research article production and cytotoxicity of...

Research ArticleProduction and Cytotoxicity of Extracellular Insoluble andDroplets of Soluble Melanin by Streptomyces lusitanus DMZ-3

D N Madhusudhan1 Bi Bi Zainab Mazhari1 Syed G Dastager2 and Dayanand Agsar1

1 A-DBT Research Laboratory Department of Microbiology Gulbarga University Gulbarga 585 106 India2NCIM Resource Centre Biochemical Sciences Division National Chemical Laboratory (CSIR) Pune 411008 India

Correspondence should be addressed to Dayanand Agsar dayanandagsargmailcom

Received 21 February 2014 Revised 14 March 2014 Accepted 14 March 2014 Published 16 April 2014

Academic Editor Leandro Rocha

Copyright copy 2014 D N Madhusudhan et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

A Streptomyces lusitanus DMZ-3 strain with potential to synthesize both insoluble and soluble melanins was detected Melaninsare quite distinguished based on their solubility for varied biotechnological applications The present investigation reveals theenhanced production of insoluble and soluble melanins in tyrosine medium by a single culture Streptomyces lusitanus DMZ-3was characterized by 16S rRNA gene analysis An enhanced production of 529 gL insoluble melanin was achieved in a submergedbioprocess following response surfacemethodology Combined interactive effect of temperature (50∘C) pH (85) tyrosine (20 gL)and beef extract (05 gL) were found to be critical variables for enhanced production in central composite design analysisAn optimized indigenous slant culture system was an innovative approach for the successful production (264mgL) of puresoluble melanin from the droplets formed on the surface of the culture Both insoluble and soluble melanins were confirmed andcharacterized by Chemical reactions UV FTIR and TLC analysis First time cytotoxic study of melanin using brine shrimps wasreported Maximum cytotoxic activity of soluble melanin was Lc

50

-040 120583gmL and insoluble melanin was Lc50

-080 120583gmL

1 Introduction

Melanin a polyphenolic polymer formed by the oxidativepolymerization of phenolic andor indolic compounds wasgenerally produced from the oxidation of L-tyrosine bytyrosinase or laccase to L-DOPA and dopaquinones finally todihydroxyindole carboxylic acid and their reduced forms [1]Melanins are commonly found in animals plants bacteriaand fungi [2] In humans they are found mainly in the skinand hair as dark colored pigments In bacteria and fungimelanins are found in their cell wall The biological melaninsare commonly known based on the color and the substratefrom which they originate Eumelanin is blackish brownPheomelanin is yellow to red and Pyomelanin is brown incolor [3] Eumelanin is the predominant pigment synthesizedin humans and microorganisms especially in bacteria andfungi [2]

Melanin is commercially extracted from cuttlefish anddepends on irregular supply of natural material and also is

expensive [4] Plenty of literature is available regarding thesynthesis and production of eumelanin by different bacteria[5 6] and fungi [7 8] The production of melanin byrecombinant E coli under optimized submerged bioprocesswas reported by Munoz et al [9] Dastager et al [10] andQuadri and Agsar [11] have reported the production ofmelanin by Streptomyces species Manivasagan et al [12] andSurwase et al [13] have reported the production of melaninby Actinoalloteichus sp and Brevundimonas sp respectivelyemploying Response Surface Method (RSM) RSM usingdifferent statistical designs is an important approach tooptimize the process condition for the enhanced productionof bioactive molecules [14] Formation of droplets on thesurface of the colonies of few sporulated microorganismsconstituting mainly enzymes or antibiotics or pigments wasreported [15ndash17]

Melanin plays an important role in humans and its lackleads to several abnormalities and diseases The reduced

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 306895 11 pageshttpdxdoiorg1011552014306895

2 BioMed Research International

melanin in neurons causes Parkinsonrsquos disease [18] Melaninalso plays an important role in microorganisms againstdamages from high temperatures chemical stress and bio-chemical threats [19] The role of biologically active melaninincludes being cytotoxic antitumor [20] antivenin [21]antiviral [22] and radio protective [23] Sun-screens con-taining water soluble melanin protect against harmful UVradiations Water soluble melanins are used in solid plasticfilms lenses paints varnishes and other surface protectionformulations to provide greater UV protections [24] AIDStreatment news [25] reveals the selective antiviral activity ofsynthetic soluble melanin against human immunodeficiencyvirus [22] However it is critical for melanin to be watersoluble for a better commercial potential in biotechnologicalapplications Insoluble melanins require sever treatmentssuch as boiling in strong alkali or the use of strong oxidantsfor making them water soluble which often damages them[26] The present investigation was undertaken to produceinsoluble melanin and soluble melanin from droplets ofStreptomyces as no literature is available regarding thisapproach The standardization of production of bioactivemolecules from such droplets is a novel criterion explainingproduction of melanin by Streptomyces in unique formFurther the cytotoxic activity of melanins was evaluatedusing brine shrimps The brine shrimp cytotoxic activityhas been found out as safe practical and economical todetermine the bioactivity of the synthetic compounds [27]which showed a significant correlation with in vitro growthcheck for human solid tumor cell lines [28]

2 Materials and Methods

21 Screening of Streptomyces Streptomyces collection pre-served in our A-DBT (Actinomycetes-Diversity and Biopro-cess Technology) research laboratory was screened by plateculture for the synthesis of melanin on starch tyrosine agar(STA) Starch 10 g K

2HPO42 g KNO

32 g NaCl 2 g Tyrosine

4 g MgSO4005 g CaCO

303 g FeSO

4001 g Agar 20 g

deionized water 1 L and pH 80 and also on tyrosine agar(TA) Gelatin 5 g Tyrosine 5 g beef extract 3 g Agar 20 gdeionized water 1 L and pH 80 [29] The plates inoculatedwith test isolates were incubated at 40∘C for 120 h andobserved for synthesis of melanin based on the intensity ofdark brown pigmentation and degree of zone of catalysis asreported by Nicolaus et al [3] and Shivaveerakumar et al[30]

22 Molecular Characterization of Streptomyces Efficientisolate for melanin synthesis was characterized by 16S rRNAanalysis [31] Genomic DNA was prepared by using Chelex-100 (Sigma-Aldrich USA) chelating ion exchange resinmethod [32] Employing about 100 nanogram DNA 16SrRNA amplified using universal F27 (51015840AGAGTTTGATCM-TGGCTCAG-) and R1525 (51015840TACGG(CT) TACCTTGT-TACGACTT) primers Accuracy of PCR product was visual-ized on agarose gel and sequenced using a BigDye Terminatorkit version 31 on an automatic ABI 3100 sequencer (AppliedBiosystems Inc) The sequences obtained were analyzed

using NCBI Blast search and EzTaxon [33] to restore closestrelatives and phylogenetic tree was obtained

23 Production of Insoluble Melanin Submerged bioprocessin tyrosine broth was standardized for the production ofmelanin using Streptomyces lusitanus DMZ-3 employingimportant process variables one at a time and keeping othersat a constant level [34] pH (70 75 80 and 85) of themedium inoculum size (from 1 times 106 to 1 times 109 withinterval of 1 times 101) incubation temperature (35 40 45 50and 55∘C) period of incubation (48 72 96 120 144 and168 h) and rate of agitation (120 140 160 180 and 200 rpm)were manually optimized The influence of various carbonsources (Starch glucose sucrose maltose and beef extractat 02 to 20 concentrations) nitrogen sources (soyabeanmeal ammonium nitrate casein and tyrosine at 02 to 20concentrations) and mineral salts (CuSO

4 MgSO

4 FeSO

4

MnSO4 and K

2HPO4at 005 to 025 concentrations) were

also optimizedResponse Surface Method (RSM) with central composite

design (CCD) was employed [12] to resolve the optimumcombination and interactive effect of critical process variableson the enhanced production of melanin The CCD of 30runs was set using the Design Expert Software USA (Version70) All the experiments were carried out in duplicate andaverage of melanin production obtained was considered asthe dependent variables or responses (119884) The predictedresponse was calculated from the second degree polynomialequation which included all the terms 119884 = 120573

0+ sum120573

119894119883119894+

sum1205731198941198941198832

119894

+sum120573119894119895119883119894119883119895 where119884 stands for the response variable

1205730is the intercept coefficient 120573

119894represents the coefficient

of the linear effect 120573119894119894the coefficient of quadratic effect

and 120573119894119895the 119894119895th interaction coefficient effect 119883

119894119883119895are input

variables which influence the response variable 119884 and 120573119894is

the 119894th linear coefficient [35] Other parameters which haveno much role in production of melanin were kept constantThe statistical and numerical analysis of the model was per-formedwith the analysis of variance (ANOVA)The statisticalsignificance of the model was analyzed by the Fisherrsquos 119865-testits associated probability 119875(119865) correlation coefficient 119877 anddetermination coefficient 1198772 which explains the quality ofpolynomial model The quadratic models were representedas contour plots (three-dimensional) and response surfacecurves were created for each variable The model was vali-dated for enhanced production of melanin at specific levelof optimized critical process variables

The extraction and purification of the melanin werecarried out as per the standard protocols described by Favaet al [36] and Harki et al [37] respectively The incubatedbroth was centrifuged at 8000 g for 15 minute to separate thecell mass and the pigment Extracted dried pigment pelletwas subjected to the dialysis in cellulose membrane againstphosphate buffer of pH 70 and purified by column usingSilica Gel material of 60ndash120 mesh size

24 Production of Soluble Melanin An indigenous methodof slant culture system was standardized and operated forthe synthesis and extraction of soluble pigment Potential

BioMed Research International 3

isolate of Streptomyceswas inoculated on the slants of tyrosineagar The 50mL capacity borosilicate glass tube with 15mLmedium was employed for the synthesis of melanin formedas clearly visible pigment droplets on the surface of theslant culture The slants inoculated were incubated at 45∘Cfor 120 h The dark brown pigment droplets present onthe entire surface of the culture were completely extractedusing micropipette and dried in hot air oven at 60∘C for1 h Replicates of three slants were considered to calculate asimple arithmeticmean of total solublemelanin produced perliter of tyrosine medium

25 Confirmation ofMelanin Thepigments obtained by bothsubmerged bioprocess and slant culture were confirmed asinsoluble and solublemelanin by following chemicalmethodUV-vis spectroscopy FT-IR spectroscopy and thin layerchromatographic techniques The solubility of pigments indeionized water 1 N HCl 1 N NaOH 1N KOH 1N NH

4OH

ethanol acetone chloroform and benzene was assessed[1 38] The reaction of the pigment with oxidizing agentH2O2(30) reducing agents H

2S and sodium hydrosulfite

(5) was observed and recorded for the confirmation ofthe pigment as a melanin The pigment was also subjectedto precipitation reaction with FeCl

3(1) ammonical silver

nitrate and potassium ferricynide UV-visible absorptionspectrum in the region of 200 to 600 nm was observed[39] for a characteristic property of a melanin using Sys-tronics 2201 double beam UV-visible spectrophotometerThe pigments were directly subjected to FT-IR Spectroscopyanalysis and spectrum was recoded at 4000 to 500 cmminus1[40] using thermo Nicolet iS5 FT-IR Spectroscopy Theconfirmation of the pigment as melanin was also performedby thin layer chromatography [41]The pigment extracts wereseparated and compared with standard melanin using silicagel chromatography plate (Merck TLC Silica Gel 60 F

254)

The separation was made using the different proportions oforganic solvents such as chloroform hexane butanol aceticacid andmethanol After optimizing the solvent proportionsseparate bands were observed staining with iodine

26 Cytotoxic Activity of Melanin The cytotoxic activities ofinsoluble and solublemelanins were determined by followingthe standard protocol ofMeyer et al [42] using brine shrimps(Artemia salina) Artificial seasaline water was prepareddissolving 20 g of NaCl per liter and pH was adjusted to85 with 01M Na

2CO3 1 g eggs of brine shrimp was added

to the 1 L seawater and incubated at 28∘C for 48 h withconstant air supply and light The hatched brine shrimpswere collected and rinsed in fresh seawater The insolubleand soluble melanin concentrations (0 1 2 4 8 16 32 and64 120583gmL) were diluted in 5mL seawater in separate tubesand incubated at 28∘C Sample with zero concentration ofmelanins was considered as control The mortality numberof brine shrimps for every 6 h up to 24 h was recorded Thepercentage ofmortality and lethal concentration value (LC

50-

120583gmL) of melanins were calculatedThemortality end pointof the bioassay was referred to as the absence of controlled

forward motion during 30 seconds of observation and theconcentration that killed 50 of brine shrimps as LC

50

Criterion of toxicity for fractions was categorized as nontoxic(LC50

values gt 1000 120583gmL) poor toxic (500ndash1000120583gmL)and toxic (lt500120583gmL) according to Deciga-Campos et al[43]

3 Results and Discussion

31 Screening and Characterization of Streptomyces Sevenisolates of Streptomyces were screened for the extracellularsynthesis of melanin on starch tyrosine and tyrosine agarStarch casein agar is a medium prescribed by Kuster andWilliams [44] for the isolation of actinobacteria Caseinwas replaced by tyrosine and used for the isolation andscreening of Streptomyces for the synthesis ofmelaninDegreeof coloration or intensity of color (brown) was conventionalmethod prescribed [45] for the detection ofmelanin synthesisby microorganisms A varied degree of synthesis of melaninby the test isolates of Streptomyces was clear from the visible(Figure 1(a)) brown pigment (melanin) intensity on starchtyrosine agar In addition to the intensity of brown pigmen-tation the formation of catalytic zone around the coloniesof the test isolates on tyrosine agar (Figure 1(b)) significantlyreveals the synthesis of melanin However tyrosine agarwas reported earlier [29] to be used for the differentiationof Streptomycetes but never recorded for the formation ofcatalytic zone indicating the melanin activity Surprisinglythis medium exhibited both high intensity of color and alsocatalytic zone (a clear zone around the colony catalyzing thetyrosine) indicating the synthesis of melanin The degrees ofcatalytic zone developed can be considered as an innovativecriterion for the selection of potential isolate targeting theproduction of melanin An isolate Streptomyces DMZ-3 wasselected as physiologically efficient and potential isolates(Figure 1(b)) for the maximum synthesis of melanin Asimilar approach was reported by Shivaveerakumar et al [30]as a novel criterion for the screening of actinobacteria aimingat the synthesis of extracellular tyrosinase

Actinobacteria can be analyzed at various molecularlevels to gain information suitable for constructing databasesand effective identification Sequence analysis of variousgenes provides a stable classification and accurate iden-tification which has become the cornerstone of modernphylogenetic taxonomy [46] The region of 16S rRNA geneis highly variable and differs significantly between specieswhere other areas are more conserved and suitable foridentification at the generic level [47] An almost com-plete 16S rRNA gene sequence of isolate DMZ-3 (1401nucleotides) was determined (Genbank NCBI Accessionnumber KF486519) A phylogenetic tree was constructedbased on 16S rRNA gene sequences to show the comparativerelationship between isolate DMZ-3 and other related Strep-tomyces species (Figure 1(c))The comparative analysis of 16SrRNA gene sequence and phylogenetic relationship revealsthat isolate DMZ-3 lies in a subclade with Streptomyces lusi-tanus sharing 997 of 16S rRNA gene sequence similarity


Streptomyces albaduncus JCM 4715TAY999757Streptomyces cinnabarinus NRRL B-12382TDQ026640

Streptomyces griseoloalbus NBRC 13046TAB184275Streptomyces heliomycini NBRC 15899TAB184712

Streptomyces thinghirensis DSM 41919TFM202482Streptomyces griseorubens NBRC 12780TAB184139

Streptomyces althioticus NRRL B-3981TAY999791Streptomyces matensis NBRC 12889TAB184221

Streptomyces malachitofuscus NBRC 13059TAB184282Streptomyces griseoflavus LMG 19344TAJ781322Streptomyces paradoxus NBRC 14887TAB184628

Streptomyces glaucescens NBRC 12774TAB184843Streptomyces viridochromogenes NBRC 3113TAB184728

Streptomyces azureus NBRC 12744TAB184837Streptomyces levis NBRC 15423TAB184670

Streptomyces minutiscleroticus NBRC 13000TAB184249Streptomyces capillispiralis NBRC 14222TAB184577

Streptomyces werraensis NBRC 13404TAB184381Streptomyces gancidicus NBRC 15412TAB184660Streptomyces pseudogriseolus NBRC 12902TAB184232Streptomyces cellulosae NBRC 13027TAB184265Streptomyces hawaiiensis NBRC 12784TAB184143Streptomyces arenae NBRC 13016TAB249977

Streptomyces purpurascens NBRC 13077TAB184859Streptomyces atrovirens NRRL B-16357TDQ026672

Streptomyces pilosus NBRC 12807TAB184161Streptomyces spinoverrucosus NBRC 14228TAB184578

DMZ3 16S Streptomyces lusitanus NBRC 13464TAB184424Streptomyces longispororuber NBRC 13488TAB184440

Streptomyces viridodiastaticus NBRC 13106TAB184317Streptomyces albogriseolus NRRL B-1305TAJ494865

Streptomyces speibonae PK-BlueTAF452714Streptomyces griseosporeus NBRC 13458TAB184419

Streptomyces thermocarboxydus DSM 44293TU94490Streptomyces indiaensis NBRC 13964TAB184553

Streptomyces lomondensis NBRC 15426TAB184673Streptomyces iakyrus NBRC 13401TAB184877

Streptomyces parvulus NBRC 13193TAB184326Streptomyces coeruleorubidus NBRC 12844TAB184849Streptomyces coerulescens ISP 5146TAY999720

Streptomyces bellus ISP 5185TAJ399476

10065

36

8854

12

59

7076

18

53

4838

6449

65

59

29

9527

9937

12

9

4915

98

3

3810

0

58163

9420

5

12

0001

DMZ-3

Streptomycesflaoiridis NBRC 12772TAB184842

Streptomyces chromofuscus NBRC 12851TAB184194

(a)

(b) (c)

Figure 1 Screening of isolates for the synthesis of melanin on starch tyrosine (a) tyrosine agar (b) and phylogenetic tree (c) of StreptomycesDMZ-3

32 Production of Insoluble and Soluble Melanin Operationof process variables usingmanually one at a time and keepingothers constant is a precondition to detect critical variablesfor the production of melanin employing response surfacemethodology 42 gL melanin was produced by Streptomyceslusitanus DMZ-3 in tyrosine medium under the optimizedsubmerged bioprocess Among all the optimized process vari-ables temperature pH tyrosine and beef extract were iden-tified as critical variables based on the level of the melaninproduction Determination of important physiochemical andnutritional factors is an important criterion for themaximumproduction of melanin in the development of a suitablebioprocess Few reports are available regarding the produc-tion of extracellular and intracellular melanins Santos andStephanopoulos [5] and Munoz et al [9] reported 375mgLand 6 gL of extracellular melanin production respectivelyby recombinant E coli Sajjan et al [6] and Youngchim et al[48] have reported the production of intracellular melanin byKlebsiella sp andAspergillus fumigatus respectively Dastageret al [10] revealed the production of extracellular melanin byStreptomyces in synthetic media

In the recent past Response Surface Method a softwarebased statistical design is the most validated method forthe production of biomolecules especially in the submergedfermentation In the present investigation an attempt wasmade to achieve the higher production of melanin by Strep-tomyces lusitanus DMZ-3 in submerged bioprocess Actualand predicted values of the degree of melanin productionwith identified critical process variables employing RSMwith CCD were shown in Table 1 Maximum response with529 gL of melanin was achieved at 9th run against a pre-dicted value of 465 gL A polynomial equation regarding theproduction of melanin (119884) based on the regression analysiswas as follows

Melanin production (119884) = 435 + 00941198831

+ 0171198832+ 095119883

3

minus 00501198834minus 057119883

2

1

minus 0341198832

2

minus 0581198832

3


Table 1 Optimization of critical process variables employing response surface method for the production of insoluble melanin byStreptomyces lusitanus DMZ-3

Run Critical process variables Melanin production (gL)1198831

119860 Temperature ∘C 1198832

119861 pH 1198833

119862 Tyrosine 1198834

119863 Beef extract Actual value Predicted value1 4250 775 125 125 435 4352 4250 775 125 125 435 4353 3500 700 050 200 108 2124 3500 850 200 200 305 3515 2750 775 125 125 212 1906 3500 850 200 050 421 3737 4250 775 125 125 435 4358 4250 775 275 125 405 3919 5000 850 200 050 529 46510 4250 925 125 125 315 33511 3500 700 200 200 311 29912 5000 700 050 050 163 15713 3500 700 200 050 241 29114 5000 850 200 200 392 45315 5750 775 125 125 216 22716 4250 775 125 125 435 43517 5000 850 050 200 155 14418 3500 850 050 200 153 13819 4250 775 125 275 448 34720 4250 775 125 125 435 43521 4250 775 125 minus025 278 36722 5000 850 050 050 189 17223 3500 850 050 050 155 17524 5000 700 200 200 320 34025 4250 775 minus025 125 0090 01226 4250 625 125 125 298 26727 4250 775 125 125 435 43528 5000 700 200 050 335 32229 5000 700 050 200 138 15830 3500 700 050 050 310 221

minus 0191198832

4

+ 01511988311198832

+ 02411988311198833+ 0024119883

11198834

+ 03211988321198833minus 0073119883

21198834

+ 004111988331198834

(1)

where 119883 was the response variables for melanin productionwith119883

111988321198833 and119883

4as coded values of temperature pH

tyrosine and beef extract respectively The model character-istic response for the production of melanin was statisticallyanalyzed (Table 2) using ANOVA The model showed a highcoefficient 1198772 value of 08832 where standard should be gt075and between 0 and 1 The model 119865 value of 810 impliesthe model as significant and the lack of fit 119865 value was 562indicating the lack of fit was not significant in relation to thepure error The ratio greater than 4 is desirable to confirm

the model as acceptable and the obtained ratio was 10462This revealed that the model can be used to navigate thedesign space The response variables 119862 1198602 1198612 and 1198622 werefound to be as significant model terms Each critical variablein the model with respect to incubation time was presentedas response surface curves by contour plots (Figure 2) Everycritical variable showed maximum melanin production ata constant middle level of the other variables Howeverincrease in the production of melanin was observed withincrease in these variables The validation of the statisticalmodel and regression analysis considering 119883

1(50∘C) 119883

2(85

pH) 1198833(2) and 119883

4(05) values were evident that the use

of RSM with CCD can be effectively used and the conditionsare ideal for the production of melanin

Temperature pH tyrosine and beef extractwere themostcritical factors to produce enhanced level (529 gL) melaninby Streptomyces lusitanus DMZ-3 in submerged bioprocessManivasagan et al [12] reported the production (8537120583gL)


Table 2 Analysis of variance (ANOVA) of model response data

Source Sum of squares Df Mean square 119865 value 119875 value prob gt 119865Model 4250 14 304 810 00001119860 temperature 021 1 021 056 04647119861 pH 069 1 069 184 01948119862 tyrosine 2157 1 2157 5755 lt00001119863 beef extract 0061 1 061 016 06923119860119861 038 1 038 100 03329119860119862 090 1 090 240 01425119860119863 9506119864 minus 003 1 9506119864 minus 003 0025 08756119861119862 161 1 161 429 00561119861119863 0086 1 0086 023 06397119862119863 0026 1 0026 0070 079431198602 881 1 881 2351 000021198612 309 1 309 824 001171198622 936 1 936 2499 000021198632 104 1 104 276 01173

Residual 562 15 037Lack of fit 562 10 056Pure error 0000 5 0000Cor total 4812 29

1198772

= 08832

ofmelanin fromActinoalloteichus spMA-32 with glycerol L-tyrosine NaCl and trace salt solution as critical process vari-ables employing response surface methodology with centralcomposite design However Surwase et al [13] revealed pHtryptone L-tyrosine and copper sulphate as critical variablesfor the production (68 gL) of melanin by a bacteriumBrevundimonas sp SGJ employing response surface methodwith Box-Behnken method It is evident from the presentinvestigation and with reported literature that a critical pro-cess variable either physicochemical or nutritional does varyfrom one organism to another irrespective of method beingemployed for the production of melanin The physiologicalandmetabolic nature of the organism involvedmight regulatethe process variables required for the production of melaninin given bioprocessThe simple ingredients of themedium foran efficient production ofmelanin are the added advantage ofthe present investigation

The preserved culture of Streptomyces lusitanus DMZ-3on tyrosine agar to our surprise could show the formation ofdark brown pigment droplets (Figure 3) on the entire surfaceof the slant culture This natural phenomenon of synthesisof dark brown pigment in the form of droplets was exploredand standardized for the production of melanin It is a novelapproach to progress the production of melanin employing aslant culture system An entire slant culture grown on 15mLmedium could generate about 1mL droplet and upon drying396mg of pigment was obtained A simple experimentdesigned statistically in triplicate revealed the production of264mg pigment per liter of tyrosine medium by Streptomyceslusitanus DMZ-3 Early literature reports the formation ofdroplets on the surface of the colonies of fungi [17] andStreptomyces [49 50] consisting broad range of enzymes

antibiotics and pigments The present investigation revealsthe formations of droplets on the surface of Streptomyceslusitanus and for the first time droplets were reported tocontain exclusively pure soluble melanin It is interesting tonote that the formation of droplets occurred only when theisolate was grown at 45∘C on tyrosine agar with a pH of 80It confirms the earlier reports [51] indicating the formationof droplets by organisms under stress conditions Martınet al [52] reported that the gene for the secretion of thiskind of metabolite droplets lies within the gene cluster forthe corresponding biosynthesis In depth investigations areessential to understand specific genes regulating the synthesisof solublemelanin droplets by Streptomyces lusitanusDMZ-3A water soluble melanin has been reported [53] in a mutantstrain of Bacillus thuringiensis However few patents revealthe production of water soluble synthetic [54] and fungalmelanin [55]

33 Confirmation and Characterization of Melanins Thepigment obtained under submerged bioprocess was purifiedby chromatographic technique using silica gel columnsThus purified pigment (pigment 1) and the pigment directlyextracted from the slant culture system (pigment 2) wereanalyzed for their characteristic chemical features (Table 3)Interestingly both pigments 1 and 2 produced by the sameorganism Streptomyces lusitanus DMZ-3 were revealed tobe insoluble and soluble in water respectively Howeversolubility of both pigments in other alkaline solutions andorganic solvents is one and the same The other chemicalreactions of both the pigments were also similar In allblackish brown color and total chemical reactions confirm


D Beef extractB pH

D Beef extract C Tyrosine

050088

125163

200

050088

125163

200

050088

125163

200

700738

775813

850

000

123

245

368

490

270

315

360

405

450

C Tyrosine A Temperature

000

123

245

368

490

35003875

42504625

5000

050088

125163

200

144

hour

s

B pHA Temperature

35003875

42504625

5000

700738

775813

850

210

268

325

383

440

144

hour

s

B pHC Tyrosine 700738

775813

850

050088

125163

200

000

123

245

368

490

144

hour

s144

hour

s

144

hour

s

D Beef extract A Temperature

050088

125163

200

35003875

42504625

5000

210

270

330

390

450

144

hour

s

Figure 2 Contour plots and response surface curves of the production of insoluble melanin by Streptomyces lusitanus

Droplets

Figure 3 Formation of dark brown pigment droplets on the surfaceof the slant culture of Streptomyces lusitanus DMZ-3

[1 56] pigments 1 and 2 as insoluble and soluble melaninrespectively

Both pigments gave maximum UV absorption between200 and 300 nm but decreased towards visible range inthe similar line of standard melanin under UV-Vis spec-trophotometer (Figure 4) revealing [57] the typical nature

of the melanin absorbance The FT-IR spectrophotometric(Figure 5) analysis of insoluble and soluble pigments showedthe absorption at 330500 cmminus1 (ndashOH and ndashNH bonds)165141 cmminus1 (aromatic stretch C=C) and 338647 cmminus1 and164448 cmminus1 respectively The absorbance at these rangesproves [58 59] the pigments as melanins and the peaks inthe soluble melanin indicate the purity Finally the pigmentswere separated after standardizing the solvent proportionwith 1 4 1 ratio of 01 N NaOH ethanol and chloroformrespectively The separated bands (Figure 6) reveal the equal119877119891with standard melanin

34 Cytotoxicity of Melanins Assessment of cytotoxicity ofchemicals using cell lines is not an uncommon procedureand is accurately correlated [60] with the assessment of


Table 3 Chemical analysis of the pigments

Color and reactions ObservationsPigment 1 Pigment 2

Color Blackishbrown

Blackishbrown

Solubility in water Insoluble SolubleSolubility in 01 NNH4OH Readily soluble Readily solubleSolubility in 01 NNaOH Soluble SolubleSolubility in 01 NKOH Soluble SolubleSolubility in organic solvents(Ethanol acetone chloroformbenzene)

Insoluble Insoluble

Reaction with sodiumdithionite and with potassiumferricyanide

Decolorizedand turned to

brown


brownReaction with H2S Reduced Reduced

1900 3316 4732 6148 7565 8980minus0089

0363

0815

1267

1720

2172

Abs

Standard melaninPigment 1Pigment 2

123

Figure 4 UV-visible spectrophotometric analysis of insoluble andsoluble melanins

cytotoxicity using brine shrimps The brine shrimp assaymethod is considered as an excellent alternate option to assessthe cytotoxic activity of the biological product [61] Fromthe beginning of its introduction to standardization [42] thisin vivo test had successfully been adopted for the bioassayof active cytotoxic and antitumor agents [62] Further thelethal concentration of brine shrimp can be correlated withthe lethal dose in mice and was explained using medicinalplants earlier [63]The number of brine shrimps survived andthe percentage ofmortality for 6 h 12 h and 24 hwas summa-rized in Table 4 After 24 h the total mortality was 100 inthe highest concentration (64120583gmL) of soluble melanin and95 mortality was observed in insoluble melanin The LC

50

value was 040 120583gmL for soluble melanin and 080120583gmLfor insolublemelanin Both pigments exhibit higher cytotoxicactivity as LC

50value of both showed less than 500120583gmL

[43] However soluble melanin could reveal 100 mortalityand greater cytotoxic activity with half of the LC

50value

when compared to insoluble melanin

4 Conclusions

A successful production of both insoluble and solublemelanins from a single isolate of Streptomyces is a significant

137241

151542

153815

163382

165141

123326

133615

164448

330500

338647

30

4000 3500 3000 2500 2000 1500 1000

35

45

55

40

50

606570

80

9095100

75

85

68567

144592

Pigment 1Pigment 2

Wavenumbers (cmminus1)

T(

)

Figure 5 FT-IR Spectroscopic analysis of pigments 1 and 2

Pigment 1 Pigment 2Standardmelanin

Figure 6 Thin layer chromatograph of insoluble and solublemelanins

observation of the present investigation Streptomyces lusi-tanusDMZ-3 characterized by 16S rRNA analysis was provedto be an efficient isolate for the synthesis of both melaninsAn increased production (529 gL) of insoluble melanin intyrosine medium under submerged bioprocess was achievedby response surface methodology The isolate had shown agreater consistency towards higher conditions of tempera-ture (50∘C) and pH (85) for the maximum production ofinsoluble melanin using simple nutritional ingredients suchas tyrosine and beef extract An indigenous slant culturesystem designed and standardized for the production of puresoluble melanin from the droplets formed on the surfaceof the culture is a novel criterion In an optimized slantculture system we were able to harvest 0264 gL pure solublemelanin Both melanin pigments produced by Streptomyceslusitanus DMZ-3 were confirmed and characterized withchemical reactions UV FTIR and TLC analysis Assessmentof cytotoxicity of natural products is normally determined byeither cell culture methods employing specific cell lines orusing laboratorymice as experimental animals In the presentwork cytotoxicity of melanins was successfully investigatedby using brine shrimps for the first time Both insoluble and


Table 4 Cytotoxic activity of the insoluble and soluble melanins

Concentration of melanin(120583gmL)

Log of concentrationof melanin

Survived brine shrimps Percent mortality Lc50 (120583gmL) at 24 h6 h 12 h 24 h 6 h 12 h 24 h

Insoluble melanin010 0 19 11 06 10 45 70

080

020 0301 17 11 07 15 45 65040 0602 17 10 06 15 50 70080 0903 17 09 04 15 55 80160 1204 16 09 04 20 55 80320 1505 16 09 03 20 55 85640 1806 16 07 01 20 65 95

Soluble melanin010 0 17 05 04 15 75 80

040

020 0301 17 05 04 15 75 80040 0602 16 05 04 20 75 80080 0903 16 05 04 20 75 80160 1204 15 05 03 25 75 85320 1505 15 04 02 25 80 90640 1806 15 03 00 25 85 100

soluble melanins have been proved to be highly toxic butsoluble melanin was more biologically active (040120583gmL) ascompared to insoluble melanin (080120583gmL)

Conflict of Interests

The authors have no conflict of interests to declare

Acknowledgment

The research Grant provided by Department of Biotechnol-ogy (BTPR13864PID065802010 Dt 05082011) Govern-ment of India New Delhi is sincerely acknowledged

References

[1] A A Bell and M H Wheeler ldquoBiosynthesis and functions offungal melaninsrdquo Annual Review of Phytopathology vol 24 pp411ndash451 1986

[2] K Tarangini and S Mishra ldquoProduction characterization andanalysis ofmelanin from isolatedmarinePseudomonas sp usingvegetable wasterdquo Research Journal of Engineering Sciences vol 2no 5 pp 40ndash46 2013

[3] R A Nicolaus M Piattelli and E Fattorusso ldquoThe structureof melanins andmelanogenesis-IV On some natural melaninsrdquoTetrahedron vol 20 no 5 pp 1163ndash1172 1964

[4] M Magarelli P Passamonti and C Renieri ldquoPurificationcharacterization and analysis of sepia melanin from commer-cial sepia ink (Sepia Officinalis)rdquo CES Medicina Veterinaria yZootecnia vol 5 no 2 pp 18ndash28 2010

[5] C N S Santos and G Stephanopoulos ldquoMelanin-based high-throughput screen for L-tyrosine production in EscherichiacolirdquoApplied and Environmental Microbiology vol 74 no 4 pp1190ndash1197 2008

[6] S Sajjan G Kulkarni V Yaligara L Kyoung and T BKaregoudar ldquoPurification and physiochemical characterizationof melanin pigment from klebsiella sp GSKrdquo Journal of Micro-biology and Biotechnology vol 20 no 11 pp 1513ndash1520 2010

[7] R Romero-Martinez M Wheeler A Guerrero-Plata GRico and H Torres-Guerrero ldquoBiosynthesis and functions ofmelanin in Sporothrix schenckiirdquo Infection and Immunity vol68 no 6 pp 3696ndash3703 2000

[8] K Rajagopal G Kathiravan and S Karthikeyan ldquoExtractionand characterization ofmelanin from Phomopsis a phellophyticfungi Isolated from Azadirachta indica A Jussrdquo African Journalof Microbiology Research vol 5 no 7 pp 762ndash766 2011

[9] V H Munoz N Cabrera-Valladares F Bolıvar G Gosset andA Martınez ldquoOptimum melanin production using recombi-nant Escherichia colirdquo Journal of Applied Microbiology vol 101no 5 pp 1002ndash1008 2006

[10] S G Dastager W-J Li A Dayanand et al ldquoSeperationidentification and analysis of pigment (melanin) production inStreptomycesrdquoAfrican Journal of Biotechnology vol 5 no 11 pp1131ndash1134 2006

[11] S R Quadri and D Agsar ldquoDetection of melanin producingthermo-alkaliphilic Streptomyces from limestone quarries ofthe Deccan trapsrdquoWorld Journal of Science and Technology vol2 no 2 pp 08ndash12 2012

[12] P Manivasagan J Venkatesan K Senthilkumar K Sivakumarand S-K Kim ldquoIsolation and characterization of biologicallyactive melanin fromActinoalloteichus sp MA-32rdquo InternationalJournal of Biological Macromolecules vol 58 pp 263ndash274 2013

[13] N S Surwase S B Jadhav S S Phugare and J P JadhavldquoOptimization of melanin production by Brevundimonas spSGJ using response surface methodologyrdquo 3 Biotech vol 3 pp187ndash194 2013

[14] M Aghaie-Khouzani H Forootanfar M Moshfegh M RKhoshayand and M A Faramarzi ldquoDecolorization of some


synthetic dyes using optimized culture broth of laccase pro-ducing ascomycete Paraconiothyrium variabilerdquo BiochemicalEngineering Journal vol 60 pp 9ndash15 2012

[15] D B Johnstone and S A Waksman ldquoProduction of strepto-mycin by Streptomyces bikinienisrdquo Journal of Bacteriology vol55 no 3 pp 317ndash236 1948

[16] B A M Rudd and D A Hopwood ldquoGenetics of actinorhodinbiosynthesis by Streptomyces coelicolor A3(2)rdquo Journal of Gen-eral Microbiology vol 114 no 1 pp 35ndash43 1979

[17] M Gareis and E-M Gareis ldquoGuttation droplets of Penicilliumnordicum and Penicillium verrucosum contain high concentra-tions of the mycotoxins ochratoxin A and Brdquo Mycopathologiavol 163 no 4 pp 207ndash214 2007

[18] M Fasano S Giraudo S Coha B Bergamasco and L LopianoldquoResidual substantia nigra neuromelanin in Parkinsonrsquos diseaseis cross-linked to 120572-synucleinrdquo Neurochemistry Internationalvol 42 no 7 pp 603ndash606 2003

[19] N G Allam and E H F Abd El-Zaher ldquoProtective role ofAspergillus fumigatus melanin against ultraviolet (UV) irradi-ation and Bjerkandera adusta melanin as a candidate vaccineagainst systemic candidiasisrdquo African Journal of Biotechnologyvol 11 no 24 pp 6566ndash6577 2012

[20] R C RDeGoncalves and S R Pombeiro-Sponchiado ldquoAntiox-idant activity of themelanin pigment extracted fromAspergillusnidulansrdquo Biological and Pharmaceutical Bulletin vol 28 no 6pp 1129ndash1131 2005

[21] Y-C Hung V Sava M-Y Hong and G S Huang ldquoInhibitoryeffects on phospholipase A2 and antivenin activity of melaninextracted from Theasinensis Linnrdquo Life Sciences vol 74 no 16pp 2037ndash2047 2004

[22] D C Montefiori and J Zhou ldquoSelective antiviral activityof synthetic soluble L-tyrosine and L-dopa melanins againsthuman immunodeficiency virus in vitrordquo Antiviral Researchvol 15 no 1 pp 11ndash25 1991

[23] E Dadachova R A Bryan X Huang et al ldquoIonizing radiationchanges the electronic properties of melanin and enhances thegrowth ofmelanized fungirdquo PLoSONE vol 2 no 5 article e4572007

[24] J Gallas and M Eisner ldquoMelanin polyvinyl alcohol plasticlaminates for optical applicationsrdquo US Patent 7029758 2006

[25] J S James ldquoSoluble melanins-Potential HIV Treatmentrdquo AidsTreatment News pp 139 November 1991

[26] G Prota Melanins and Melanogenesis Academic Press NewYork NY USA 1992

[27] P A De Almeida T M S Da Silva and A EchevarrialdquoMesoionic 5-alkyl-13-dithiolium-4-thiolates synthesis andbrine shrimp toxicityrdquo Heterocyclic Communications vol 8 no6 pp 593ndash600 2002

[28] T M S Silva R J B Nascimento M M Batista M F Agraand C A Camara ldquoBrine shrimp bioassay of some speciesof Solanum from Northestern Brazilrdquo Brazilian Journal ofPharmacognosy vol 17 no 1 pp 35ndash38 2007

[29] RM Atlas Tyrosine Agar Hand Book ofMicrobiological MediaASM Press Washington DC USA 4th edition 2010

[30] S Shivaveerakumar D N Madhusudhan D Agsar andM Kontro ldquoCatalatyc zone a novel screening approach forthe extracellular tyrosinase by Streptomyces vinaceusdrappusDSV5rdquo Journal of Pure and Applied Microbiology vol 7 no 4pp 2799ndash2808 2013

[31] W GWeisburg S M Barns D A Pelletier and D J Lane ldquo16Sribosomal DNA amplification for phylogenetic studyrdquo Journalof Bacteriology vol 173 no 2 pp 697ndash703 1991

[32] F J Laurent F Provost and P Boiron ldquoRapid identification ofclinically relevant Nocardia species to genus level by 16S rRNAgene PCRrdquo Journal of Clinical Microbiology vol 37 no 1 pp99ndash102 1999

[33] J Chun J-H Lee Y Jung et al ldquoEzTaxon a web-based toolfor the identification of prokaryotes based on 16S ribosomalRNA gene sequencesrdquo International Journal of Systematic andEvolutionary Microbiology vol 57 no 10 pp 2259ndash2261 2007

[34] B-L Liu and Y-M Tzeng ldquoOptimization of growth mediumfor the production of spores from Bacillus thuringiensis usingresponse surface methodologyrdquo Bioprocess Engineering vol 18no 6 pp 413ndash418 1998

[35] B Sajeena Beevi P P Jose and G Madhu ldquoOptimization ofprocess parameters affecting biogas production from organicfraction of municipal solid waste via anaerobic digestionrdquoWorld Academy of Science Engineering and Technology Interna-tional Journal of Environmental Earth Science and Engineeringvol 8 no 1 2014

[36] F Fava D D Gioia and L Marchetti ldquoCharacterization ofa pigment produced by Pseudomonas fluorescens during 3-chlorobenzoate co-metabolismrdquoChemosphere vol 27 no 5 pp825ndash835 1993

[37] E Harki T Talou and R Dargent ldquoPurification characterisa-tion and analysis of melanin extracted from Tuber melanospo-rum Vittrdquo Food Chemistry vol 58 no 1-2 pp 69ndash73 1997

[38] M Thomas ldquoMelaninsrdquo in Modern Methods of Plant Analysispp 661ndash675 Springer Berlin Germany 1955

[39] G M Gadd ldquoEffects of media composition and light on colonydifferentiation and melanin synthesis inMicrodochium bolleyirdquoTransactions of BritishMycological Society vol 78 no 1 pp 115ndash122 1982

[40] J P Ravishankar V Muruganandam and T S SuryanaryananldquoIsolation and characterization of melanin from a marinefungusrdquo Botanica Marina vol 38 no 5 pp 413ndash416 1995

[41] O A Borowik and M D Engstrom ldquoChromosomal evolutionand biogeography of collared lemmings (Dicrostonyx) in theeastern and High Arctic of Canadardquo Canadian Journal ofZoology vol 71 no 8 pp 1481ndash1493 1993

[42] B N Meyer N R Ferrigni and J E Putnam ldquoBrine shrimpa convenient general bioassay for active plant constituentsrdquoPlanta Medica vol 45 no 1 pp 31ndash34 1982

[43] M Deciga-Campos I Rivero-Cruz M Arriaga-Alba et alldquoAcute toxicity and mutagenic activity of Mexican plants usedin traditional medicinerdquo Journal of Ethnopharmacology vol 110no 2 pp 334ndash342 2007

[44] E Kuster and S T Williams ldquoStarch-casein medium forisolation of StreptomycetesrdquoNature vol 202 pp 928ndash929 1964

[45] T A Nurudeen and D G Ahearn ldquoRegulation of melaninproduction by Cryptococcus neoformansrdquo Journal of ClinicalMicrobiology vol 10 no 5 pp 724ndash729 1979

[46] G Muyzer S Hottentrager A Teske and C Wawer ldquoDenatur-ing gradient gel electrophoresis of PCR-amplified 16S rDNAmdasha new molecular approach to analyze the genetic diversity ofmixed microbial communitiesrdquo inMolecular Microbial EcologyManual A D L Akkermans J D van Elsas and F J de BruijnEds pp 1ndash23 Kluwer Academic Publishers Dordrecht TheNetherlands 1996

[47] R Amann and W Ludwig ldquoRibosomal RNA-targeted nucleicacid probes for studies in microbial ecologyrdquo FEMS Microbiol-ogy Reviews vol 24 no 5 pp 555ndash565 2000


[48] S Youngchim R Morris-Jones R J Hay and A J HamiltonldquoProduction of melanin by Aspergillus fumigatusrdquo Journal ofMedical Microbiology vol 53 no 3 pp 175ndash181 2004

[49] ldquoThe family of Streptomycetaceae part II molecular biologyrdquoin The Prokaryotes H Schrempf M Dworkin S Falkow ERosenberg K H Schleifer and E Stackebrandt Eds vol 3 pp605ndash622 Springer 2007

[50] K F Chater S Biro K J Lee T Palmer and H SchrempfldquoThe complex extracellular biology of Streptomycesrdquo FEMSMicrobiology Reviews vol 34 no 2 pp 171ndash198 2010

[51] D H Jennings ldquoThe role of droplets in helping to maintain aconstant growth rate of aerial hyphaerdquo Mycological Researchvol 95 no 7 pp 883ndash884 1991

[52] J F Martın J Casqueiro and P Liras ldquoSecretion systems forsecondarymetabolites how producer cells send outmessages ofintercellular communicationrdquoCurrent Opinion inMicrobiologyvol 8 no 3 pp 282ndash293 2005

[53] A E Aghajanyan A A Hambardzumyan A S Hovsepyan RA Asaturian A A Vardanyan and A A Saghiyan ldquoIsolationpurification and physicochemical characterization of water-soluble Bacillus thuringiensis melaninrdquo Pigment Cell Researchvol 18 no 2 pp 130ndash135 2005

[54] S J Orlow and J M Pawelek ldquoSoluble melaninrdquo US Patent no5218079A June 2003

[55] R K Seshagiri P Jalmi and P Bodke ldquoA process for productionofwater solublemelanin using a strain of the fungusGliocephalotrichumrdquo Patent no WO 2010064262 A2 June 2010

[56] W Yuan S H Burleigh and J O Dawson ldquoMelanin biosynthe-sis by Frankia strain CeI5rdquo Physiologia Plantarum vol 131 no2 pp 180ndash190 2007

[57] V E Coyne and L Al-Harthi ldquoInduction of melanin biosynthe-sis inVibrio choleraerdquoApplied and Environmental Microbiologyvol 58 no 9 pp 2861ndash2865 1992

[58] S L Hoti and K Balaraman ldquoFormation of melanin pigmentby a mutant of Bacillus thuringiensis H-14rdquo Journal of GeneralMicrobiology vol 139 no 10 pp 2365ndash2369 1993

[59] P Selvakumar S Rajasekar K Periasamy and N Raaman ldquoIso-lation and characterization of melanin pigment from Pleurotuscystidiosus (telomorph of Antromycopsis macrocarpa)rdquo WorldJournal of Microbiology and Biotechnology vol 24 no 10 pp2125ndash2131 2008

[60] J L Carballo Z L Hernandez-Inda P Perez andM D Garcıa-Gravalos ldquoA comparison between two brine shrimp assays todetect in vitro cytotoxicity in marine natural productsrdquo BMCBiotechnology vol 2 no 17 pp 1ndash5 2002

[61] E L Quignard A M Pohlit S M Nunomura et al ldquoScreeningof plants found in Amazonas state for lethality towards brineshrimprdquo Acta Amazon vol 33 no 1 pp 93ndash104 2003

[62] S Pisutthanan P Plianbangchang N Pisutthanan S Ruanruayand O Muanrit ldquoBrine shrimp lethality activity ofThai medici-nal plants in the familyMeliaceaerdquoNaresuanUniversity Journalvol 12 no 2 pp 13ndash18 2004

[63] A Lagarto Parra R Silva Yhebra I Guerra Sardinas and LIglesias Buela ldquoComparative study of the assay ofArtemia salinaL and the estimate of the medium lethal dose (LD50 value)in mice to determine oral acute toxicity of plant extractsrdquoPhytomedicine vol 8 no 5 pp 395ndash400 2001

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


melanin in neurons causes Parkinsonrsquos disease [18] Melaninalso plays an important role in microorganisms againstdamages from high temperatures chemical stress and bio-chemical threats [19] The role of biologically active melaninincludes being cytotoxic antitumor [20] antivenin [21]antiviral [22] and radio protective [23] Sun-screens con-taining water soluble melanin protect against harmful UVradiations Water soluble melanins are used in solid plasticfilms lenses paints varnishes and other surface protectionformulations to provide greater UV protections [24] AIDStreatment news [25] reveals the selective antiviral activity ofsynthetic soluble melanin against human immunodeficiencyvirus [22] However it is critical for melanin to be watersoluble for a better commercial potential in biotechnologicalapplications Insoluble melanins require sever treatmentssuch as boiling in strong alkali or the use of strong oxidantsfor making them water soluble which often damages them[26] The present investigation was undertaken to produceinsoluble melanin and soluble melanin from droplets ofStreptomyces as no literature is available regarding thisapproach The standardization of production of bioactivemolecules from such droplets is a novel criterion explainingproduction of melanin by Streptomyces in unique formFurther the cytotoxic activity of melanins was evaluatedusing brine shrimps The brine shrimp cytotoxic activityhas been found out as safe practical and economical todetermine the bioactivity of the synthetic compounds [27]which showed a significant correlation with in vitro growthcheck for human solid tumor cell lines [28]

2 Materials and Methods

21 Screening of Streptomyces Streptomyces collection pre-served in our A-DBT (Actinomycetes-Diversity and Biopro-cess Technology) research laboratory was screened by plateculture for the synthesis of melanin on starch tyrosine agar(STA) Starch 10 g K

2HPO42 g KNO

32 g NaCl 2 g Tyrosine

4 g MgSO4005 g CaCO

303 g FeSO

4001 g Agar 20 g

deionized water 1 L and pH 80 and also on tyrosine agar(TA) Gelatin 5 g Tyrosine 5 g beef extract 3 g Agar 20 gdeionized water 1 L and pH 80 [29] The plates inoculatedwith test isolates were incubated at 40∘C for 120 h andobserved for synthesis of melanin based on the intensity ofdark brown pigmentation and degree of zone of catalysis asreported by Nicolaus et al [3] and Shivaveerakumar et al[30]

22 Molecular Characterization of Streptomyces Efficientisolate for melanin synthesis was characterized by 16S rRNAanalysis [31] Genomic DNA was prepared by using Chelex-100 (Sigma-Aldrich USA) chelating ion exchange resinmethod [32] Employing about 100 nanogram DNA 16SrRNA amplified using universal F27 (51015840AGAGTTTGATCM-TGGCTCAG-) and R1525 (51015840TACGG(CT) TACCTTGT-TACGACTT) primers Accuracy of PCR product was visual-ized on agarose gel and sequenced using a BigDye Terminatorkit version 31 on an automatic ABI 3100 sequencer (AppliedBiosystems Inc) The sequences obtained were analyzed

using NCBI Blast search and EzTaxon [33] to restore closestrelatives and phylogenetic tree was obtained

23 Production of Insoluble Melanin Submerged bioprocessin tyrosine broth was standardized for the production ofmelanin using Streptomyces lusitanus DMZ-3 employingimportant process variables one at a time and keeping othersat a constant level [34] pH (70 75 80 and 85) of themedium inoculum size (from 1 times 106 to 1 times 109 withinterval of 1 times 101) incubation temperature (35 40 45 50and 55∘C) period of incubation (48 72 96 120 144 and168 h) and rate of agitation (120 140 160 180 and 200 rpm)were manually optimized The influence of various carbonsources (Starch glucose sucrose maltose and beef extractat 02 to 20 concentrations) nitrogen sources (soyabeanmeal ammonium nitrate casein and tyrosine at 02 to 20concentrations) and mineral salts (CuSO

4 MgSO

4 FeSO

4

MnSO4 and K

2HPO4at 005 to 025 concentrations) were

also optimizedResponse Surface Method (RSM) with central composite

design (CCD) was employed [12] to resolve the optimumcombination and interactive effect of critical process variableson the enhanced production of melanin The CCD of 30runs was set using the Design Expert Software USA (Version70) All the experiments were carried out in duplicate andaverage of melanin production obtained was considered asthe dependent variables or responses (119884) The predictedresponse was calculated from the second degree polynomialequation which included all the terms 119884 = 120573

0+ sum120573

119894119883119894+

sum1205731198941198941198832

119894

+sum120573119894119895119883119894119883119895 where119884 stands for the response variable

1205730is the intercept coefficient 120573

119894represents the coefficient

of the linear effect 120573119894119894the coefficient of quadratic effect

and 120573119894119895the 119894119895th interaction coefficient effect 119883

119894119883119895are input

variables which influence the response variable 119884 and 120573119894is

the 119894th linear coefficient [35] Other parameters which haveno much role in production of melanin were kept constantThe statistical and numerical analysis of the model was per-formedwith the analysis of variance (ANOVA)The statisticalsignificance of the model was analyzed by the Fisherrsquos 119865-testits associated probability 119875(119865) correlation coefficient 119877 anddetermination coefficient 1198772 which explains the quality ofpolynomial model The quadratic models were representedas contour plots (three-dimensional) and response surfacecurves were created for each variable The model was vali-dated for enhanced production of melanin at specific levelof optimized critical process variables

The extraction and purification of the melanin werecarried out as per the standard protocols described by Favaet al [36] and Harki et al [37] respectively The incubatedbroth was centrifuged at 8000 g for 15 minute to separate thecell mass and the pigment Extracted dried pigment pelletwas subjected to the dialysis in cellulose membrane againstphosphate buffer of pH 70 and purified by column usingSilica Gel material of 60ndash120 mesh size

24 Production of Soluble Melanin An indigenous methodof slant culture system was standardized and operated forthe synthesis and extraction of soluble pigment Potential




4OH






254)





50-



50

























10065

36

8854

12

59

7076

18

53

4838

6449

65

59

29

9527

9937

12

9

4915

98

3

3810

0

58163

9420

5

12

0001

DMZ-3



(a)

(b) (c)





+ 0171198832+ 095119883

3

minus 00501198834minus 057119883

2

1

minus 0341198832

2

minus 0581198832

3





119861 pH 1198833

119862 Tyrosine 1198834


minus 0191198832

4

+ 01511988311198832

+ 02411988311198833+ 0024119883

11198834

+ 03211988321198833minus 0073119883

21198834

+ 004111988331198834

(1)


111988321198833 and119883




1(50∘C) 119883

2(85

pH) 1198833(2) and 119883








1198772

= 08832






D Beef extractB pH


050088

125163

200

050088

125163

200

050088

125163

200

700738

775813

850

000

123

245

368

490

270

315

360

405

450


000

123

245

368

490

35003875

42504625

5000

050088

125163

200

144

hour

s

B pHA Temperature

35003875

42504625

5000

700738

775813

850

210

268

325

383

440

144

hour

s


775813

850

050088

125163

200

000

123

245

368

490

144

hour

s144

hour

s

144

hour

s


050088

125163

200

35003875

42504625

5000

210

270

330

390

450

144

hour

s


Droplets









Color Blackishbrown

Blackishbrown


Insoluble Insoluble



brown



1900 3316 4732 6148 7565 8980minus0089

0363

0815

1267

1720

2172

Abs


123



50




50value


4 Conclusions


137241

151542

153815

163382

165141

123326

133615

164448

330500

338647

30

4000 3500 3000 2500 2000 1500 1000

35

45

55

40

50

606570

80

9095100

75

85

68567

144592

Pigment 1Pigment 2


T(

)











080

020 0301 17 11 07 15 45 65040 0602 17 10 06 15 50 70080 0903 17 09 04 15 55 80160 1204 16 09 04 20 55 80320 1505 16 09 03 20 55 85640 1806 16 07 01 20 65 95

Soluble melanin010 0 17 05 04 15 75 80

040

020 0301 17 05 04 15 75 80040 0602 16 05 04 20 75 80080 0903 16 05 04 20 75 80160 1204 15 05 03 25 75 85320 1505 15 04 02 25 80 90640 1806 15 03 00 25 85 100




Acknowledgment


References










































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




4OH






254)





50-



50

























10065

36

8854

12

59

7076

18

53

4838

6449

65

59

29

9527

9937

12

9

4915

98

3

3810

0

58163

9420

5

12

0001

DMZ-3



(a)

(b) (c)





+ 0171198832+ 095119883

3

minus 00501198834minus 057119883

2

1

minus 0341198832

2

minus 0581198832

3





119861 pH 1198833

119862 Tyrosine 1198834


minus 0191198832

4

+ 01511988311198832

+ 02411988311198833+ 0024119883

11198834

+ 03211988321198833minus 0073119883

21198834

+ 004111988331198834

(1)


111988321198833 and119883




1(50∘C) 119883

2(85

pH) 1198833(2) and 119883








1198772

= 08832






D Beef extractB pH


050088

125163

200

050088

125163

200

050088

125163

200

700738

775813

850

000

123

245

368

490

270

315

360

405

450


000

123

245

368

490

35003875

42504625

5000

050088

125163

200

144

hour

s

B pHA Temperature

35003875

42504625

5000

700738

775813

850

210

268

325

383

440

144

hour

s


775813

850

050088

125163

200

000

123

245

368

490

144

hour

s144

hour

s

144

hour

s


050088

125163

200

35003875

42504625

5000

210

270

330

390

450

144

hour

s


Droplets









Color Blackishbrown

Blackishbrown


Insoluble Insoluble



brown



1900 3316 4732 6148 7565 8980minus0089

0363

0815

1267

1720

2172

Abs


123



50




50value


4 Conclusions


137241

151542

153815

163382

165141

123326

133615

164448

330500

338647

30

4000 3500 3000 2500 2000 1500 1000

35

45

55

40

50

606570

80

9095100

75

85

68567

144592

Pigment 1Pigment 2


T(

)











080

020 0301 17 11 07 15 45 65040 0602 17 10 06 15 50 70080 0903 17 09 04 15 55 80160 1204 16 09 04 20 55 80320 1505 16 09 03 20 55 85640 1806 16 07 01 20 65 95

Soluble melanin010 0 17 05 04 15 75 80

040

020 0301 17 05 04 15 75 80040 0602 16 05 04 20 75 80080 0903 16 05 04 20 75 80160 1204 15 05 03 25 75 85320 1505 15 04 02 25 80 90640 1806 15 03 00 25 85 100




Acknowledgment


References










































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















10065

36

8854

12

59

7076

18

53

4838

6449

65

59

29

9527

9937

12

9

4915

98

3

3810

0

58163

9420

5

12

0001

DMZ-3



(a)

(b) (c)





+ 0171198832+ 095119883

3

minus 00501198834minus 057119883

2

1

minus 0341198832

2

minus 0581198832

3





119861 pH 1198833

119862 Tyrosine 1198834


minus 0191198832

4

+ 01511988311198832

+ 02411988311198833+ 0024119883

11198834

+ 03211988321198833minus 0073119883

21198834

+ 004111988331198834

(1)


111988321198833 and119883




1(50∘C) 119883

2(85

pH) 1198833(2) and 119883








1198772

= 08832






D Beef extractB pH


050088

125163

200

050088

125163

200

050088

125163

200

700738

775813

850

000

123

245

368

490

270

315

360

405

450


000

123

245

368

490

35003875

42504625

5000

050088

125163

200

144

hour

s

B pHA Temperature

35003875

42504625

5000

700738

775813

850

210

268

325

383

440

144

hour

s


775813

850

050088

125163

200

000

123

245

368

490

144

hour

s144

hour

s

144

hour

s


050088

125163

200

35003875

42504625

5000

210

270

330

390

450

144

hour

s


Droplets









Color Blackishbrown

Blackishbrown


Insoluble Insoluble



brown



1900 3316 4732 6148 7565 8980minus0089

0363

0815

1267

1720

2172

Abs


123



50




50value


4 Conclusions


137241

151542

153815

163382

165141

123326

133615

164448

330500

338647

30

4000 3500 3000 2500 2000 1500 1000

35

45

55

40

50

606570

80

9095100

75

85

68567

144592

Pigment 1Pigment 2


T(

)











080

020 0301 17 11 07 15 45 65040 0602 17 10 06 15 50 70080 0903 17 09 04 15 55 80160 1204 16 09 04 20 55 80320 1505 16 09 03 20 55 85640 1806 16 07 01 20 65 95

Soluble melanin010 0 17 05 04 15 75 80

040

020 0301 17 05 04 15 75 80040 0602 16 05 04 20 75 80080 0903 16 05 04 20 75 80160 1204 15 05 03 25 75 85320 1505 15 04 02 25 80 90640 1806 15 03 00 25 85 100




Acknowledgment


References










































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





119861 pH 1198833

119862 Tyrosine 1198834


minus 0191198832

4

+ 01511988311198832

+ 02411988311198833+ 0024119883

11198834

+ 03211988321198833minus 0073119883

21198834

+ 004111988331198834

(1)


111988321198833 and119883




1(50∘C) 119883

2(85

pH) 1198833(2) and 119883








1198772

= 08832






D Beef extractB pH


050088

125163

200

050088

125163

200

050088

125163

200

700738

775813

850

000

123

245

368

490

270

315

360

405

450


000

123

245

368

490

35003875

42504625

5000

050088

125163

200

144

hour

s

B pHA Temperature

35003875

42504625

5000

700738

775813

850

210

268

325

383

440

144

hour

s


775813

850

050088

125163

200

000

123

245

368

490

144

hour

s144

hour

s

144

hour

s


050088

125163

200

35003875

42504625

5000

210

270

330

390

450

144

hour

s


Droplets









Color Blackishbrown

Blackishbrown


Insoluble Insoluble



brown



1900 3316 4732 6148 7565 8980minus0089

0363

0815

1267

1720

2172

Abs


123



50




50value


4 Conclusions


137241

151542

153815

163382

165141

123326

133615

164448

330500

338647

30

4000 3500 3000 2500 2000 1500 1000

35

45

55

40

50

606570

80

9095100

75

85

68567

144592

Pigment 1Pigment 2


T(

)











080

020 0301 17 11 07 15 45 65040 0602 17 10 06 15 50 70080 0903 17 09 04 15 55 80160 1204 16 09 04 20 55 80320 1505 16 09 03 20 55 85640 1806 16 07 01 20 65 95

Soluble melanin010 0 17 05 04 15 75 80

040

020 0301 17 05 04 15 75 80040 0602 16 05 04 20 75 80080 0903 16 05 04 20 75 80160 1204 15 05 03 25 75 85320 1505 15 04 02 25 80 90640 1806 15 03 00 25 85 100




Acknowledgment


References










































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





1198772

= 08832






D Beef extractB pH


050088

125163

200

050088

125163

200

050088

125163

200

700738

775813

850

000

123

245

368

490

270

315

360

405

450


000

123

245

368

490

35003875

42504625

5000

050088

125163

200

144

hour

s

B pHA Temperature

35003875

42504625

5000

700738

775813

850

210

268

325

383

440

144

hour

s


775813

850

050088

125163

200

000

123

245

368

490

144

hour

s144

hour

s

144

hour

s


050088

125163

200

35003875

42504625

5000

210

270

330

390

450

144

hour

s


Droplets









Color Blackishbrown

Blackishbrown


Insoluble Insoluble



brown



1900 3316 4732 6148 7565 8980minus0089

0363

0815

1267

1720

2172

Abs


123



50




50value


4 Conclusions


137241

151542

153815

163382

165141

123326

133615

164448

330500

338647

30

4000 3500 3000 2500 2000 1500 1000

35

45

55

40

50

606570

80

9095100

75

85

68567

144592

Pigment 1Pigment 2


T(

)











080

020 0301 17 11 07 15 45 65040 0602 17 10 06 15 50 70080 0903 17 09 04 15 55 80160 1204 16 09 04 20 55 80320 1505 16 09 03 20 55 85640 1806 16 07 01 20 65 95

Soluble melanin010 0 17 05 04 15 75 80

040

020 0301 17 05 04 15 75 80040 0602 16 05 04 20 75 80080 0903 16 05 04 20 75 80160 1204 15 05 03 25 75 85320 1505 15 04 02 25 80 90640 1806 15 03 00 25 85 100




Acknowledgment


References










































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


D Beef extractB pH


050088

125163

200

050088

125163

200

050088

125163

200

700738

775813

850

000

123

245

368

490

270

315

360

405

450


000

123

245

368

490

35003875

42504625

5000

050088

125163

200

144

hour

s

B pHA Temperature

35003875

42504625

5000

700738

775813

850

210

268

325

383

440

144

hour

s


775813

850

050088

125163

200

000

123

245

368

490

144

hour

s144

hour

s

144

hour

s


050088

125163

200

35003875

42504625

5000

210

270

330

390

450

144

hour

s


Droplets









Color Blackishbrown

Blackishbrown


Insoluble Insoluble



brown



1900 3316 4732 6148 7565 8980minus0089

0363

0815

1267

1720

2172

Abs


123



50




50value


4 Conclusions


137241

151542

153815

163382

165141

123326

133615

164448

330500

338647

30

4000 3500 3000 2500 2000 1500 1000

35

45

55

40

50

606570

80

9095100

75

85

68567

144592

Pigment 1Pigment 2


T(

)











080

020 0301 17 11 07 15 45 65040 0602 17 10 06 15 50 70080 0903 17 09 04 15 55 80160 1204 16 09 04 20 55 80320 1505 16 09 03 20 55 85640 1806 16 07 01 20 65 95

Soluble melanin010 0 17 05 04 15 75 80

040

020 0301 17 05 04 15 75 80040 0602 16 05 04 20 75 80080 0903 16 05 04 20 75 80160 1204 15 05 03 25 75 85320 1505 15 04 02 25 80 90640 1806 15 03 00 25 85 100




Acknowledgment


References










































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Color Blackishbrown

Blackishbrown


Insoluble Insoluble



brown



1900 3316 4732 6148 7565 8980minus0089

0363

0815

1267

1720

2172

Abs


123



50




50value


4 Conclusions


137241

151542

153815

163382

165141

123326

133615

164448

330500

338647

30

4000 3500 3000 2500 2000 1500 1000

35

45

55

40

50

606570

80

9095100

75

85

68567

144592

Pigment 1Pigment 2


T(

)











080

020 0301 17 11 07 15 45 65040 0602 17 10 06 15 50 70080 0903 17 09 04 15 55 80160 1204 16 09 04 20 55 80320 1505 16 09 03 20 55 85640 1806 16 07 01 20 65 95

Soluble melanin010 0 17 05 04 15 75 80

040

020 0301 17 05 04 15 75 80040 0602 16 05 04 20 75 80080 0903 16 05 04 20 75 80160 1204 15 05 03 25 75 85320 1505 15 04 02 25 80 90640 1806 15 03 00 25 85 100




Acknowledgment


References










































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







080

020 0301 17 11 07 15 45 65040 0602 17 10 06 15 50 70080 0903 17 09 04 15 55 80160 1204 16 09 04 20 55 80320 1505 16 09 03 20 55 85640 1806 16 07 01 20 65 95

Soluble melanin010 0 17 05 04 15 75 80

040

020 0301 17 05 04 15 75 80040 0602 16 05 04 20 75 80080 0903 16 05 04 20 75 80160 1204 15 05 03 25 75 85320 1505 15 04 02 25 80 90640 1806 15 03 00 25 85 100




Acknowledgment


References










































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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