, Egypt3Dept. of Microbiology, Faculty of Pharmacy, Cairo University, Egypt

*For Correspondence - [email protected]

Studies on the production of actinomycin by Nocardioides luteus

Current Trends in Biotechnology and PharmacyVol. 5 (3) 1282-1297 July 2011, ISSN 0973-8916 (Print), 2230-7303 (Online)

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Micromonospora and Actinoplanes (4).Although Candida spp. occur among normalbody flora in mouth and gastrointestinal tract,they can be responsible for many types ofdiseases in both humans and animals rangingfrom superficial mucocutaneous illness to deeplife-threatening invasive, systemic diseases thatcan occur in different organs thus, causingdifferent signs and symptoms. There are morethan 20 species of Candida, of which Candidaalbicans is the most common. Yet, all Candidaspp. cause the same spectrum of diseaseshowever, differences in disease severity andsusceptibility to antifungal agents were reported.

Among the increase in the incidence ofnosocomial fungal infections, those caused byCandida albicans, has increased proportionally.This problem appears to be a global one; in large,university-affiliated hospitals (5).

Therefore, new sources and strategies arerequired to find new broad-spectrum antifungalmolecules to combat these pathogens. For thisreason, intensive program searching for newantibiotics is running worldwide mainly byscreening for members of the cladeactinomycetes as being a rich, unrivaled sourceof antibiotics.

Impressed by these facts, our work aims toinvestigate the potentialities of some locallyisolated actinomycetes to produce bioactivecompounds acting against Candida albicanspathogen. The optimal fermentation conditionsfor bioactive compound production by the mostactive actinomycetes isolate have beeninvestigated. In addition, trials to identify theactive substances were reported.

Materials and MethodsIsolation of actinomycetes : Stone samples werecrushed to a fine powder. Stone powder wasdispersed in sterile 1/4 strength Ringer’s solutionsupplemented with 0.001 % Tween 80 and

shaken vigorously for up to one hour (6).Suspensions were then serially diluted inphosphate buffer. Spread plate method wasapplied for actinomycetes isolation; 0.1 ml ofthe appropriate dilution was spread evenly overthe corresponding overnight dried mediumplates. Five different isolation media known tosupport the growth of actinomycetes were usednamely; starch casein medium (7), arginineglycerol salts medium (9), starch nitrate medium,M3 medium (9) and ISP medium 5 (Glycerol-Asparagine Agar) (10).

After incubation, representatives of eachcolony form were picked off and streaked toobtain a pure culture. They were finally storedon starch casein slopes at 4 °C, and as sporesuspensions in 20 % glycerol at 20 °C (11).

Target organisms : The isolated strain activitieswere tested against: Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC 27953and Alcaligenes faecalis NRRL B-170 asexamples for Gram negative pathogens,Staphylococcus aureus ATCC 29213, Bacillussubtilis NRRL B-4219 and Micrococcus luteusNRRL B-287 as examples for Gram positivepathogens, Aspergillus niger NRRL 599,Aspergillus flavus NRRL 1957 and Trichophytonmentagrophytes ATCC 9533 as examples forfungal pathogens and Candida albicans ATCC10231, Saccharomyces cerevisiae ATCC 10275as examples for yeasts.

The challenged fungi were maintained andcultured on Dox’s agar medium that has thefollowing composition (g/l): sucrose, 20; NaNO

3,

2; MgSO4.7H

2O, 0.5; KCl, 0.5; FeSO

4.7H

2O, 0.1;

agar, 20. The pH of the medium was adjusted to7.0-7.2 before sterilization.

The tested yeasts were maintained andcultured on malt yeast medium that has thefollowing composition (g/l): glucose, 4; yeastextract, 4; malt extract, 20; agar, 20. The pH of

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the medium was adjusted to 7.0-7.2 beforesterilization. The used bacteria were maintainedand cultured on nutrient agar medium that hasthe following composition (g/l): glucose, 20;peptone, 10 and agar, 20. The pH of the mediumwas adjusted to 7.0-7.2 before sterilization.

Screening of actinomycetes isolates forantimicrobial activity : Five days old slants wereharvested by scratching with sterile distilledwater and poured into 250 ml Erlenmeyer flaskscontaining 50 ml sterile starch casein nitratebroth. The production flasks were incubated in arotary shaker at 150 rpm and 30 °C ± 2 °C for 7days. At the end of the incubation period, thecultured broths were filtered and assayed for theiractivity against the target organisms using agarwell diffusion method. 0.1 ml of each culturefiltrate was placed in holes (0.9 cm diameter)made in seeded agar plates. The agar plates wereincubated at 30 °C for 24 hours for bacteria andfor 48 hours for fungi and yeasts. The diametersof the resulting inhibition zones were thenmeasured and considered to estimate the isolatesantimicrobial potentialities.

Characterization of actinomycete isolate ofchoice : Only the actinomycetes isolate of thehighest activity was subjected to identificationat the genus and species level. A wide range ofmorphological, physiological and biochemicalcriteria were used to characterize this isolate.These criteria include morphological features,pigmentation, utilization of organic compoundsand resistance to antibiotics as well asdiaminopimelic acid in whole cell hydrolysate.

Cultivation factors affecting the production ofantifungal compound(s) : Unless otherwisestated, the basal fermentation conditions were:4 ml of distilled water was added to a 5 day oldslant and a spore suspension in 250 mlErlenmeyer flask containing 50 ml of starch

casein nitrate medium. The flask was incubatedin a rotary shaker at 150 rpm, 30 °C± 2 for 2days. 2 ml was used to inoculate 50 ml of starchcasein nitrate medium in 250 ml Erlenmeyerflasks. The flasks were incubated on anincubatory shaker at 30 °C at 150 rpm for 7 days.

At the end of the fermentation process, theantifungal activity was tested against Candidaalbicans using agar well diffusion method. Also,the cell dry weight in the fermentation broth wasdetermined by filtering the flask content on a pre-weighed filter paper (Whatman no.1). The filterpapers were dried at 60-70 °C until a constantweight. The difference between the weights ofthe filter papers after and before filtration shouldcorrespond to the cell dry weight.

Effect of fermentation period, inoculum age,size : The effect of prolongation of incubationfor maximum growth and antibiotic productionwas observed up to 12 days of incubation. Todetermine the effect of the age of inocula, 2 mlof the inoculum broth were taken at time intervals2, 3, 4, 5, 6, 7 days to inoculate separate flasksof the fermentation media. In addition, differentinoculum volumes (ranging from 2-8 % v/v) wereused to inoculate the fermentation media todetermine the most suitable volume forproducing antifungal compound(s).

Carbon and nitrogen supplements : Six carbonsources namely: starch, fructose, maltose,glucose, mannose, and sucrose were tested; eachat 1 % level. After selection of the best source,different concentrations (5, 10, 15, 20 gm/l) weretested.

Different organic and inorganic nitrogensources were individually added as sole nitrogensources in fermentation media to replace KNO

3

and casein. These are: peptone, yeast extract,corn steep liquor, sodium nitrate and ammoniumchloride. They were added according to nitrogen



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equivalent basis. After selection of the bestsource, different concentrations 2.5, 3, 3.76, 4,4.5 g/l were investigated.

Mineral salts : Different concentrations (0-4 %)of NaCl were used in the cultivations todetermine the best for antifungal production.Different concentrations (0-1.6 %) of K

2HPO

4

in the culture medium were tested.

Different pH and shaking rate : Varied initialpH values were tested using different bufferingsolutions. A control flask was done by adjustingthe pH at 7 by 1M HCl and 1M NaOH. The buffersolutions used were: Citrate phosphate buffer atpH 6, 6.5 and 7, Phosphate buffer at pH 7, 7.5and 8 and Tris-maleate buffer at pH 8 and 8.5.Different rpm were investigated (100, 150, 200)as well as static conditions were maintained.

Extraction of the antifungal bioactivecompound (s) : After fermentation of the media,the culture broth was filtered in vacuo and thefiltrate was extracted with equal volume of ethylacetate. The latter was dried under reducedpressure using a rotary evaporator at atemperature not exceeding 50 °C, yielding anorange-yellow residue (270 mg).

Thin Layer chromatography (TLC) : Ethylacetate extract of the culture filtrate wasdeveloped on TLC plate using solvent systemconsisting of Benzene: Ethyl acetate: Acetic acid(1: 3.5: 0.5 v/v) to evaluate the secondarymetabolite constituents of Nocardioides luteus.

The chromatogram was detected under aUV lamp and each spot was scratched from theTLC plate and dissolved in ethyl acetate. Silicagel was removed by centrifugation at 2000 rpmfor 10 min. The supernatant was air-dried andthe constituents were tested by paper disc assaymethod (12).

Isolation of the active compound : The residue(270 mg) was dissolved in 5 ml ethyl acetate and

adsorbed on silica gel G for columnchromatography. The mixture was added at thetop of a column chromatography (45 cm long ×4 cm wide) using silica gel G as an adsorbent,benzene/ ethyl acetate (1:1) was used as aneluent. Fractions of each equal to 15 ml, werecollected individually. Then, the polarity wasincreased after 53 fractions to benzene/ ethylacetate (1:2) for 97 fractions followed by ethylacetate for 25 fractions then, 5 % methanol inethyl acetate for 11 fractions and finally methanolfor 39 fractions.

Fractions were grouped in accordance totheir TLC profiles and tested for theirantimicrobial activity (Bioassay- guidedfractionation). The TLC plates revealed that thefractions were to be pooled into 8 fractions, only5 of which were active against C.albicans. Themost active was subjected to further purificationby re-fractionation using columnchromatography.

The elution with benzene/ ethyl acetate(1:2) afforded 97 fractions. The fractions from45 to 70 were collected together to give rise to80 mg of an orange - yellow mixture. It was re-fractionated on flash silica gel column (45 cmlong × 4 cm wide) using chloroform/ methanol(9:1) as eluent. Fractions, each equal to 10 ml,were collected individually. The fractions from27 to 34 were collected together once again andpurified on RP-18 column (30 cm long × 4 cmwide) using 20 % water in methanol to afford 10mg of semi-pure compound. Further purificationfailed due to the instability of the compound.

The partially purified active materialobtained was subjected to various examinationsby ultraviolet, infrared, 1H-NMR 500MHz and13C-NMR.

Cytotoxicity assay to human tumor cell lines:Sulphorhodamine-B (SRB) assay of cytotoxicactivity (13):

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These tests were done at National CancerInstitute, Pharmacology Unit- Tissue CultureDepartment.

Cell lines: HEPG2 (liver carcinoma), HeLa(cervix carcinoma), MCF7 (breast carcinoma),HCT116 (colon carcinoma), HEP2 (larynxcarcinoma), CACO (colon carcinoma and HFB4(normal melanocytes) were maintained andcultured on RPMI-1640 medium supplementedwith penicillin/streptomycin (100 units/mlPenicillin and 2 mg/ml Streptomycin) and 10 %fetal bovine serum (heat inactivated at 56 °C for30 min).

Cells at exponential phase cultures wereharvested by trypsinization and seeded in 96-wellmicrotiter plates at a concentration of 5x104-105

cell/well for 24 hrs. Cells were then incubatedwith different concentrations (0, 1, 2.5, 5 and 10ìg/ml in DMSO) of both the culture filtrate andthe partially-purified compound. The volumewas completed to total volume of 200 ìl/wellusing fresh medium and incubated for 24, 48 and72 hrs in triplicate. Control cells were treatedwith vehicle alone. After incubation, the cellswere fixed with 50 ìl cold 50 % trichloroaceticacid for 1 hr at 4 °C, washed with distilled waterand then stained for 30 min at room temperaturewith 50 ìl 0.4 % SRB dissolved in 1 % aceticacid. The wells were washed with 1 % aceticacid to dissolve unbound SRB, the plates wereair-dried and the dye was solubilized with 100ìl/well of 10 mM tris base (pH 10.5) for 5 minon a shaker at 1600 rpm. The optical density(O.D.) of each well was measuredspectrophotometrically at 564 nm with an ELISAmicroplate reader. The relation between survivalfraction and extract concentration was plotted togive the survival curve of each tumor cell line,and the IC

50 was calculated.

Survival fraction = O.D. (treated cells)/O.D. (control cells).

Results and DiscussionIn the current study, a total of 127 pure

actinomycetes isolates were isolated from thestone of monuments in Tell BastaTombs, Zagazig city. Bacteria isolated fromweathered rock samples in the Mediterraneanbasin were, in the majority of cases,actinomycetes and other Gram-positive bacteria.The other possible reason for these high countswas the ability of actinomycetes to cope withdry habitats. They were frequently isolated fromstone environments and many of their membersexisted for extended periods as restingarthrospores that germinated in the occasionalpresence of exogenous nutrients (14,15).

In view of the decline in the discovery ofnew antifungal compounds, the isolatedactinomycetes were assayed for theirantimicrobial activities, specially theiranticandidal activity.

Among the isolated 127 actinomycetes,only 10 isolates showed promising activity.However, relatively higher anticandidal activitywas exhibited by a culture referred to as isolateno.9. The antimicrobial activities offered by thisisolate are recorded in Table 1.

According to the plan of this work, theisolate with highest activity was only consideredfor identification. The criteria used in theidentification were micromorphology, detectionof diaminopimelic acid (DAP) isomers in thewhole cell hydrolysate, whole cell diagnosticsugars and presence of mycolic acids (16). Basedon these criteria, the isolate was found to belongto the genus Nocardioides. The isolate wasfurther classified to the species level bymorphological, physiological andchemotaxonomic features. This identificationwas according to Bergey’s Manual of SystematicBacteriology (17) and Bergey’s Manual ofDeterminative Bacteriology (18). The criteria



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Table 1 The antimicrobial activities of actinomycetes isolate No.9

Tested microbial pathogen Diameter of inhibition zone (mm)Escherichia coli ATCC 25922 20Bacillus subtilis NRRL B-4219 30Pseudomonas aeruginosae ATCC 27953 -Staphylococcus aureus ATCC 29213 20Candida albicans ATCC 10231 30Saccharomyces cerevisiae ATCC 10275 25Aspergillus niger NRRL 599 30Aspergillus flavus NRRL 1957 -Alcaligenes faecalis NRRL B-170 30Micrococcus luteus NRRL B-287 10Trycophyton mentagrophytes ATCC 9533 14

Table 2 Identification criteria of actinomycetes isolate no. 9 (Nocardioides luteus)

Experimental Parameter Character Experimental Parameter Character

Aerial mycelia color Grey L-diaminopimelic acid in whole cell hydrolysate +Vegetative mycelia color Yellow-Brown Growth on sole carbon sources (1.0 % w/v)Melanin on tyrosine agar + Sucrose +Degradation of - Lactose +Casein + Salicin +Xanthene - D-Fructose +Hypoxanthene- Galactose +Urea + D-Mannose +Growth at D-Mannitol +45oC - Rafinose +10oC + D-Xylose +pH 4.3 + L-Arabinose +Growth in the presence of (%w/v) CellobiosemNaCl (4) + Inositol +NaCl (7) + Rhamnose +NaCl (10) + Glucose +Crystal violet (0.0001) - Sodium acetate (0.1 %) +Phenol (0.1) Sodium citrate (0.1%) +Resistance to antibiotics Sodium propionate (0.1 % ) +Gentamicin (10 µg) Growth on sole nitrogen source (0.1 %)Streptomycin (10 µg) - L-Arginine +Neomycin (10 µg) - L-Histidine +Penicillin G (10 units) - L-Cysteine +Rifampicin (5 µg) - L-Valine +Tetracycline (30 µg) - L-Phenyl alanine +Tobramycin (10 µg) + Potassium nitrate +Erythromycin (15 µg) - L-Threonine +Bacitracin (10 µg) - L-Serine +Chloramphenicol (30 µg) - L-Methionine +

Novobiocin (5 µg) -

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used in the identification were listed in materialsand methods, results of which are given in Table2.

The genus Nocardioides is easilydifferentiated from all other actinomycetes onthe basis of the diaminopimelic acid type in thecell wall peptidoglycan, menaquinone profile,and the cellular fatty acid profile (19,20).

The genus Nocardioides is one of a fewgenera that have LL-2,6-diaminopimelic acid(LL-DAP) as the diagnostic diamino acid in thecell wall peptidoglycan layer(21). This feature,together with glycine in the cell wallpeptidoglycan, indicates that this genus is wallchemotype I (22). This chemical markerdistinguishes the genus Nocardioides from manyother actinomycetes genera that possess othertypes of diamino acids in the cell wallpeptidoglycan.

Regarding the bioactivities of theNocardioides spp., only a few Nocardioidesstrains were found to produce antibiotics(23,24,25). Strain ATCC 39419, which produceda novel anti-tumor antibiotic, sandramycin, wasoriginally assigned to Nocardioides (26) but haslater been classified as a member of a new genusKribbella (27).

However, to our knowledge, this is the firststudy to investigate the ability of a Nocardioidessp. to produce antibiotics in detail; regarding thephysiological parameters that affect theproduction followed by an attempt to isolate andidentify the active compound(s).

Cultivation parameters : Since antibiotics aresecondary metabolites synthesized by pathways,which are often connected and influenced byprimary metabolism, thus, frequently anintermediate metabolite from primarymetabolism serves as precursor for thebiosynthesis of the antibiotic. Therefore, the

composition of the culture medium, closelyconnected with the metabolic capacities of theproducing organism, greatly influences thebiosynthesis of antibiotics. Changes in the natureand concentration of carbon and nitrogensources, phosphorus concentration and traceelements have been reported to affect antibioticbiosynthesis in different organisms. Results (Fig.1) showed that activity and growth graduallyincreased with increasing the incubation periodtill the activity reached maximum after 7 days



of incubation then it began to decline with timewhile the growth nearly remained constant after7 days. These results revealed that theanticandidal metabolite was early produced andreached maximum at the stationary phase ofgrowth. The cessation of growth in the stationaryphase is most commonly caused by theexhaustion of the essential nutrients of themedium as well as the accumulation ofundesirable metabolites. Similarly, the optimalincubation period required for maximum growthand antibiotic yields by Streptomyces sp. 201 wassix days (28). On the other hand, the highestbiomass and antibiotic production ofStreptomyces sp. KEH23 is observed after 96 hof incubation. However, after 108 h, the diameterof the inhibition zones dropped gradually (29).

Fig. 1. Effect of different fermentation periods onanticandidal activity of Nocardioides luteus

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The performance of a microbial culture canbe strongly influenced by the type of theinoculum as shown in Fig. 2. Thus, the age andsize of the inoculum should be optimized toimprove the performance of the fermentationprocess. Our results showed that maximuminhibition zone, corresponding to maximumanticandidal activity, was observed at inoculumage 48-72 hrs, while both the biomass yieldremained unaffected. On the other hand, the cellgrowth increased by increasing the inoculumsize, increase in anticandidal activity was notobserved on using more than 2 ml inoculum/50ml production medium.

The anticandidal activity production(Fig.3A) seems to be no way correlated to thebiomass yield. Starch showed maximuminhibition zone, whereas maximum growth wasproduced by maltose. Minimum inhibition zonewas produced by fructose while minimumbiomass was recorded by sucrose. These findingswere in agreement with the fact that secondarymetabolite production in actinomycetes is oftenstimulated by slowly assimilated complexcarbohydrates or oils in the production media,while it decreased when more rapidly utilizedmonosaccharide such as glucose were present(30). A possible explanation of this phenomenon

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Fig. 2. The growth and anticandidal activity as affected by N.luteus inoculum

Fig. 3. Role of carbon nutrition on the growth and anticandidal activities of N.luteusA. Effect of carbon source B. Effect of starch concentration

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is that glucose or other simple carbon sourcescause catabolite repression in which theproduction of an enzyme of secondary metabolitebiosynthesis is inhibited.

The growth profile and antibacterialbiosynthesis by the tested organism was not onlyaffected by the nature of the used carbon source,but also with its level. Results (Fig. 3B) showedthat the activity increased with increasing thestarch concentration till it reached a maximumof 30 mm at 10 gm/l after which it remainedconstant while the dry weight values keptincreasing with increasing starch concentrations.Antibiotic production from alkaliphilicactinomycetes isolate A2D, identified as S.tanashiensis strain A2D, was higher in mediumhaving glucose (1 %) as carbon source whilehighest biomass resulted on using 1.5 %(w/v)glucose concentration (31). However, maximumgrowth and antibiotic production byStreptomyces sp. 201, were obtained in mediumsupplemented with mannitol as a sole carbonsource followed by sucrose and glycerol (32).

Our results showed that organic nitrogensources such as peptone, corn steep liquor andspecially yeast extract were highly favorable foranticandidal activity although this doesn’tnecessarily correlate to the growth ofNocardioides luteus, as shown in Fig. 4A. Onthe other hand, the tested inorganic nitrogensources resulted in a decreased anticandidalproduction that also doesn’t correlate withgrowth. Maximum inhibition zone was observedon using yeast extract. Therefore, it was chosenas a sole nitrogen source instead of casein andKNO

3 in the proceeding work. The activity

increased with increasing the nitrogen sourceconcentration till it reached a maximum at theconcentration 3.76 gm/l, then constant throughthe concentration 4 gm/l. Thereafter, it began todecrease. The dry weight increased gradually aswell by increasing the yeast extractconcentrations until reaching 4 gm/l, after whichit started to decrease, Fig. 4B. The superiority ofthe organic nitrogen sources has been reportedby many investigators (30-33).



Fig. 4. Role of nitrogen nutrition on the growth and anticandidal activity of N.luteusA. Effect of nitrogen source B. Effect of nitrogen concentration

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Evidently, the activity was maximal, withan inhibition zone diameter of 37.7 mm, on usingsodium chloride at concentration 1 gm %. Theantifungal activity at a greater NaClconcentration decreased to reach 30 mm at 4gm% concentration (Fig. 5).

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Antibiotic biosynthesis by certainhalophytic actinomycetes species was found tobe optimized at specific NaCl levels (34,35).Similarly, the optimum salt requirement of thestrain Streptomyces tanashiensis strain A2D forantibiotic production in medium containingcarbon source (glucose) and nitrogen source(soybean). They found that 2 % NaCl wasoptimum for maximum growth (4.9 mg/ml) aswell as antimicrobial compound production (20.9mg/ml). Further increase in salt concentrationreduced the antimicrobial agent biosynthesis(36).

Similarly, the role of K2HPO

4 on the growth

and anticandidal activity was investigated. Theproductivity of the experimental organism wasoptimized at 0.1 gm% (Fig. 6). However thedepletion of the tested salt from the fermentationmedium resulted in a decrease in the antifungalactivity. The dry weight yield increased graduallywith increasing K

2HPO

4 concentration until 1.2

gm% after which it remained constant. It was

Fig. 5 Effect of NaCl concentration

Fig. 6. Growth and anticandidal activity ofN.luteus as affected by K

2HPO

4

reported that phosphate ions seemed to stimulatethe process of primary metabolite production atthe expense of secondary metabolite. Also, theyreported that the highest dipotassium hydrogenphosphate concentrations studied at 1.6 g/lproved to provide non limiting growth conditionswithout affecting the antibiotic production (33).It was also noted that the excess PO

4 altered the

biochemical composition of the myceliumprotoplasm and affected the physiologicalfunction of the cell and decreases thebiosynthesis of the antibiotic (37). Conversely,phosphate at all concentrations inhibited theproduction of inhibitory substance(s) byStreptomyces (Ds-104) strain (36).

The pH value of the cultivation medium isvery important for growth of the microorganism,characteristic of their metabolism and hence forbiosynthesis of metabolites. The hydrogen ionconcentration may have direct effect on the cellor it may indirectly affect it by varying thedissociation degree of the medium components.Results showed that both production and growthwere maximal in the neutral medium either onusing buffers or by initial adjustment with 1MHCl and 1M NaOH. It is also noted that tris-maleate buffer, although not causing a

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remarkable decrease in activity yet, it resultedin a great decrease in the cell biomass (Data notshown). Increasing the pH value of the culturemedia of Streptomyces spp. led to an increase inthe antifungal production up to certain limit (pH7.0) above which any increase in the pH valuewas accompanied by a decrease in the antifungalproduction and consequently the antagonisticactivity. Maximum antifungal activity recordedby Streptomyces strain Ds-104 occurred on usingfermentation medium at pH range 7.0-7.5. Itafforded an inhibition zone diameter rangebetween 12 and 18 mm. The results obtained byother scientists also assured that the neutral rangeof pH value enhanced the formation of

anticandidal agents from different Streptomycesspp. (36-39).

With respect to the role of the agitation rate,the obtained results revealed that the maximumactivity was obtained at 200 rpm, though cellbiomass yields decreased with increasing theagitation speed being maximal at static state (Fig.7). Antifungal antibiotic production by the strainStreptomyces rimosus MY02 increased slightlycorresponding to agitation rate. But antifungalantibiotic production had no significantdifferences at 150–200 rpm. Finally, theyconcluded that the best agitation rate was 180rpm (40).



Table 4. The antitumour activities exhibited by N. luteus bioactive extract

Doxorubicin Crude Extract Semi-pure Tested cell linecompound

3.96 µg/ml 3.43 µg/ml 2.82 µg/ml HFB4 (normal melanocytes)5.5 µg/ml 2.51 µg/ml 2.99 µg/ml HEPG2 (liver carcinoma cell line)2.97 µg/ml 3.28 µg/ml 2.86 µg/ml MCF7 (breast carcinoma cell line)4.89 µg/ml 3.13 µg/ml 2.4 µg/ml HEP2 (larynx carcinoma cell line)3.64 µg/ml 2.67 µg/ml 2.82 µg/ml HELA (cervical carcinoma cell line)3.43 µg/ml 2.82 µg/ml 2.97 µg/ml CACO (colon carcinoma cell line)3.743 µg/ml 3.74 µg/ml 2.97 µg/ml HCT116 (colon carcinoma cell line)

-Table 3. The TLC profile of the different constituents responsible for the anticandidalactivity of N.luteus

Band Retention factor (Rf) Inhibition zone (mm)

1 0.07 202 0.20 21.53 0.47 264 0.6 195 0.68 13.56 0.82 17.5

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Isolation, purification and identification of theanticandidal component (s) : The TLCchromatogram for ethyl acetate extract of theculture filtrate of Nocardioides luteus developedas 6 major bands. By testing their activity againstC.albicans using paper disc assay method, theyall showed considerable activity as shown inTable 3.

The compound was subjected several timesto chromatographic purification but it could notbe obtained as a pure sample. The semi-purecompound was identified from its UV, IR, 1H-NMR and 13C-NMR spectra and comparing thesespectra with published data (41) as derivative ofthe actinomycins class of compounds. Its UVspectrum showed maximum absorption (ë

max in

CH3OH) at 224, 427 and 444 nm.

Its IR spectrum showed the characteristicpeaks of NH group at 3433 cm-1, aliphatichydrogens (CH, CH

2 and CH

3) at 2966, 2924 and

2855 cm-1. The amide group (CONH) signalappeared at 1648 cm-1.

The 1H-NMR measurement showed thecharacteristic signals of the phenoxazonechromophore at δ 2.21 (CH

3 at position 4), 2.52

(CH3 at position 6), 7.34 (d, H-7) and 7.61 (d,

H-8). The presence of two peptide rings in theactinomycin structure was established by thepresence of 3 amide signals at δ 8.19, 8.01 and7.73. Also, the methyl groups of valine andsarcosine amino acids gave the signals at δ 2.9(one methyl) and 2.86 (two methyl groups).

The 13C-NMR measurement showed thecharacteristic signals of the phenoxazonechromophore at δ 7.8 (CH

3 at position 4) and δ

15.11 (CH3 at position 6), in addition to the

carbonyl group at δ 173.39 (C-3). The carbonylsignals in between δ110-140 were attributed tothe other carbons of the phenoxazone group. Thesignals corresponding to the amide and lactonegroups of the peptide system were detected inbetween δ 160-172

The actinomycins are a family ofchromopeptide antibiotics, differing only in theamino acids present in the pentapeptide chains,which are elaborated by certain members of orderActinomycetales. Actinomycin was first isolatedfrom a culture of Streptomyces antibioticus byWaksman and Woodruff (40,41). It was the firstantibiotic to be produced after penicillin. Thevarious products have been designated A, B, C,D, I, J, and X(42,43,44). It has been shown,however, that these actinomycins were nothomogeneous substances but consisted of anumber of closely related biologically activecomponents. Evidence also suggested that theA, B, D, and X complexes might consist ofidentical components but differ in the relativeamount of each component present. Structuralstudies have revealed that the actinomycinmolecule is composed of two polypeptides linkedto a chromophoric quinonoid moiety which isthe same for all components. Differences in thecomponents of an actinomycin complex or ofdifferent complexes are due to variations in thenumber, arrangement, and kinds of amino acidspresent in the peptides.

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Fig. 7. Growth and anticandidal activity of N.luteus asaffected by the agitation rate.

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Since its discovery by Waksman andWoodruff in 1940, actinomycin was obtainedfrom the culture media of a number ofactinomycetes species; Streptomyces,Actinoplanes and Micromonospora by variousinvestigators and was isolated in crystalline form.Yet, this is the first time it was reported to beisolated from a Nocardioides sp not to mention,Nocardioides luteus.

Cytotoxicity assay : Due to the broad spectrumof the activity of the culture filtrate extract andits activity against Saccharomyces cerevisiae,which is used as biological model to establishthe cytotoxicity and cytostaticity of natural and/or synthetic chemical compounds (43,44) as wellas our discovery that it belonged to actinomycinsclass, we carried out cytotoxicity tests againstdifferent human tumor cell lines to support theprevious data. Knowing that antitumorantibiotics produced by actinomycetes are amongthe most important cancer chemotherapeuticagents including members of the anthracycline,bleomycin, actinomycin, mitomycin and aureolicacid families (45,46), we extended our schemeto test the antitumor activity of the extractproduced by the experimental Nocardioidesluteus. It showed significant antitumor activityagainst the tested human cancer cell linessurpassing doxorubicin’s activity in most cases.The IC

50 of crude extract, semi-pure compound

on different human tumor cell lines compared todoxorubicin is illustrated in Table 4.

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