Research Article Biochemical Evaluation and Green ...downloads.hindawi.com/journals/jchem/2015/486948.pdf · Using Peroxidase from Euphorbia (Euphorbia amygdaloides ) and Its Antibacterial

Research ArticleBiochemical Evaluation and Green Synthesis of Nano SilverUsing Peroxidase from Euphorbia (Euphorbia amygdaloides)and Its Antibacterial Activity

Semra Cicek12 Azize AlaylJ Gungor3 Ahmet Adiguzel4 and Hayrunnisa Nadaroglu15

1Department of Nanoscience and Nanoengineering Faculty of Engineering Ataturk University 25240 Erzurum Turkey2Department of Agricultural Biotechnology Faculty of Agriculture Ataturk University 25240 Erzurum Turkey3Department of Chemical Technology Erzurum Vocational Training School Ataturk University 25240 Erzurum Turkey4Department of Molecular Biology and Genetics Faculty of Science Ataturk University 25240 Erzurum Turkey5Department of Food Technology Erzurum Vocational Training School Ataturk University 25240 Erzurum Turkey

Correspondence should be addressed to Hayrunnisa Nadaroglu hnisa25atauniedutr

Received 29 June 2015 Accepted 24 August 2015

Academic Editor Mallikarjuna N Nadagouda

Copyright copy 2015 Semra Cicek et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Silver nanoparticles are used an increased attention for various biomedical and medical applications In this study green synthesisof silver nanoparticles was made with simple method by using peroxidase enzyme partially purified from Euphorbia (Euphorbiaamygdaloides) plant Optimum pH temperature and time period were determined to obtain silver nanoparticles using theperoxidase enzyme The result shows that higher silver nanoparticle was synthesized for 4 hours and at 20∘C and pH 8 Alsooptimal concentration ofmetal ions was found as 05mMThe synthesized silver nanoparticles were characterized byUV spectrumscanning electron microscope (SEM) and X-ray diffraction Antibacterial activity of silver nanoparticles was measured againstsome microorganisms such as Serratia marcescens Yersinia pseudotuberculosis Klebsiella pneumoniae Staphylococcus aureusStaphylococcus epidermidis Streptococcus pyogenes Pseudomonas aeruginosa Salmonella typhimurium Listeriamonocytogenes andEscherichia coli Synthesized silver nanoparticles have wide spectrum antibacterial activity in low concentration and may be a goodalternative therapeutic approach in medicine and pharmaceutical fields in future

1 Introduction

Metal nanoparticles (gold silver platinum etc) have featuressuch as crystal structures and very strong metallic bonds andthey are commonly used in many fields such as industrialproducts machinery industry biomedical andmedical areasdiagnosis biomarkers cell labeling antimicrobial agentsdrug administration environmental pollution control drugdelivery systems cancer therapy biosensors and materialchemistry [1 2] Silver nanoparticles (Ag-NPs) have potentialfor use in many fields such as mainly the medical fieldfood cosmetics materials science medicine environmentalremediation nonlinear optics biolabeling and optical recep-tor because of their features such as good electrical activitychemical stability and catalytic and antibacterial activity [34]

Physical and chemical methods such as UV radiationmicrowave radiation [5] chemical reducing [6] photochem-ical method [7] and heat evaporation are available in theproduction of nanoparticlesHowever thesemethods includethe use of toxic chemical which can cause serious problemsin terms of human animal and environment as opposedto a growing environmental awareness And more than onereaction step increases high cost depending on high energyrequirements In addition difficulties in the purification steplead to obtaining low yields of nanoparticles

Due to this reason economic and environmentallyfriendly methods have been developed to contribute to greenchemistry For this purpose using of plant extracts andbiological microorganisms (bacteria fungi and yeast) inorder to perform enzymatical synthesis of nanoparticles hascome into prominence [8 9]

Hindawi Publishing CorporationJournal of ChemistryVolume 2015 Article ID 486948 7 pageshttpdxdoiorg1011552015486948

2 Journal of Chemistry

Some studies have been made for the production ofnanoparticles by using plant extracts Achillea biebersteinii[10] Cassia auriculata [11] Acacia farnesiana [12] Gloriosasuperba [13] Alternanthera dentata [14] Cocos nucifera [15]and so forth However very few studies were found forsynthesis of nanoparticles by using peroxidase enzyme

Peroxidase (EC 11117 donor hydrogen-peroxide oxi-doreductase) is an oxidoreductase that catalyzes the reactionbetween the compounds which tend to give its hydrogenatoms with H

2O2receiving these atoms Peroxidases have

wide usage area in clinical biochemistry immunoassays foodprocessing detergent formulations wastewater treatmentand synthesis of aromatic compounds [16]

In this research peroxidase (POX) enzyme was partiallypurified by ammonium sulfate precipitation from plant ofEuphorbia (Euphorbia amygdaloides) and silver nanoparticleswere synthesized using the purified peroxidase At the sametime the obtained nanoparticles were characterized by UVspectrum scanning electron microscope (SEM) and X-raydiffraction The antibacterial activity of newly synthesizednano silver NPs was assayed

2 Experimental

21 Collection of Plant Sample and Preparation of PlantsExtract Used Euphorbia (Euphorbia amygdaloides) in thestudy was collected from near the town of Hasankale ofErzurum Turkey and it was identified with the helping oftaxonomists Plants were washed with distilled water severaltimes for cleaning dust and soil on plants Then plants werecut into small pieces Small pieces (50 g) were thoroughlyshattered to form a homogeneous mixture in blander using250mL 10mMsodiumphosphate buffer (pH 6)Then it wascentrifuged at 5000timesg for 10min and the supernatant wasused for enzyme purification [17]

22 Partial Purification of the Peroxidase Enzymewith Ammo-nium Sulphate Precipitation Prepared Euphorbia (Euphorbiaamygdaloides) plant homogenate was saturated from 60 to80 with ammonium sulphate then the peroxidase enzymewas precipitated by centrifuging at 8000timesg 10min Obtainedprecipitate was dissolved at 10mM sodium phosphate buffer(pH 60) and was incubated at 4∘C for further analysis [17]

23 Peroxidase Enzyme Activity Test Determination of per-oxidase activity was made by substrate of 1mM 221015840-azino-bis(3-ethylbenzthiazoline-sulfonic acid) diammonium salt(ABST) prepared in 01M phosphate buffer at pH 6 Forthis purpose 28mL ABST was transferred to a test tubethen the reaction mixture was formed by the addition 100 120583Lof 80 enzyme and 100 120583L of 32mM H

2O2solution into

the test tube The change in absorbance was monitored at412 nmusingUV-visible spectrophotometer at 1min intervalsfor 3min Blank test tube was prepared using distilled waterinstead of enzyme in the reaction mixture

24 Synthesis of Silver Nanoparticles 100 120583L of purified per-oxidase enzyme from Euphorbia (Euphorbia amygdaloides)

plant was added in sample of silver nitrate solution (AgNO3)

(29mL 10mM) and incubated in a closed space for 4 hoursThe solution was becoming dark brown which indicates thepresence of Ag nanoparticles Then water was removed byhelping of evaporatory and synthesized silver nanoparticlesthat were dried at 70∘C for 24 hours [18 19]

25 Characterization of Silver Nanoparticles Synthesized sil-ver nanoparticles were characterized with scanning at rangeof 200ndash1000 nm by using UV-VIS spectrophotometer (PGInstrument T80UV-VIS spectrophotometer) Determinationof topography silver nanoparticles was performed by SEM(scanning electron microscope) In addition XRD analysis(X-ray diffraction analysis) was performed to determine thesize of Ag nanoparticles

26 Biochemical Properties of Silver Nanoparticles SynthesisContact time pH temperature and metal ion concentrationwere determined for the purpose of optimization synthesizedAg nanoparticles

261 The Optimum Contact Time For determination of theoptimum contact time samples were spectrophotometricallymeasured between 0 and 240min with 3min intervals

262 The Determination of Optimum pH Synthesis of silvernanoparticles was performed in sodium phosphate buffer atpH 2-3 sodium acetate buffer at pH 4ndash6 sodium phosphatebuffer at pH 7 8 and sodium carbonate buffer at pH 9ndash11and the values of absorbance were measured by UV-visiblespectrophotometer pH was adjusted by using 01 N HCI and01 N NaOH

263TheDetermination of OptimumTemperature Synthesisof nanoparticles was separately carried out from 10∘C to 90∘Crespectively and changes in absorbans of the samples weremeasured by UV-visible spectrophotometer

264 The Determination of Optimum Concentration of MetalIon Synthesis of silver nanoparticles was performed by usingsilver nitrate solution at 05mM 1mM 3mM 5mM and7mM and the absorbance of samples was measured by UV-visible spectrophotometer

27 Antibacterial Activity of Silver Nanoparticles againstBacteria in Petri Dish Antagonistic activity of synthesizedsilver nanoparticles was determined against bacteria [20 21]Sterile nutrient agar (NA-Oxoid) was prepared and sterilizedthroughout one nightThediscs 6mm in diameter taken fromthe pathogenic bacteria culture growing on NA were placedin the center of petri dishes containing nutrient agar mediaAnd bacterial cultures of 1 times 108 cellsmL concentration weredrawn around these pathogen-inoculated petri dishes Eachbacterium was studied in 3 replications and the averageinhibition zone created by bioagent was identified with thehelp of the values obtained

Journal of Chemistry 3

Table 1 The purification process of purified peroxidase enzyme from Euphorbia (Euphorbia amygdaloides)

Enzymefraction

Volume Activity Total activity Protein Specific activity PurificationmL EUmL EU (mgmL) EUmg fold

Crude extract 50 2165 plusmn 13 1083 times 103 100 333 plusmn 02 0272 mdash(NH4

)2

SO4

(40ndash80) 20 1757 plusmn 102 351 times 103 324 388 plusmn 038 812 2985

250 300 350 400 450 500 550 600200Wavelength (nm)

0

1

2

3

4

Abso

rban

ce

Figure 1 UV-vis spectra of silver nanoparticles

3 Results and Discussion

Peroxidase enzyme was partially purified from Euphorbia(Euphorbia amygdaloides) For this purpose the highestcollapse of peroxidase enzyme was observed in the rangeof 60ndash80 ammonium sulphate saturation by performingammonium sulphate precipitation in the range of 0ndash90for Euphorbia (Euphorbia amygdaloides) plant homogenateThe enzyme was purified with a yield of 324 and 299purification-fold from Euphorbia (Euphorbia amygdaloides)plant (Table 1)

100 120583L purified peroxidase enzyme (5mg proteinmL)from Euphorbia (Euphorbia amygdaloides) plant was addedto the AgNO

3sample (29mL 1mM) When it was stirred

color of the reaction mixture was changed from colorness todark brown This color change showed formation of silvernanoparticles

31 Characterization of the Synthesized Silver NanoparticlesThe absorbance values of synthesized silver nanoparticleswere measured against distilled water using UV-VIS spec-trophotometer UV-visible spectra of silver nanoparticlessynthesized by reduction of the compound AgNO

3and also

catalyzed with purified peroxidase enzyme from Euphorbia(Euphorbia amygdaloides) plant were showed in Figure 1 (atroom temperature for 4 hours) It was observed that the colorof the reaction medium was changed from colorness to darkbrown due to excitation of surface plasmon vibration of silvernanoparticles The peaks of synthesized silver nanoparticlesby peroxidase enzyme were in the range of 390 to 440 nmand the sharpest peak of these nanoparticles was observedat 412 nm (Figure 1) The sharp peak at 412 nm exhibited thecharacteristic peak of synthesized silver nanoparticles

0 15 30 45 60 75 90Ab

sorb

ance

(273

nm

)Temperature (∘C)

0

1

2

3

4

2h1h

4h3h

Figure 2 The effect of temperature on the synthesis of silvernanoparticles using peroxidase enzyme from Euphorbia (Euphorbiaamygdaloides)

A number of studies belong to silver nanoparticlesthat were identified and their characteristic UV-VIS peakswere also around 400 nm For example the peaks of silvernanoparticles were at wavelength of 390 nm and 420 nm inthe synthesis of silver nanoparticle with redox reactions forsaccharose and maltose respectively Ag NPs synthesized byusing olive leaf extracts showed characteristic peak in therange of 440ndash458 nm and they supported our experiments[20 21]

311 Effect of Temperature UV-visible spectra of silvernanoparticle prepared at different temperatures were given inFigure 2 It was observed that the temperature increases andthe absorbance increases in Figure 2

It has been observed that synthesis of silver nanoparticleswas performed with high amounts at room temperaturewith catalyzed peroxidase enzyme purified from Euphor-bia (Euphorbia amygdaloides) When the synthesis of Agnanoparticle was done at room temperature the great disad-vantages could be prevented spending extra energy and lossof its activity by denaturing of protein structure of peroxidaseenzyme at high temperature

312 Effect of pH The effect of pH was investigated in thesynthesis of Ag nanoparticles UV-visible spectra of silvernanoparticles prepared at different pHwere given in Figure 2For this purpose the synthesis of Ag nanoparticles was per-formed in the range of pH 3ndash11 Different chemical reagents


345

678

91011

0

1

2

3

4

Abso

rban

ce (2

73 n

m)

1 2 3 4 50Time (h)

Figure 3 The effect of pH on the synthesis of silver nanoparti-cles using peroxidase enzyme from Euphorbia (Euphorbia amyg-daloides)

were used for preparing appropriate buffer solutions (pH 2-3phosphate buffers pH4-5-6 acetate buffers pH7-8 phosphatebuffers and pH 9-10-11 carbonate buffers) It was shown inFigure 3 that the pH values were affected a little on synthesisof nanoparticle but the highest synthesis of Ag NPs occurredat pH 6 In a study conducted by Gungor et al [17] optimumpH of purified peroxidase enzyme from Euphorbia (Euphor-bia Amygdaloides) plant was found as 60 Considering thissituation it was determined that synthesis of nanoparticlecatalyzed by obtained peroxidase enzyme from Euphorbia(Euphorbia Amygdaloides) plant was performed at the highestrate at pH 60 which was the optimum pH of the enzymeThesynthesis of AgNPswas also performed at room temperaturepH60 (Figure 3) It was determined that the reaction ratewasincreased after 4 hours andAg+ almost completely reduced toAg0

In the synthesis of Ag NPs performed with olive leafextracts optimum pH was found to be pH 8 the most stablepH of olive leaf extract as antioxidative [21 22]

313 Effect of Contact Time at RoomTemperature Thereduc-tion reaction ofAg+ by catalyzed peroxidase enzymeobtainedfrom Euphorbia (Euphorbia amygdaloides) plants wasobserved for 4 hours The graphic of absorbance changesdepending on time of synthesis reaction of Ag NPs was givenin Figure 4

The absorbance values were monitored at 412 nm andshow increase with timeThe reactions of synthesis of AgNPswere completed rate of 97 after 4 hours followed duringone day From the obtained results it was determined that AgNPs were prepared using green synthesis method they werenot aggregate and obtained nanoparticles were stable Alsonanoparticles were stable and were not degrading during oneweek

Some studies show longer stability than one week forsyntheses of Ag NPs These findings supported our research[19 23]

0000

1000

2000

3000

4000

Abso

rban

ce (2

70 n

m)

100 200 3000Time (min)

Figure 4 UV-visible spectra of Ag nanoparticles as function of timeat room temperature and pH 6

[AgNO3] (mM)1 2 3 40

0

1

2

3

4

Abso

rban

ce (2

73 n

m)

0513

57

Figure 5 The effect of concentration of AgNO3

on the synthesisof silver nanoparticles using peroxidase enzyme from Euphorbia(Euphorbia amygdaloides)

314 Effect of AgNO3Concentration The effect of AgNO

3

concentration was investigated on the synthesis of Ag NPsusing peroxidase enzyme For this purpose Ag NPs weresynthesized at different concentration (05 1 3 5 ve 7mM)of AgNO

3using the same amount of enzyme

The increasing concentration of AgNO3increased syn-

thesis ofAgNPs for 4 hours in Figure 5However the reactionsynthesis of Ag NPs was performed at concentration of 1mMAgNO

3concentration the stability of reaction was increased

as more stable (Figure 5) In general its concentration of1mM seems appropriate for both measured and followedreaction So 1mM concentration of AgNO

3was used in all

performed studies [17]

32 XRD Studies XRD and crystallographic analysis ofproducedAgNPs by catalyzed peroxidase enzymewere givenin Figure 6

The characteristic peaks of silver from XRD spectrum at2120579 = 38∘ 43∘ 63∘ 82∘ can be indexed at (111) (200) (220)and (311) facets which agree with the values reported for facecentered cubic (fcc) silver nanocrystals [24]


(200)(220)

(311)

(111)

20 40 60 80 1002120579

0

200

400

600

800

1000

Inte

nsity

Figure 6 X-ray diffraction pattern of the synthesis of silvernanoparticles using peroxidase enzyme from Euphorbia (Euphorbiaamygdaloides)

Figure 7 SEM image of the synthesized silver nanoparticles

33 Surface Characterization of AG NPs Chemical andmineralogical compositions of green synthesized Ag NPswere determined by scanning electron microscopy Scanningelectron microscope (SEM) was used to examine the surfaceof adsorbent Images of AgNPsweremagnified 5000 times byMetek Apollo prime active area 10mm2 Microscope inspectS50 and SE detector R580 (Figure 7) It is observed fromFigure 7 that most of the Ag nanoparticles were spherical inshape Figure 7 shows well dispersed Ag NPs have identifiedin the sizes range of 7ndash20 nm

Many synthesizedAgNPs using plant extractmainlyweredetermined as of spherical shape and 5ndash35 nm in size [24 25]

34 Antibacterial Activity Antibacterial effect of silver isknown Antibacterial activity of synthesized Ag NPs bycatalyzed peroxidase enzyme from Euphorbia (Euphorbiaamygdaloides) plant was studied using the disc diffusionmethod against Serratia marcescens Yersinia pseudotubercu-losis Klebsiella pneumoniae Staphylococcus aureus Staphy-lococcus epidermidis Streptococcus pyogenes Pseudomonasaeruginosa Salmonella typhimurium Listeriamonocytogenesand Escherichia coli O157H7

Table 2 Diameter zone of inhibition by AgNPs and AgNO3

againsthuman pathogenic bacteria

Serialnumber Pathogenic bacteria Inhibition zone (mm)

Ag NPs AgNO3

1 Serratia marcescens 180 5

2 Yersinia pseudotuberculosis 185 5

3 Klebsiella pneumoniae 190 5

4 Staphylococcus aureus 110 5

5 Staphylococcus epidermidis 110 5

6 Streptococcus pyogenes 170 5

7 Pseudomonas aeruginosa 160 5

8 Salmonella typhimurium 185 5

9 Listeria monocytogenes 110 5

10 Escherichia coli O157H7 175 5

The diameter of inhibition zones (mm) around each wellwith silver nanoparticles solution is represented in Table 2The silver nanoparticles synthesized by peroxidase enzymefrom Euphorbia (Euphorbia amygdaloides) (10mL) are foundto have highest antimicrobial activity against Klebsiella pneu-moniae (190mm) Yersinia pseudotuberculosis (185mm)and Salmonella typhimurium (185mm) Then negative con-trol (DMSO and distilled water) showed activity against allthe microbial strains tested and no activity was observedagainst pathogens The positive control (AgNO

3) showed

activity against all themicrobial strains tested and the activityagainst all bacteria was observed as 50mm

Ag NPs gain the ability to easily pass through the cellmembrane of bacteria with the increase in surface area dueto the fact that size of Ag NPs is small Thus the antibacterialactivity of Ag NPs increases From obtained results it wasconcluded that synthesizedAgNPs could easily pass into bac-teria and have high interaction with its surface SynthesizedAg NPs were quite effective on microorganisms Findings ofsome studies supported our work The form surface whichhas high antimicrobial activity could be possibly synthesizedwith produced silver nanoparticles [26 27]

4 Conclusion

The sizes of Ag NPs synthesized by catalyzed peroxidaseenzyme obtained from Euphorbia (Euphorbia amygdaloides)plants were in the range from 7 to 20 nm and in the sphericalform Ag NPs were characterized using UV-vis spectroscopySEM and XRD The synthesis of Ag NPs was confirmedwith performed analysis and characterization of Ag NPs wascarried out in terms of size quantity direction andmorphol-ogy In addition the Ag NPs showed excellent antibacterialactivity Thus synthesized Ag NPs are believed to havepotential for use in the creation of new antibacterial floorand surface and the preparation of antimicrobial packagingmaterials adhesive plasters and dressings


Conflict of Interests

The authors have declared that no conflict of interests exists

Acknowledgments

This research was performed under Project no 2014173and supported by the Research Development Centre ofAtaturk University The authors acknowledge the support ofAtaturkUniversity Turkey for this workThe SEM EDX andFTIR study of this research was carried out in the AtaturkUniversity Faculty of Science and XRD study of this researchwas carried out in Ataturk University Faculty of EngineeringSo the authors thank Professor Doctor Umit Demir andProfessor Doctor Yasar Totik respectively

References

[1] J Gupta ldquoNanotechnology applications in medicine and den-tistryrdquo Journal of Investigative and Clinical Dentistry vol 2 no2 pp 81ndash88 2011

[2] H Xie M M Mason and J P Wise Sr ldquoGenotoxicity of metalnanoparticlesrdquo Reviews on Environmental Health vol 26 no 4pp 251ndash268 2011

[3] F Martinez-Gutierrez P L Olive A Banuelos et al ldquoSynthesischaracterization and evaluation of antimicrobial and cytotoxiceffect of silver and titanium nanoparticlesrdquo NanomedicineNanotechnology Biology andMedicine vol 6 no 5 pp 681ndash6882010

[4] V K Sharma R A Yngard and Y Lin ldquoSilver nanoparticlesgreen synthesis and their antimicrobial activitiesrdquo Advances inColloid and Interface Science vol 145 no 1-2 pp 83ndash96 2009

[5] M N Nadagouda T F Speth and R S Varma ldquoMicrowave-assisted green synthesis of silver nanostructuresrdquo Accounts ofChemical Research vol 44 no 7 pp 469ndash478 2011

[6] O Y Golubevaa O V Shamova D S Orlov T Y PazinaA S Boldina and V N Kokryakov ldquoStudy of antimicrobialand hemolytic activities of silver nanoparticles prepared bychemical reductionrdquo Glass Physics and Chemistry vol 36 no5 pp 628ndash634 2010

[7] M Harada C Kawasaki K Saijo M Demizu and Y KimuraldquoPhotochemical synthesis of silver particles using water-in-ionic liquid microemulsions in high-pressure CO

2

rdquo Journal ofColloid and Interface Science vol 343 no 2 pp 537ndash545 2010

[8] E C Njagi H Huang L Stafford et al ldquoBiosynthesis of ironand silver nanoparticles at room temperature using aqueoussorghum bran extractsrdquo Langmuir vol 27 no 1 pp 264ndash2712011

[9] M F Zayed W H Eisa and A A Shabaka ldquoMalva parvi-flora extract assisted green synthesis of silver nanoparticlesrdquoSpectrochimica Acta Part A Molecular and Biomolecular Spec-troscopy vol 98 pp 423ndash428 2012

[10] F Namvar J Baharara T Ramezani et al ldquoGreen synthesis ofsilver nanoparticles using Achillea biebersteinii flower extractand its anti-angiogenic properties in the rat aortic ring modelrdquoMolecules vol 19 no 4 pp 4624ndash4634 2014

[11] R Sahana S C G Kiruba Daniel S G Sankar G ArchunanS J Vennison and M Sivakumar ldquoFormulation of bactericidalcold cream against clinical pathogens using Cassia auriculataflower extract-synthesized Ag nanoparticlesrdquo Green ChemistryLetters and Reviews vol 7 no 1 pp 64ndash72 2014

[12] J Manjanna S Yallappa S K Peethambar et al ldquoGreensynthesis of silver nanoparticles using Acacia farnesiana (Sweetacacia) seed extract under microwave irradiation and theirbiological assessmentrdquo Journal of Cluster Science vol 24 pp1081ndash1092 2013

[13] S Ashokkumar S Ravi and S Velmurugan ldquoGreen synthesisof silver nanoparticles from Gloriosa superba L leaf extract andtheir catalytic activityrdquo Spectrochimica Acta Part A Molecularand Biomolecular Spectroscopy vol 115 pp 388ndash392 2013

[14] D A Kumar V Palanichamy and S M Roopan ldquoGreensynthesis of silver nanoparticles using Alternanthera dentataleaf extract at room temperature and their antimicrobial activ-ityrdquo Spectrochimica Acta Part A Molecular and BiomolecularSpectroscopy vol 127 pp 168ndash171 2014

[15] R Mariselvam A J A Ranjitsingh A Usha Raja NanthiniK Kalirajan C Padmalatha and P Mosae Selvakumar ldquoGreensynthesis of silver nanoparticles from the extract of the inflo-rescence of Cocos nucifera (Family Arecaceae) for enhancedantibacterial activityrdquo Spectrochimica ActamdashPart A Molecularand Biomolecular Spectroscopy vol 129 pp 537ndash541 2014

[16] S Akhtar A A Khan and Q Husain ldquoPartially purifiedbitter gourd (Momordica charantia) peroxidase catalyzed decol-orization of textile and other industrially important dyesrdquoBioresource Technology vol 96 no 16 pp 1804ndash1811 2005

[17] A A Gungor Y Demir and N Demir ldquoPurification of per-oxidase from latex of Euphorbia (Euphorbia amygdaloides) andinvestigation of kinetic propertiesrdquo Asian Journal of Chemistryvol 20 no 1 pp 477ndash482 2008

[18] K Vahabi and S K Dorcheh ldquoBiosynthesis of silver nano-particles by Trichoderma and its medical applicationsrdquo inBiotechnology and Biology of Trichoderma V K Gupta MSchmoll A Herrera-Estrella R S Upadhyay I Druzhininaand M G Tuohy Eds chapter 29 pp 393ndash404 ElsevierLondon UK 2014

[19] M R Bindhu and M Umadevi ldquoSynthesis of monodispersedsilver nanoparticles using Hibiscus cannabinus leaf extract andits antimicrobial activityrdquo SpectrochimicaActa PartAMolecularand Biomolecular Spectroscopy vol 101 pp 184ndash190 2013

[20] E Filippo A Serra A Buccolieri and D Manno ldquoGreensynthesis of silver nanoparticles with sucrose and maltosemorphological and structural characterizationrdquo Journal of Non-Crystalline Solids vol 356 no 6ndash8 pp 344ndash350 2010

[21] M M H Khalil E H Ismail K Z El-Baghdady and DMohamed ldquoGreen synthesis of silver nanoparticles using oliveleaf extract and its antibacterial activityrdquo Arabian Journal ofChemistry vol 7 no 6 pp 1131ndash1139 2014

[22] R Meenatchiammal and G V Bai Journal of PharmaceuticalChemical and Biological Sciences vol 4 pp 101ndash111 2014

[23] S P Chandran M Chaudhary R Pasricha A Ahmad and MSastry ldquoSynthesis of gold nanotriangles and silver nanoparticlesusing Aloe vera plant extractrdquo Biotechnology Progress vol 22no 2 pp 577ndash583 2006

[24] S S Shankar A Rai A Ahmad andM Sastry ldquoRapid synthesisof Au Ag and bimetallic Au core-Ag shell nanoparticles usingNeem (Azadirachta indica) leaf brothrdquo Journal of Colloid andInterface Science vol 275 no 2 pp 496ndash502 2004

[25] S Ashokkumar S Ravi V Kathiravan and S VelmuruganldquoSynthesis of silver nanoparticles using A indicum leaf extractand their antibacterial activityrdquo Spectrochimica Acta Part AMolecular and Biomolecular Spectroscopy vol 134 pp 34ndash392015


[26] C Baker A Pradhan L Pakstis D J Pochan and S I ShahldquoSynthesis and antibacterial properties of silver nanoparticlesrdquoJournal of Nanoscience and Nanotechnology vol 5 no 2 pp244ndash249 2005

[27] J R Morones J L Elechiguerra A Camacho et al ldquoThebactericidal effect of silver nanoparticlesrdquo Nanotechnology vol16 no 10 pp 2346ndash2353 2005

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Applied ChemistryJournal of



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Spectroscopy

Analytical ChemistryInternational Journal of


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Organic Chemistry International

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CatalystsJournal of


Some studies have been made for the production ofnanoparticles by using plant extracts Achillea biebersteinii[10] Cassia auriculata [11] Acacia farnesiana [12] Gloriosasuperba [13] Alternanthera dentata [14] Cocos nucifera [15]and so forth However very few studies were found forsynthesis of nanoparticles by using peroxidase enzyme

Peroxidase (EC 11117 donor hydrogen-peroxide oxi-doreductase) is an oxidoreductase that catalyzes the reactionbetween the compounds which tend to give its hydrogenatoms with H

2O2receiving these atoms Peroxidases have

wide usage area in clinical biochemistry immunoassays foodprocessing detergent formulations wastewater treatmentand synthesis of aromatic compounds [16]

In this research peroxidase (POX) enzyme was partiallypurified by ammonium sulfate precipitation from plant ofEuphorbia (Euphorbia amygdaloides) and silver nanoparticleswere synthesized using the purified peroxidase At the sametime the obtained nanoparticles were characterized by UVspectrum scanning electron microscope (SEM) and X-raydiffraction The antibacterial activity of newly synthesizednano silver NPs was assayed

2 Experimental

21 Collection of Plant Sample and Preparation of PlantsExtract Used Euphorbia (Euphorbia amygdaloides) in thestudy was collected from near the town of Hasankale ofErzurum Turkey and it was identified with the helping oftaxonomists Plants were washed with distilled water severaltimes for cleaning dust and soil on plants Then plants werecut into small pieces Small pieces (50 g) were thoroughlyshattered to form a homogeneous mixture in blander using250mL 10mMsodiumphosphate buffer (pH 6)Then it wascentrifuged at 5000timesg for 10min and the supernatant wasused for enzyme purification [17]

22 Partial Purification of the Peroxidase Enzymewith Ammo-nium Sulphate Precipitation Prepared Euphorbia (Euphorbiaamygdaloides) plant homogenate was saturated from 60 to80 with ammonium sulphate then the peroxidase enzymewas precipitated by centrifuging at 8000timesg 10min Obtainedprecipitate was dissolved at 10mM sodium phosphate buffer(pH 60) and was incubated at 4∘C for further analysis [17]

23 Peroxidase Enzyme Activity Test Determination of per-oxidase activity was made by substrate of 1mM 221015840-azino-bis(3-ethylbenzthiazoline-sulfonic acid) diammonium salt(ABST) prepared in 01M phosphate buffer at pH 6 Forthis purpose 28mL ABST was transferred to a test tubethen the reaction mixture was formed by the addition 100 120583Lof 80 enzyme and 100 120583L of 32mM H

2O2solution into

the test tube The change in absorbance was monitored at412 nmusingUV-visible spectrophotometer at 1min intervalsfor 3min Blank test tube was prepared using distilled waterinstead of enzyme in the reaction mixture

24 Synthesis of Silver Nanoparticles 100 120583L of purified per-oxidase enzyme from Euphorbia (Euphorbia amygdaloides)

plant was added in sample of silver nitrate solution (AgNO3)

(29mL 10mM) and incubated in a closed space for 4 hoursThe solution was becoming dark brown which indicates thepresence of Ag nanoparticles Then water was removed byhelping of evaporatory and synthesized silver nanoparticlesthat were dried at 70∘C for 24 hours [18 19]

25 Characterization of Silver Nanoparticles Synthesized sil-ver nanoparticles were characterized with scanning at rangeof 200ndash1000 nm by using UV-VIS spectrophotometer (PGInstrument T80UV-VIS spectrophotometer) Determinationof topography silver nanoparticles was performed by SEM(scanning electron microscope) In addition XRD analysis(X-ray diffraction analysis) was performed to determine thesize of Ag nanoparticles

26 Biochemical Properties of Silver Nanoparticles SynthesisContact time pH temperature and metal ion concentrationwere determined for the purpose of optimization synthesizedAg nanoparticles

261 The Optimum Contact Time For determination of theoptimum contact time samples were spectrophotometricallymeasured between 0 and 240min with 3min intervals

262 The Determination of Optimum pH Synthesis of silvernanoparticles was performed in sodium phosphate buffer atpH 2-3 sodium acetate buffer at pH 4ndash6 sodium phosphatebuffer at pH 7 8 and sodium carbonate buffer at pH 9ndash11and the values of absorbance were measured by UV-visiblespectrophotometer pH was adjusted by using 01 N HCI and01 N NaOH

263TheDetermination of OptimumTemperature Synthesisof nanoparticles was separately carried out from 10∘C to 90∘Crespectively and changes in absorbans of the samples weremeasured by UV-visible spectrophotometer

264 The Determination of Optimum Concentration of MetalIon Synthesis of silver nanoparticles was performed by usingsilver nitrate solution at 05mM 1mM 3mM 5mM and7mM and the absorbance of samples was measured by UV-visible spectrophotometer

27 Antibacterial Activity of Silver Nanoparticles againstBacteria in Petri Dish Antagonistic activity of synthesizedsilver nanoparticles was determined against bacteria [20 21]Sterile nutrient agar (NA-Oxoid) was prepared and sterilizedthroughout one nightThediscs 6mm in diameter taken fromthe pathogenic bacteria culture growing on NA were placedin the center of petri dishes containing nutrient agar mediaAnd bacterial cultures of 1 times 108 cellsmL concentration weredrawn around these pathogen-inoculated petri dishes Eachbacterium was studied in 3 replications and the averageinhibition zone created by bioagent was identified with thehelp of the values obtained



Enzymefraction



)2

SO4


250 300 350 400 450 500 550 600200Wavelength (nm)

0

1

2

3

4

Abso

rban

ce








3and also


0 15 30 45 60 75 90Ab

sorb

ance

(273

nm

)Temperature (∘C)

0

1

2

3

4

2h1h

4h3h







345

678

91011

0

1

2

3

4

Abso

rban

ce (2

73 n

m)

1 2 3 4 50Time (h)







0000

1000

2000

3000

4000

Abso

rban

ce (2

70 n

m)

100 200 3000Time (min)


[AgNO3] (mM)1 2 3 40

0

1

2

3

4

Abso

rban

ce (2

73 n

m)

0513

57




3







3was used in all





(200)(220)

(311)

(111)

20 40 60 80 1002120579

0

200

400

600

800

1000

Inte

nsity









Ag NPs AgNO3












3) showed



4 Conclusion





Acknowledgments


References








2
































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



Enzymefraction



)2

SO4


250 300 350 400 450 500 550 600200Wavelength (nm)

0

1

2

3

4

Abso

rban

ce








3and also


0 15 30 45 60 75 90Ab

sorb

ance

(273

nm

)Temperature (∘C)

0

1

2

3

4

2h1h

4h3h







345

678

91011

0

1

2

3

4

Abso

rban

ce (2

73 n

m)

1 2 3 4 50Time (h)







0000

1000

2000

3000

4000

Abso

rban

ce (2

70 n

m)

100 200 3000Time (min)


[AgNO3] (mM)1 2 3 40

0

1

2

3

4

Abso

rban

ce (2

73 n

m)

0513

57




3







3was used in all





(200)(220)

(311)

(111)

20 40 60 80 1002120579

0

200

400

600

800

1000

Inte

nsity









Ag NPs AgNO3












3) showed



4 Conclusion





Acknowledgments


References








2
































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


345

678

91011

0

1

2

3

4

Abso

rban

ce (2

73 n

m)

1 2 3 4 50Time (h)







0000

1000

2000

3000

4000

Abso

rban

ce (2

70 n

m)

100 200 3000Time (min)


[AgNO3] (mM)1 2 3 40

0

1

2

3

4

Abso

rban

ce (2

73 n

m)

0513

57




3







3was used in all





(200)(220)

(311)

(111)

20 40 60 80 1002120579

0

200

400

600

800

1000

Inte

nsity









Ag NPs AgNO3












3) showed



4 Conclusion





Acknowledgments


References








2
































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


(200)(220)

(311)

(111)

20 40 60 80 1002120579

0

200

400

600

800

1000

Inte

nsity









Ag NPs AgNO3












3) showed



4 Conclusion





Acknowledgments


References








2
































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of




Acknowledgments


References








2
































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of













Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

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Research Article Biochemical Evaluation and Green ...downloads.hindawi.com/journals/jchem/2015/486948.pdf · Using Peroxidase from Euphorbia (Euphorbia amygdaloides ) and Its Antibacterial