Biodegradation of the endocrine disrupting chemical o -phenylenediamine using intracellular enzymes from Citrobacter freundii and its kinetic studies

Research ArticleReceived: 1 May 2014 Revised: 23 July 2014 Accepted article published: 27 September 2014 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jctb.4558

Biodegradation of the endocrine disruptingchemical o-phenylenediamineusing intracellular enzymes from Citrobacterfreundii and its kinetic studiesSaranya Paranji, Muneeswari Rajasekaran and Sekaran Ganesan*

Abstract

BACKGROUND: Endocrine disrupting chemicals are widely distributed in environment. o-Phenylenediamine (OPD), an endocrinedisruptor, is widely used in the leather, dyeing and polymer industries. There have been studies on the photocatalytic andbiological degradation of OPD but there is no single report on the enzymatic degradation of OPD.

RESULTS: In the present investigation, purified mixed intracellular enzymes (MIE) were used for the degradation of aqueousOPD from Citrobacter freundii, a marine bacterium. The degradation rate of OPD (concentration, 333 ppm) by MIE was achievedwith maximum removal of 93%. The degrading efficiency using MIE was enhanced by Zn2+ to about 95.87%. The degrada-tion of OPD using MIE of C. freundii followed pseudo-second-order rate kinetics. Fourier transform infrared spectroscopy,ultraviolet–visible spectroscopy, fluorescence spectroscopy, high-performance liquid chromatography and cyclic voltammetrywere used to characterise the degradation of OPD by enzymatic treatment. Nuclear magnetic resonance spectroscopy and gaschromatography–mass spectrometry were used for confirmation of the end product of OPD in enzymatic treatment.

CONCLUSION: The endocrine disruptor OPD was degraded into pyruvic acid, a non-toxic end product, using MIE.© 2014 Society of Chemical Industry

Supporting information may be found in the online version of this article.

Keywords: intracellular enzymes; Citrobacter freundii; o-phenylenediamine; pyruvic acid

INTRODUCTIONThere is a growing concern about the apparently increasingincidence of reproductive disorders in mammals, abnormal devel-opment in wild animals, and reduced fertility in males. Endocrinedisrupting chemicals in the environment have been consideredthe cause for the above societal problems.1 –3 Endocrine disrupt-ing chemicals are now regarded as the most dangerous pollutantsby environmental protection agencies around the world.4 Aro-matic amines are commonly used in the manufacture of dyes,rubber, textiles and generated from gasoline and coal combus-tion. They represent a considerable long-term threat to aquaticand, ultimately, human life because of their low water solubilityand high toxicity.5

o-Phenylenediamine (OPD) is an aromatic amine, used as a com-ponent of polymers, pesticides, drugs, and dyeing intermediatecompounds.6 – 9 OPD is characterised by density (1.031 g cm−3)boiling point (252 ∘C), melting point (102–104 ∘C), high solubilityin hot water and sparingly soluble in cold water. Despite industrialapplications of OPD, the hazardous nature of this compound toliver, kidney, nervous system10 and hormone system demands itscomplete removal from the environment. Mutagenicity of OPD isdue to its diamino functional groups substituted with the benzenering.11

Biological methods are usually considered to be the most eco-nomically effective and environmentally sound for the treatmentof pollutants. However, the removal of OPD from wastewater wasnot successful, showing 33% degradation after 5 days using anacclimated activated sludge inoculum;12 this might be due to themutagenic character of OPD onto microbes used in biologicaltreatment systems. Hence, alternative technology to biologicaltreatment of OPD containing wastewater has been explored.Existing methods for the removal of aromatic amines from waterinclude adsorption, chemical oxidation, electrochemical tech-niques and irradiation.13 Oxidation of OPD in wastewater byhydrogen peroxide was catalysed by horseradish peroxidase andcobaltoxime(II) derivatives.14,15

Recently, there has been considerable interest in the utili-sation of advanced oxidation processes for the destruction of

∗ Correspondence to: Sekaran Ganesan, Environmental Technology Division,Council of Scientific and Industrial Research (CSIR) – Central Leather ResearchInstitute (CLRI), Adyar, Chennai 600 020, Tamilnadu, India. E-mail: [email protected]

Environmental Technology Division, Council of Scientific and IndustrialResearch (CSIR) – Central Leather Research Institute (CLRI), Adyar, Chennai600 020, Tamilnadu, India

J Chem Technol Biotechnol (2014) www.soci.org © 2014 Society of Chemical Industry

www.soci.org S Paranji, M Rajasekaran, S Ganesan

organic compounds in contaminated water.16 Among them, theUV/oxidation process, which involves ultraviolet irradiation inconjunction with hydrogen peroxide and heterogeneous photo-catalysis are very promising.16 The key advantages of the former isits inherent destructive nature. It does not involve mass transfer;it can be carried out under ambient conditions and may lead tocomplete mineralisation of organic carbon into CO2. Moreover,photocatalytic process is receiving increasing attention becauseof its low cost while using sunlight as the source of irradiation.17

Although advanced oxidation processes have shown to be ade-quate for the degradation of persistent organic compounds, theymay involve considerable energy consumption, related to the UVlamps that provide photons to the system. However, all of thesemethods have their own limitations to their use at the commercialscale besides they involve high investment and maintenancecosts, incompleteness of degradation, formation of hazardousby-products, and low efficiency.18

Intracellular enzymes are very specific to the metabolic reactionsthat facilitate the degradation of recalcitrant chemicals. The intra-cellular enzymes that mediate hydroxylation of a wide range ofaniline homologues have been explored for the remediation ofharmful aromatic amine contaminants.19 However, there is no sin-gle report on the degradation of aromatic amine using mixed intra-cellular enzymes without the application of chemicals and heavymetal catalysts.

Hence, the focal theme of the present investigation was onextraction and characterisation of intracelluar enzymes from anisolated organism and identification of end products in the enzy-matic degradation of OPD using the intracellular enzymes.

MATERIALS AND METHODSMaterialo-Phenylenediamine (diamino benzene) of high purity wasobtained from Sigma–Aldrich–Fluka (Bangalore, India).

Culture enrichment and isolationThe OPD-degrading bacterium was first enriched from the marinesoil from a beach in Chennai, India. Enrichment was carried out inM9 Minimal media (HiMedia) of volume 100 mL with samples con-taining 5 mg of OPD as the sole carbon source. OPD concentrationin minimal media was increased from 5 to 50 mg with an incremen-tal increase of 5 mg. Only one isolate (OPD-M) from acclimatisedsoil was observed to degrade OPD in the solution.

Identification of the strains and phylogenetic analysisThe isolate (OPD-M) was identified by 16S rDNA sequencing andphylogenetic analysis. The genomic DNA was isolated according tothe procedure of Marmur.20 Phylogenetic analysis was performedby subjecting the deduced sequence to the 16S rDNA databaseto obtain the similar sequences and the phylogenetic tree wasconstructed using the Phylip package.21

Culture conditions for intracellular enzyme productionThe OPD-M culture was aseptically inoculated into 1000 mL Erlen-meyer flasks containing 25 mg OPD as the substrate in 500 mL ofminimal media for intracellular enzyme production. Culture flaskswere incubated on the shaker at 150 rpm, pH 7 and 35 ∘C for 4days. The bacterial cells were separated from the culture flasks bycentrifugation at 4274 × g for 20 min. The bacterial biomass waswashed twice with the phosphate buffer (pH 7.0) and stored at−20 ∘C.

Extraction of intracellular enzymesThe method for intracellular protein extraction was adapted fromNaiem and Jai22 with some modifications. The bacterial cells weresonicated with 1 mL of 0.1 mol L−1 phosphate buffer. The extractswere centrifuged at 14 000× g for 30 min. The cell-free supernatantwas collected and dialysed against 0.1 mol L−1 phosphate buffer(pH 7.0) for overnight. The purified mixed intracellular enzymes(MIE) were lyophilised and used for further analysis and for thedegradation of OPD.

Assays of intracellular enzymesThe lyophilised enzyme extract was analysed for various intracel-lular enzymes present. The enzymes involved in the degradationof OPD were determined using the respective assay procedures:catechol dioxygenase,23 dehydrogenase,24 phenol hydroxylase,25

alkaline phosphatase26 and acetate kinase.27 Protein concentra-tion was determined by the Lowry method,28 employing bovineserum albumin as the standard. The intracellular enzymes werequantified and used as a mixture of intracellular enzymes for thedegradation of OPD.

Molecular weight determination of purified intracellularenzymesThe molecular weight of enzymes was determined by usingsodium dodecyl sulfate–polyacrylamide gel electrophoresis(SDS-PAGE) according to the method followed by Lammeli,29

on a 5% stacking gel and 12% resolving gel. A protein markerranging from 14.3 to 94.7 kDa was used as a standard marker fordetermination of molecular weight.

Confirmation of molecular weight of the intracellularenzymes using MALDI-TOFThe intracellular protein was subjected to SDS-PAGE and the pro-tein band on the gel was excised after staining with Coomassieblue and subjected to a tryptic digestion. The protein bands

Figure 1. SDS-PAGE of intracellular enzymes from Citrobacter freundii. Lane1, molecular weight marker; lane 2, purified intracellular enzymes.

wileyonlinelibrary.com/jctb © 2014 Society of Chemical Industry J Chem Technol Biotechnol (2014)

Enzymatic biodegradation of o-phenylene diamine www.soci.org

were subjected to amino acid sequencing. The processed spec-tral data were obtained after the analysis. Proteins were identi-fied using the public domain Mascot search engine by incorpo-rating the standard parameters (http://www.matrixscience.com).The database used was Swissprot and trypsin was used as prote-olytic enzyme by the random cleavage. The peptide mass valueswere MH+ and mono-isotopic and mass tolerance was limited to60 ppm.30

Statistical optimisation for degradationof o-phenylenediamine by mixed intracellular enzymesThe various parameters such as time (0.5–3 h), pH (1–10), tem-perature (20–70 ∘C) and the intracellular enzyme concentration(0.1–0.5%, w/v) for the degradation of OPD by MIE were optimisedusing one parameter at a time for the degradation of OPD (5 mgin 100 mL) in buffered solution. The most significant parameters,such as time (1.5–2.5 h), pH (6–8), temperature (25–35 ∘C) andMIE concentration (0.1–0.3%, w/v) were further optimised usingresponse surface methodology.

Response surface methodology was used to optimise the mostsignificant parameters for improving the degradation of OPD byMIE. The four independent parameters were studied at threedifferent levels and a set of 30 experiments was carried out.

The analysis was done according to the following modelequation [Eqn (1)]:

Y = 𝛼0 +k∑

i=1

𝛼ixi +k∑

i=1

𝛼ix2i +

∑

i<j

𝛼ijxixj (1)

where Y is the predicted response, k is the number of parameters,𝛼0 is the design factor of interest, 𝛼i and 𝛼ij are coefficients. Thesecond-degree term (xi

2) gives the optimal values of the responseand the interactive term (xixj) represents the influence of oneparameter over the other. The accuracy and general ability of theabove polynomial model could be evaluated by the coefficientof determination R2. Each experimental design was carried out intriplicate, and the mean values were given.

Design expert, version 8.0.7 (Statease Inc., Minneapolis, MN, USA)was used for the experimental designs and regression analysisof the experimental data. Statistical analysis of the model wasperformed to evaluate the analysis of variance (ANOVA). Thequality of the polynomial model equation was judged statisticallyby the coefficient of determination R2 and its statistical significancewas determined by an F-test.

Assay for o-phenylenediamineThe effect of MIE on degradation of OPD was expressed in terms ofpercentage decolourisation of OPD and was determined by mon-itoring the decrease in absorbance at a wavelength of 420 nm.31

The reaction mixture without MIE was used as control.

Effect of metal ions on the degradationof o-phenylenediamineFive milligrams of OPD was added to the solution of vol-ume 100 mL containing 1 mmol L−1 of KCl, CaCl2·2H2O, ZnCl2,MgCl2·6H2O, FeSO4·7H2O, CuSO4·5H2O, or EDTA with 0.1 mol L−1

phosphate-buffered (pH 7.0) MIE solution and incubated at 35 ∘Cfor 1 h to determine the stimulatory or inhibitory effects of metalions on degradation of OPD by MIE.

Table 1. Coded and real values of the factors tested in the responsesurface methodology experimental design

Levels of the factors

Factor −1 0 +1

A Time (h) 1.5 2 2.5B pH 6 7 8C Temperature (∘C) 25 30 35D Concentration of enzyme (%, w/v) 0.1 0.2 0.3

Table 2. Analysis of variance for the second-order polynomial modelfor degradation of o-phenylenediamine

SourceDegrees of

freedomMean

square F-valueP-value

prob. > F

Model 14 879.41 27.61 <0.0001 (significant)Residual 15 31.85 – –Lack of fit 10 31.85 – –Pure error 5 0.000 – –Total 29 – – –R2 – – – 0.9626

Identification of the degradation productsof o-phenylenediamineThe unknown fragmented compounds were detected onUV–visible spectra and they were supported by the combinationof techniques including high-pressure liquid chromatography(HPLC) and fluorescence spectra. The final end product of enzy-matic breakdown of OPD was also confirmed using Fouriertransform infrared spectroscopy (FTIR), nuclear magnetic res-onance spectroscopy (NMR) and gas chromatography–massspectrometry (GC-MS).

Ultraviolet–visible spectroscopyThe UV–visible scans of OPD and the fragmented products ofOPD by MIE were evaluated in the range 200–800 nm using aUV–visible spectrophotometer (Cary varion; Agilent Technologies,Middleburg, Netherlands). The enzymatic degradation of OPD wasstudied by measuring the absorbance at a wavelength of 420 nm.

Fluorescence spectroscopyOPD is fluorescence active and the fluorescence spectrum wasrecorded using a fluorescence spectrophotometer in the wave-length range 200–800 nm (Cary Eclipse; Agilent Technologies,Middleburg, Netherlands). The OPD degraded by MIE was alsoanalysed. The enzymatic reaction mixtures were pre-scannedusing the fluorescence spectrophotometer to study the excitationand emission characteristics of both OPD and the degradationproducts.

Cyclic voltametryA three-electrode system consisting of a platinum disc work-ing electrode of diameter 2 mm (CH Instruments, Austin,USA), an Ag/AgCl reference electrode (CH Instruments), and aplatinum-wire counter-electrode was used to determine thereduction potential of OPD. The degradation of OPD by MIE wasconfirmed using the reduction potential of OPD using cyclicvoltametry.

J Chem Technol Biotechnol (2014) © 2014 Society of Chemical Industry wileyonlinelibrary.com/jctb


Table 3. Response surface methodology experimental design for the degradation of o-phenylene diamine by mixed intracellular enzymes

Factor 1 Factor 2 Factor 3 Factor 4 Response 1*Run no. A: Time (h) B: pH C: Temperature (∘C) D: Concentration of enzyme (%, w/v) Decolourisation activity (%)

1 1.5 8 35 0.3 812 1 7 30 0.2 813 2 7 30 0.2 934 2 7 30 0.2 935 1.5 6 25 0.3 836 2 7 20 0.2 887 2 7 30 0.2 938 2.5 8 35 0.3 899 2 7 30 0 010 2 9 30 0.2 4911 1.5 8 35 0.1 5212 2 7 30 0.2 9313 1.5 8 25 0.1 5414 2 5 30 0.2 5315 2.5 8 25 0.1 5616 1.5 6 35 0.3 7917 1.5 6 25 0.1 6418 2.5 6 35 0.3 8519 3 7 30 0.2 8820 2 7 30 0.2 9321 2 7 40 0.2 8722 2 7 30 0.4 8723 2.5 8 35 0.1 6024 2.5 6 35 0.1 5425 1.5 6 35 0.1 5126 2.5 6 25 0.1 6327 2.5 8 25 0.3 7928 2 7 30 0.2 9329 1.5 8 25 0.3 7630 2.5 6 25 0.3 89

*Experiments were done in triplicates.

Fourier transform infrared spectroscopy for confirmationof degradation of o-phenylenediamine by mixed intracellularenzymesThe functional groups present in the initial OPD and the degradedOPD were identified using an FTIR spectrophotometer (PerkinElmer, Massachusetts, USA). The samples were lyophilised andpellets were prepared with spectroscopic grade KBr of 10 mmdiameter and 1 mm thickness. The spectrum was analysed in thespectral range of 400–4000 cm−1.

High-performance liquid chromatographyThe degraded compounds of OPD were quantified using HPLCwith a UV detector at a wavelength of 220 nm (LC Agilent 20;Agilent Technologies, Santa Clara, CA, USA). The injectionvolume of 100 μL sample was separated using cartridge con-taining C18 column. The mobile phase used was methanol andwater in the ratio 95:5 and was delivered at a flow rate of1 mL min−1.

Nuclear magnetic resonance spectroscopyThe sample preparation for NMR analysis was carried out bydissolving 5–30 mg of lyophilised samples in 650𝜇L of deuterateddimethylsulfoxide (Brucker NMR FT 500 mHz; Bruker, Ettlingen,

Germany). The degradation products of OPD were identified using1H and 13C NMR analyses.

Gas chromagraphy–mass spectrometry analysisGC-MS analysis of OPD and the OPD degraded by MIE with therespective masses of the products was carried out. Mass spectrawere analysed in the range of 60–650 atom mass units (amu) atthe rate of 90 scans min−1 for a run time of 40 min.

Nonlinear kinetic model for the degradationof o-phenylenediamine using mixed intracellular enzymesThe kinetic rate constants for the degradation of OPD using MIEwere determined using the nonlinear kinetic models. The pseudo-first-order32 and pseudo-second-order33 models employed were:

rt = re

[1 − exp

(−k1t

)](2)

rt =k2r2

e t

1 + k2ret(3)

where re and rt are the amounts of OPD degraded (mg mg−1 ofenzymes) at equilibrium and at time t, and k1 and k2 are the first-and second-order rate constants.



Figure 2. Response surface curve for degradation of o-phenylenediamine (OPD) (%) by mixed intracellular enzymes (MIE) as the function of (a) time (h)and temperature (∘C), (b) time (h) and concentration of enzymes (%, w/v), (c) pH and concentration of enzymes (%, w/v), (d) time (h) and pH, and (e)predicated versus actual values of the experiments.

Table 4. Effect of metal ions on the degradation of o-phenylenediamine by mixed intracellular enzymes

Compound (1 mmol L−1) Degradation efficiency (%)

ZnCl2 95.87

FeSO4· 7H2O 0

MgCl2· 6H2O 13.72

CuSO4· 5H2O 36.57

EDTA 16.32

KCl 9.8

CaCl2·2H2O 0

RESULTS AND DISCUSSIONMicrobial identification and phylogenetic analysisThe soil selected for isolation of bacteria was acclimatised witho-phenylenediamine for about 45 days. The acclimatised soil sam-ple was inoculated into minimal media (M9, HiMedia) with OPD asthe sole carbon source. After 3 days of incubation, the above sam-ple was used as inoculum for the fresh medium containing OPD.The process was repeated several times in order to isolate a robustmicroorganism for degradation of OPD with high efficiency. Theguanine–cytosine richness in the bacterial genome, an importantfactor in characterising a species, was about 20–75%.34 The iso-lated microorganism was found to be highly stable with 53.23%guanine–cytosine content. The microorganism was optimised for



Figure 3. Mechanism of Zn2+ in activating (a) alkaline phosphatase and (b) dehydrogenase to enhance the degradation of o-phenylenediamine (OPD).

the production of intracellular enzymes. The 16S rDNA sequencingdata confirmed that the isolated organism was Citrobacter freundii.The nucleotide sequence has been assigned an accession numberKC791704 from the NCBI Gene bank database. C. freundii is a bac-terium that grows at pH 7 and a temperature of 35 ∘C.

Extraction and purification of mixed intracellular enzymesThe intracellular enzyme extract obtained from C. freundii waspurified and run on SDS-PAGE to determine the molecular weight.The SDS-PAGE analysis showed that there are several bands ofproteins and enzymes present with different molecular weights,as shown in Fig. 1. The MIE was found to contain the following

enzymes: catechol dioxygenase (2.65 U mg−1), phenol hydroxylase(1.59 U mg−1), dehydrogenase (1.3 U mg−1), alkaline phosphatase(2.8 U mg−1) and acetate kinase (0.35 U mg−1). The MIE collectedfrom C. freundii were used for the degradation of OPD.

Identification of mixed intracellular enzymes and their rolein the degradation of o-phenylenediamineThe various enzymes present in the mixed intracellular enzymeswere analysed using matrix-assisted laser-desorption–time offlight (MALDI-TOF) MS. The molecular weights of extractedenzymes were as follows: 1,2-dioxygenase, 48 kDa; dehydroge-nase, 53 kDa; alkaline phosphatase, 70 kDa; phenol hydroxylase,



Figure 4. (A) UV–visible spectra, and (B) fluorescence spectra of (a) of o-phenylenediamine (OPD) and (b) OPD after treatment with mixed intracellularenzymes (MIE) in the presence of Zn2+ ion.

28 kDa; and acetate kinase, 46 kDa. Dehydrogenase may catalysethe dehydrogenation of OPD, which spontaneously decarboxy-lates to catechol.35 C. freundii transforms catechol by cleavageof the C—C bond in the aromatic ring between first and sec-ond position with the participation of catechol 1,2-dioxygenaseand phenol hydroxylase.36 The alkaline phosphatase medi-ates the hydrolysis of simple monoalkyl phosphates whichwould be formed as an intermediate during the degradationof OPD.37

Statistical optimisation of the degradationof o-phenylenediamineResponse surface methodology using a central composite designwas employed to determine the optimal levels of the four signif-icant parameters that affected enzyme activity. Three levels withthe coded levels for the parameters are shown in Table 1. Based onthe regression analysis of the data from the Table 2, the effects offour parameters on MIE were predicted by the second-order poly-nomial function as:

decolourisation activity (%) = +93.00 + 2.04A − 1.21B

− 0.63C + 15.87D + 0.44AB + 0.94AC + 0.69AD

+ 2.94BC − 0.062BD + 1.69CD − 1.61A2

− 9.99B2 − 0.86C2 − 11.86D2

where A, B, C, and D are time, pH, temperature, and enzymeconcentration, respectively.

Analysis of variance for the response surface quadratic modelAnalysis of variance (partial sum of squares, type III)The statistical significance of equation was checked by theF-test and ANOVA for the second-order polynomial model asshown in Table 2. The analysis of factor (F-test) showed that thesecond-order polynomial model was well adjusted to the exper-imental data (Table 3) and the coefficient of variation, indicatedthe degree of precision of the experiment.

In general, the higher the value of coefficient of variation,the lower is the reliability of the experiment. In the presentinvestigation the lower value of coefficient of variation (7.67) witha regression coefficient of 0.9626 indicated a better precision andreliability of experiments.38 Linear and quadratic terms were bothsignificant at the 1% level.

Localisation of optimum conditionsThe three-dimensional response surface plots described by theregression model were drawn to illustrate the effects of the inde-pendent parameters and interactive effects of each independentparameter on the response factors. Also, it showed that the opti-mum conditions required for the maximum degradation of OPDby MIE were concentration of OPD, 5 mg in 100 mL; reaction time,2 h; temperature, 30 ∘C; pH, 7.0; and enzyme concentration, 0.2%(w/v) (Fig. 2). Each figure presented the effect of two parameterswhile the other parameter was held at the zero level.

At the optimised conditions (time, 2 h; temperature, 30 ∘C;pH, 7.0; and enzyme concentration, 0.2% w/v), the maximumdecolourisation of OPD obtained was about 93%.



Figure 5. (A) Redox potential of (a) o-phenylenediamine (OPD) and (b) OPD after treatment with MIE; (B) FTIR spectrum of (a) OPD and (b) OPD aftertreatment with mixed intracellular enzymes (MIE).

Effect of metal ions on the degradationof o-phenylenediamineThe effects of metal ions independently on the degradation ofOPD using MIE were investigated (Table 4). Zn2+ ion showedan enhancing effect on degradation of OPD using MIE to about95.87%. This is in accord with the results obtained by Yamasakiet al.,39 who showed that zinc is an important trace elementrequired for enzymatic activity and for maintaining its structuralconformation. The degradation of OPD using MIE was carried outin the presence of Zn2+ ion and confirmed using FTIR, UV andfluorescence spectroscopy.

Effect of zinc on the enzymatic degradation of aqueouso-phenylenediamineZinc ion plays an important role in structural and functionalcomponent in many cellular proteins and enzymes. In thebiosynthetic–secretory pathway, zinc helps in the proper folding

of proteins and enzymes and makes them functional beforereaching the final target.40 The intracellular enzymes fromC. freundii, such as dehydrogenase and alkaline phosphatase,could be activated by the presence of zinc ion.41

Zinc ion binds with nitrogen and sulfur readily and also showslower coordination numbers despite its size. Zinc ion tends to formfour-, five- and six-dentate coordinate complexes with the proteinmoiety.42 The Zn2+ activates alkaline phosphatase by binding tothe ligands three histidine and two water molecules, as shown inFig. 3a, while Zn2+ activates dehydrogenase enzyme by bindingwith two cysteine, one histidine and one water molecule, asillustrated in Fig. 3b.

The zinc ion in the active site of intracellular enzymes causespolarisation of hydrogen–oxygen bond, making the oxygenslightly more negative, thereby weakening the peptide thepresence of Zn2+ ion.



Figure 6. HPLC chromatogram of (a) o-phenylenediamine (OPD) and (b) OPD after treatment with mixed intracellular enzymes (MIE).

Instrumentation techniques for studying the degradationof o-phenylenediamineOPD was treated with MIE at the optimised conditions and thedegradation was studied using the following instrumental tech-niques.

Ultraviolet–visible spectroscopyUV–visible spectrophotometric scans of degradation of OPD withZn2+ was measured in wavelength range of 200–800 nm. InFig. 4A.a. the peak at a wavelength of 420 nm corresponds toOPD. After degradation of OPD using MIE from C. freundii thepeak at a wavelength of 420 nm disappeared and a new peak at awavelength of 290 nm appeared. The findings of UV–visible spec-troscopy suggest that the OPD has been degraded.

Fluorescence spectroscopyThe fluorescence spectrum of fragmented OPD in the presenceof Zn2+ ion is shown in Fig. 4B. The spectrum of OPD degradedby MIE in the presence of Zn2+ showed that there is no emissionpeak. The absence of an emission peak may be due to completedestruction of the fluorescence active OPD and the formation of

non-fluorescence compounds (organic acids). Thus, the fluores-cence spectrum confirms the degradation of OPD by MIE in thepresence of Zn2+ ion.

Cyclic voltammetryCyclic voltagrams of initial OPD and treated OPD were acquired byscanning from −2.0 V to +2.0 V. The redox cycle of OPD is shownin Fig. 5A.a. The oxidation and reduction peaks were recordedat −0.75 V and at 0.75 V. Figure 5A.b shows that the enzymeshave modified OPD, indicating that only a forward reaction waspossible, and re-formation of OPD was not allowed. Also thefigure indicates that further oxidation of OPD is possible, indicatedby the peak at −0.75 V. Thus, degradation of OPD by MIE isconfirmed.

Fourier transform infrared spectrum of o-phenylenediamineand degraded o-phenylenediamineThe FTIR spectrum of OPD (Fig. 5B.a) showed the characteristicpeaks. The spectrum shows major stretching bands owing to thepeptide group occurred in the spectral region 1200–1700 cm−1.The bands at 1501.85 cm−1 and 1592.20 cm−1 are due to the



Figure 7. 1H NMR spectra of (a) o-phenylenediamine (OPD) and (b) OPD after treatment with mixed intracellular enzymes (MIE).

aromatic ring stretching vibrations of C C—C. The band at748.37 cm−1 is attributed to the aromatic C—H bending out ofplane 1,2-disubstitution (ortho). The bands at 3364.91 cm−1 and3386.58 cm−1 are due to the N—H stretching in aromatic primaryamine. The bands at 1336.16 cm−1, 1321.06 cm−1, 1274.38 cm−1 aredue to the C—N stretching in aromatic primary amine. The stretch-ing of aromatic C—H was observed at 3056.07 cm−1, 3038.19 cm−1

and 3027.68 cm−1. The bending of primary amine N—H was foundat 1633.55 cm−1.

The FTIR spectrum of the OPD degraded by MIE of C. freundiiclearly shows that the OPD was degraded (Fig. 5B.b). The bands at2922.26 cm−1 and 2853.03 cm−1 are attributed to the asymmetricand symmetric stretching of CH3 group respectively. The peak at1751 cm−1 may be due to aliphatic ketone group and the asym-metric stretching of carboxylate ion was found at 1650.06 cm−1.The bands at 1450.3 cm−1 and 1380 cm−1 are attributed to asym-metric and symmetric bending of CH3 group, respectively. Theband at 2953.6 cm−1 is due to the stretching of the O—H group.The peak at 1082.15 cm−1 may be attributed to stretching of the

C—O group. The FTIR spectrum confirms the presence of func-tional groups in pyruvic acid.

Results of high-performance liquid chromatographyThe HPLC chromatogram of OPD before and after degradation isshown in Fig. 6. The HPLC chromatogram of the initial OPD showsa compound with retention time (Rt) at 6.895 min. Figure 6b showsthat the retention time of the parent compound disappearedand a new peak with retention time (Rt) of 3.52 appeared, whichcorresponds to pyruvic acid.43 The fragmentation of OPD to theend product, pyruvic acid, by MIE is further confirmed with H1 and13C NMR spectroscopy.

1H and 13C nuclear magnetic resonance spectroscopyThe 1H NMR spectrum of OPD was scanned between 0 and12 ppm. The 1H NMR spectrum showed signals in the aromaticregion (6–7 ppm) which indicates a 1,2-di-symmetrically sub-stituted benzene skeleton. The chemical shift around 𝛿 4.3 ppmcan be assigned to the amine-bonded protons (Fig. 7a). The



Figure 8. 13C NMR spectra of (a) o-phenylenediamine (OPD) and (b) OPD after treatment with mixed intracellular enzymes (MIE).

enzyme-degraded OPD shows the presence of aliphatic protonat 𝛿 = 3.4 ppm and 𝛿 = 2.5 ppm. The compound after degradationhas been identified as pyruvic acid containing aliphatic hydrogenatoms.

The 13C NMR spectrum of OPD was scanned between 𝛿 = 0.0and 200 ppm. The chemical shift around 𝛿 = 130–140 ppm indi-cates the evidence of carbon in the aromatic ring (Fig. 8a). Theoccurrence of chemical shift around 𝛿 = 117.8 ppm confirms thepresence of carbon atoms attached to amine groups. The 13CNMR spectrum of OPD degraded sample shows that the peaks at𝛿 = 110–140 ppm were disappeared. The chemical shift around𝛿 = 31.15 ppm indicates the evidence for methyl carbon. Theketone carbon showed a chemical shift 𝛿 = 207 ppm (Fig. 8b).The absence of carboxylic peak 𝛿 = 180 ppm may be due to themasking effect of the solvent. This confirms the presence ofpyruvic acid after the degradation of OPD using MIE.

Gas chromatography–mass spectrometry analysisof metabolic products of o-phenylenediamineGC-MS analysis was used to the identify end products of OPDdegradation. The molecular ion [M+] at m/z = 108 with a retention

time of 6.08 min was found to be identical to the mass spectralproperties of the authentic OPD (Fig. 9a). The mass spectrum dataof OPD shows the fragment peak at C6H4

+ (i.e. 80). The degradedsample showed the presence of pyruvic acid, giving a molecularion peak at m/z = 88 with the retention time of 1.75 min. Pyruvicacid, on fragmentation of the COOH group, yielded CH3CO+,corresponding to a peak at m/z = 43 and on elimination of theCH3 group gave a peak corresponding to m/z = 33 (Fig. 9b). Thefragments with molecular ions confirm the presence of pyruvicacid as the product of degradation of OPD by MIE.

Nonlinear kinetic model for the degradationof o-phenylenediamineThe first-order rate constant k1 and the second-order rate con-stant k2 for degradation of OPD using MIE from C. freundii were0.012 min−1 and 1.54× 10−3 L mol−1 min−1, respectively. Moreover,the results confirmed that the degradation of OPD using intracellu-lar enzymes from C. freundii obeyed the second-order rate kineticmodel (0.987) as greater R2 values were observed than for thefirst-order rate kinetic model (0.960).



Figure 9. GC-MS spectra of (a) o-phenylenediamine (OPD) and (b) OPD after treatment with mixed intracellular enzymes (MIE).

CONCLUSIONSIn the present investigation, an attempt was made to degradethe endocrine disrupting chemical o-phenylenediamine usingmixed intracellular enzymes obtained from the marine bacteriumCitrobacter freundii. To our knowledge, this is the first report ondegradation of endocrine disrupting chemical using MIE. Themixture of intracellular enzymes from C. freundii was purifiedand identified using MALDI-TOF analysis. The various conditionsfor the degradation of OPD using MIE were optimised usingresponse surface methodology. The optimum conditions for thedegradation of OPD by MIE were time, 2 h; pH, 7.0; temperature,30 ∘C; and an enzyme concentration of 0.2% w/v. The degradationefficiency of OPD using the intracellular enzymes was about 93%.In the presence of a metal ion (Zn2+) the degradation efficiencyof OPD was enhanced to about 95.87%. The evidence for thedegradation of OPD was confirmed by UV–visible spectroscopy,

fluorescence spectroscopy, cyclic voltammetry, FTIR spectroscopyand HPLC analysis. The degraded products were identified by NMRspectroscopy and GC-MS analysis. The final degradation productof OPD by MIE was identified as pyruvic acid. The degradationof OPD using intracellular enzymes from C. freundii followedpseudo-second-order kinetics.

ACKNOWLEDGEMENTSP. Saranya is grateful to the Council of Scientific and IndustrialResearch (CSIR), India. The financial assistance under the STRAIT(CSC0201) programme is also gratefully acknowledged.

Supporting InformationSupporting information may be found in the online version of thisarticle.



REFERENCES1 Ashby J, Houthoff E, Kennedy SJ, Stevens J, Bars R, Jekat FW, et al., The

challenge posed by endocrine-disrupting chemicals. Environ HealthPerspect 105:164–169 (1997).

2 Jobling S, Nolan M, Tyler CR, Brighty G and Sumpter JP, Widespreadsexual disruption in wild fish. Environ Sci Technol 32:2498–2506(1998).

3 Sonnenschein C and Soto AM, An updated review of environmentalestrogen and androgen mimics and antagonists. J Steroid BiochemMol Biol 65:143–150 (1998).

4 Subhankar C and Petr K, Removal of the endocrine disruptor butylbenzyl phthalate from the environment. Appl Microbiol Biotechnol87:61–73 (2010).

5 Guanghua L, Junjie H and Xu T, Biodegradation kinetics of substitutedanilines by domesticated algal–bacterial system. Adv Biomed Eng6:465–469 (2012).

6 Wang Q, Tang H, Xie Q, Jia X, Zhang Y, Tan L, et al., The preparationand characterization of poly(o-phenylenediamine)/gold nanoparti-cles interface for immunoassay by surface plasmon resonance andelectrochemistry. Colloids Surf B 63:254–261 (2008).

7 Lelieveld P, Middeldorp RJF and VanPutten LM, Effectiveness ofp-aminobenzyolo-phenylenediamine (Goe 1734) against mouse,rat, and human tumor cells. J Chemotrapy Pharmacol 15:88–90(1985).

8 Sorensen HN, Kirk O, Lolck R and Dester T, Diaminobenzoic acidderivatives as dye precursors. US Patent 6231621 (2001).

9 Sun JC, Treatment and utilization of o-phenylenediamine in wastewa-ter. Anhui Huagong 92:31–33 (1998).

10 GDCh-Advisory Committee on Existing Chemicals of Environmen-tal Relevance (eds), trans. by Coleman H, Phenylenediamines (1,2-diaminobenzene 1, 3-diaminobenzene, 1, 4-diaminobenzene), BUAReport 97. S. Hirzel, Stuttgart (1995).

11 Chung KT and Cerniglia CE, Mutagenicity of azo dyes:Structure–activity relationships. Mutat Res 277:201–220 (1992).

12 O-Phenylene diamine, GuideChem. [Online]. (2010) Available: http://www.guidechem.com/reference/dic-1336.html [16 October 2014].

13 Klibanov AM and Mass B, Removal of combined organic substancesfrom aqueous solutions. US Patent 4623465 (1986).

14 Liu, H, Wang Z, Liu Y, Xiao J and Wang C, Enthalpy change andmechanism of oxidation of o-phenylenediamine by hydrogenperoxide catalyzed by horseradish peroxidase. Thermochim Acta443:173–178 (2006).

15 Nemeth S and Simandi L, Homogeneous catalytic oxidation ofo-phenylenediamine by dioxygen in the presence of cobaloxime(II)derivatives: synthesis of substituted 2H-benzimidazoles. J Mol Catal14: 87–93 (1982).

16 Nezamzadeh-Ejhieh A and Salimi Z, Solar photocatalytic degradationof o-phenylenediamine by heterogeneous CuO/X zeolite catalyst.Desalination 280:281–287 (2011).

17 Gao J, Luan X, Wang J, Wang B, Li K, Li Y, et al., Preparation ofEr3+:YAlO3/Fe-doped TiO2 –ZnO and its application in photocat-alytic degradation of dyes under solar light irradiation. Desalination268:68–75 (2011).

18 Par𝚤lt𝚤 NB and Akten D, Application of Box–Wilson experimen-tal design method for the solar photocatalytic degradation oftextile dyestuff with Fe(III)/H2O2/solar UV process. Desalination260:193–198 (2010).

19 Ee Lui A, Jeffrey Obbard P and Huimin Z, Directed evolution of anilinedehydrogenase for enhanced bioremediation of aromatic amines.Appl Microbiol Biotechnol 81:1063–1070 (2009).

20 Marmur JA, Procedure for the isolation of deoxyribonucleic acids frommicroorganisms. J Mol Biol 3:208–218 (1961).

21 Felsenstein J, Phylip, phylogenetic inference package, version 3.51c.Bacteriology 98:756–766 (1993).

22 Naiem HN and Jai SG, Purification and characterization of catechol 1,2-dioxygenase from Rhodococcus sp. NCIM 2891. Res J Environ EarthSci 3:608–613 (2011).

23 Cenci G and Caldini G, Catechol dioxygenase expression in a Pseu-domonas fluorescens strain exposed to different aromatic com-pounds. Appl Microbiol Biotechnol 47:306–308 (1997).

24 Mayer KM and Arnold FH, A colorimetric assay to quantify dehydroge-nase activity in crude cell lysates. J Biomol Screen 7:13 5–140 (2002).

25 Neujahr HY and Gaal A, Phenol hydroxylase from yeast. Purificationand properties of the enzyme from Trichosporon cutaneum. Eur JBiochem 35:386–390 (1973).

26 Fernley KN, The Enzymes Boger, vol. 4, ed. by Boyer PD. Academic Press,New York, pp. 417–447 (1971).

27 Bergmeyer HU, Methods of Enzymatic Analysis, vol. 2. Verlag Chemie,Weinheim, pp. 127–128 (1983).

28 Lowry OH, Rosebrough NJ, Farr AL and Randal J, Protein measure-ment with the Folin phenol reagent. J Biol Chem 193:265–275(1951).

29 Laemmli UK, Cleavage of structural proteins during assembly of thehead of bacteriophage T4. Nature 227:680–685 (1970).

30 Suresh Pakala B, Purushotham Gorla G, Aleem Basha P, Ravi Kumar K,Rajasekhar B, Mahesh Y, et al., Biodegradation of methyl parathionand p-nitrophenol: Evidence for the presence of a p-nitrophenol2-hydroxylase in a Gram-negative Serratia sp. strain DS001. ApplMicrobiol Biotechnol 73:1452–1462 (2007).

31 Kothari Charmy R, Microbial degradation of organopollutants,PhD thesis, Department of Biosciences, Saurashtra University(2006).

32 Lagergren S and Svenska BK, The theory of adsorption on geloestersubstances. Veternskapsakad Handlingar 24:1–39 (1898).

33 Ho YS and Mckay G, Kinetic models for the sorption of dye fromaqueous solution by wood. Transl Chem E 76B:183–191 (1998).

34 Hildebrand F, Meyer A and Eyre-Walker A, Mail evidence of selectionupon genomic GC-content in bacteria. PLoS Genet 6:1–9 (2010).

35 Reiner AM, Purification and properties of the catechol-formingenzyme 3,5-cyclohexadiene-1,2-diol-1-carboxylic acid (NAD+)oxidoreductase (decarboxylating). J Biol Chem 247:4960–4965(1972).

36 Santos V and Linardi V, Phenol degradation by yeasts isolated fromindustrial effluents. J Gen Appl Microbiol 44:213–221 (2001).

37 Singh BK and Walker A, Microbial degradation of organophosphoruscompounds. FEMS Microbiol Rev 30:428–471 (2006).

38 Box GEP, Hunter WG and Hunter JS, Statistics for Experimenters. Wiley,New York (1978).

39 Yamasaki S, Sakata-Sogawa K, Hasegawa A, Suzuki T, Kabu K, SatoE, et al., Zinc is a novel intracellular second messenger. J Cell Biol177:637–645 (2007)

40 Fukunaka A, Kurokawa Y, Teranishi F, Sekler I, Oda K, Ackland ML, et al.,Tissue nonspecific alkaline phosphatase is activated via a two-stepmechanism by zinc transport complexes in the early secretorypathway. J Biol Chem 286:16363–16373 (2011).

41 Vallee BL and Auld DS, Active-site zinc ligands and activated H2O ofzinc enzymes. Biochemistry 87:220–224 (1990).

42 Glusker JP, Katz AK and Bock CW, Metal ions in biological systems.Rigaku J 16:8–17 (1999).

43 George C, Chris R, Dang N, Malika C and Yi W, Detection and formationscenario of citric acid, pyruvic acid, and other possible metabolismprecursors in carbonaceous meteorites. Proc Natl Acad Sci U S A 108:14015–14020 (2011).


Documents

Biodegradation of the endocrine disrupting chemical o -phenylenediamine using intracellular enzymes from Citrobacter freundii and its kinetic studies