Immobilization of thermotolerant intracellular enzymes on functionalized nanoporous activated carbon and application to degradation of an endocrine disruptor: kinetics, isotherm and thermodynamics studies

Article abstract of DOI:10.1039/c5ra11279f

A bacterium, Serratia marcescens capable of degrading the endocrine disruptor, 2-nitrophloroglucinol (NPG) was isolated from tannery wastewater contaminated soil. The mixed intracellular enzymes (MICE) produced from S. marcescens were extracted and characterized. The functionalized nanoporous activated carbon matrix (FNAC) was prepared to immobilize MICE. The optimum conditions for the immobilization of MICE on FNAC were found to be time, 2.5 h; pH, 7.0; temperature, 40°C; concentration of MICE, 4 mg; particle size of FNAC, 300 μm and mass of FNAC, 1 g. The FNAC materials before and after immobilization of MICE were characterized using scanning electron microscopy, Fourier transform-infrared spectrophotometry and an X-ray diffractometer. The thermal behaviour of the free and the immobilized MICE was studied using thermogravimetric analysis. The immobilization of MICE on FNAC obeyed the Freundlich model and the immobilization process followed a pseudo second order kinetic model. MICE-FNAC matrix was used to degrade NPG in aqueous solution. The degradation of NPG by MICE-FNAC was optimum at contact time, 3 h; pH, 7.0; temperature, 40°C; concentration of NPG, 20 μM and agitation speed, 70 rpm. The degradation of NPG was found to be enhanced in the presence of Zn²⁺, Cu²⁺, Ca²⁺ and V³⁺ ions. The degradation of NPG by MICE-FNAC was studied using UV-visible, fluorescence and FTIR spectroscopy. The degradation of NPG by MICE-FNAC was confirmed using HPLC, NMR and GC-MS spectroscopy.

Full text of DOI:10.1039/c5ra11279f

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Immobilization of thermotolerant intracellular
enzymes on functionalized nanoporous activated
carbon and application to degradation of an
endocrine disruptor: kinetics, isotherm and
thermodynamics studies
Cite this: RSC Adv., 2015, 5, 66239
*
Saranya P, Ranjitha S and Sekaran G
A bacterium, Serratia marcescens capable of degrading the endocrine disruptor, 2-nitrophloroglucinol
(NPG) was isolated from tannery wastewater contaminated soil. The mixed intracellular enzymes (MICE)
produced from S. marcescens were extracted and characterized. The functionalized nanoporous
activated carbon matrix (FNAC) was prepared to immobilize MICE. The optimum conditions for the
immobilization of MICE on FNAC were found to be time, 2.5 h; pH, 7.0; temperature, 40 ꢀC;
concentration of MICE, 4 mg; particle size of FNAC, 300 mm and mass of FNAC, 1 g. The FNAC materials
before and after immobilization of MICE were characterized using scanning electron microscopy, Fourier
transform-infrared spectrophotometry and an X-ray diffractometer. The thermal behaviour of the free
and the immobilized MICE was studied using thermogravimetric analysis. The immobilization of MICE on
FNAC obeyed the Freundlich model and the immobilization process followed a pseudo second order
kinetic model. MICE-FNAC matrix was used to degrade NPG in aqueous solution. The degradation of
NPG by MICE-FNAC was optimum at contact time, 3 h; pH, 7.0; temperature, 40 ꢀC; concentration of
NPG, 20 mM and agitation speed, 70 rpm. The degradation of NPG was found to be enhanced in the
presence of Zn2+, Cu2+, Ca2+ and V3+ ions. The degradation of NPG by MICE-FNAC was studied using
UV-visible, fluorescence and FTIR spectroscopy. The degradation of NPG by MICE-FNAC was confirmed
using HPLC, NMR and GC-MS spectroscopy.
Received 13th June 2015
Accepted 27th July 2015
DOI: 10.1039/c5ra11279f
www.rsc.org/advances
could be the source for the presence of NPG in tannery wastewater.
NPG is characterized by its density (1.771 g cmꢁ3), boiling point
(343 ꢀC), and melting point (189–193 ꢀC).
1. Introduction
Endocrine disrupting compounds are a group of environmental
pollutants mimicking natural hormones, disrupting biosignal-
ling pathways or modifying hormone receptors.1 The endocrine
disrupting chemicals have received increased attention in water
quality management and healthcare management due to their
persistence in the environment and toxic activity even at low
concentration.2
Nitro phloroglucinol (NPG) has been widely used in the
chemical industry as a raw material for manufacturing dyes and in
the explosives industry as an ingredient for priming compositions,
percussion caps and detonator formulations.3,4 NPG is a synthetic
intermediate in the manufacture of benzoxazoles and benzothia-
zoles as selective ligands for human b-estrogen receptor (ER-b)5
and thus it has the ability to bind with the estrogen receptor. In the
leather industry, the use of soluble dyes such as sulphur black 1,
sulphur brown 14, sulphur red 6 and other sulphur based dyes
Nitroaromatic compounds are among the largest and most
important groups of industrial chemicals in use. The ability of
the nitro group to delocalize p electrons of the phenyl ring to
satisfy its own charge deciency and impart charge to the
molecule. The nitro group is strongly deactivated towards
electrophilic aromatic substitution of the benzene ring.6
The stability and recalcitrant nature of NPG are due to both
the conjugation state and resonance properties of nitro groups
attached to aromatic rings.6 The conventional biological treat-
ment of nitro compounds has certain disadvantages such as high
sludge production, high energy demand and frequent mainte-
nance requirement7 besides large land area requirement and less
exibility in design & operation.8 Other methods such as
adsorption, photolysis, chemical oxidation and electrochemical
technique, are widely used for the treatment of nitro compounds
containing wastewater.9 However, all these methods have their
own limitations in the eld level implementation due to high
capital cost, large area requirement and high electrical energy
demand.9
Environmental Technology Division, CSIR-Central Leather Research Institute (CLRI),
Adyar, Chennai, India. E-mail: ganesansekaran@gmail.com; Fax: +91-44-24410232;
Tel: +91-44-24452941
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Advanced oxidation processes (AOPs), based on the in situ physical connement to a certain region of space, retained its
generation of non selective and highly reactive species such as catalytic activity with the capacity to be used continuously.27 Few
hydroxyl radicals (cOH), superoxide anion radicals (O2cꢁ), and research studies have employed certain support matrices such
hydrogen peroxide (H2O2) as initiators of the oxidative degra- as epoxy activated matrix, sepabeads, curdlan matrix for the
dation of refractory organics, have been proposed as an alter- enzyme immobilization.28 The immobilization of enzymes on
native approach to the conventional methods in recent years.10 activated carbon was observed to be nontoxic, fast, inexpensive,
AOP with ozone (O3), ultraviolet (UV), hydrogen peroxide (H2O2) easy to handle and stable against denaturation by oxidants.29
and/or catalyst offered a powerful water treatment solution for However, there is no single report on the immobilization of
the reduction and/or removal of refractory organic intracellular enzymes and thereof for the degradation of NPG in
compounds.11–13 The main disadvantages of AOP are high wastewater. Hence, in the present investigation, mixed intra-
operation costs due to chemicals and/or energy input, forma- cellular enzymes (MICE) were extracted from Serratia marces-
tion of oxidation intermediates potentially toxic and compli- cens and characterized. MICE was immobilized on
cated design procedures.14
functionalized nanoporous activated carbon (FNAC) and used
The biodegradation of pollutants in wastewater mainly for the degradation of NPG. The kinetics and thermodynamics
depends on microorganisms that attack the pollutants enzy- for the immobilization of MICE on FNAC have been evaluated.
matically and convert them into nonhazardous products.15
Bacteria that inhabit chemical waste contaminated sites
possess an astonishing ability of evolving new pathways for
catabolism of recalcitrant organic compounds. Such pathways
are accompanied by the appearance of transcriptional regula-
2. Materials and methods
2.1. Material
2-Nitrophloroglucinol of high purity 98% was obtained from
Alfa Aesar (India). Ethylenediamine and glutaraldehyde of
purity 99.5% were purchased from Sigma-Aldrich-Fluka
Chemical Co., India.
tory circuits that adjust the level of biodegradative capacity of
the bacteria. Transcriptional regulation mediated by the
binding of organic effector on a regulatory protein that acts on
specic protein seems to be late evolutionary development as
compared to metabolic operons and enzymes.16 Biodegradation
of aromatic compounds has therefore, gained an increasing
attention in pollution prevention.17,18
2.2. Culture enrichment and isolation
In biodegradation of wastewater, toxic pollutants were
eliminated either through intracellular accumulation or
through enzymatic transformation into less/nontoxic
compounds,19 applicable only to labile organic compounds.
Sometimes the secondary metabolites of the pollutants are
more toxic and persistent than the parent compounds.20 Hence,
there has been constant research on the development of cost
effective techniques for the treatment of nitro organic
compounds in wastewater. The removal of aromatic nitro
groups by dioxygenase was rst reported by Ecker et al.21
Additional support for the reaction mechanism came from the
observation recorded by Sander et al.22 that the enzyme from a
Pseudomonas sp. could catalyze the elimination of the nitro
group from 2,4,5-trichloronitrobenzene.
The intracellular enzymes were explored for wastewater
treatment as they were highly specic and extremely efficient
catalysts23 and could degrade the target pollutant. The general
disadvantages observed with the application of free enzymes for
the bioremediation of wastewater containing refractory organic
compounds were periodic addition increased the operation
cost, applied free enzyme increased COD load, and sometimes
enzyme activity was lost due to the uctuating environmental
The NPG degrading bacterium was isolated from the terrestrial
soil acclimatized with NPG. Enrichment was carried out in M9
Minimal media (HiMedia) of volume 100 mL with samples
containing 150 mM of NPG as the sole carbon source. NPG
concentration in minimal media was increased from 30 to 150
mM with an incremental increase of 5 mM. A pigmented bacte-
rium isolated from the acclimatized soil was observed to
degrade NPG in the solution.
2.3. Identication of the strains and phylogenic analysis
The pigmented bacterium was identied by 16S ribosomal DNA
(16S rDNA) sequencing and phylogenetic analysis. The genomic
DNA was isolated according to the procedure of Marmur.30
Phylogenetic analysis was performed by subjecting the deduced
sequence to the 16S rDNA database to obtain the similar
sequences and the phylogenetic tree was constructed using the
Phylip package.31
2.4. Culture conditions for intracellular enzyme production
conditions. Hence, immobilization technology has been widely The bacterium was aseptically inoculated into Erlenmeyer
used for improving the activity, stability, specicity, selectivity asks of volume 1000 mL containing 75 mM NPG as the
and decreased inhibition of enzymes.24,25 The function of an substrate in 500 mL of minimal media of pH 7 for intracellular
intracellular enzyme depends largely on its conformation enzyme production. The culture asks were incubated on the
geometry. The stereo chemical conguration of an intracellular shaker at 150 rpm and at 35 ꢀC for 72 h. The bacterial cells were
enzyme changes with harsh conditions such as high tempera- separated from the culture asks by centrifugation at 6000 rpm
ture, very low/high pH, high concentration of target pollutants for 20 min. The bacterial biomass was washed twice with
and high ionic strength.26 The immobilized enzyme in its phosphate buffer (pH 7.0) and stored at ꢁ20 C.
ꢀ
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2.5. Extraction of intracellular enzymes
2.10. Immobilization of MICE on FNAC
The method for intracellular protein extraction was adapted The optimum conditions for the immobilization of MICE on
from Naiem and Jai32 with some modications. The bacterial FNAC were determined by varying the parameters such as time
cells were sonicated with 1 mL of 0.1 M phosphate buffer. The (30 to 210 min), pH (1.0–10.0), temperature (20 to 60 ꢀC),
extracts were centrifuged at 14 000 ꢂ g for 30 min. The cell-free concentration of MICE (1–5 mg), particle size of FNAC (100, 300,
supernatant was collected and dialyzed against 0.1 M phos- 400 & 600 mm) and mass of FNAC (0.5–2.5 g). The immobiliza-
phate buffer (pH 7.0) for overnight. The puried mixed intra- tion of MICE was carried out by equilibrating 1 g of FNAC with
cellular enzymes (MICE) were lyophilized and used for further phosphate buffer of volume 15 mL at pH 7 containing a known
analysis and for the degradation of NPG.
amount of lyophilized MICE. A mg of lyophilized MICE contains
532.9 mg of protein. Immobilization capacity was measured in
terms of residual protein content as shown by the mathematical
expression.
2.6. Assays of intracellular enzymes
The lyophilized enzyme was analyzed for various intracellular
enzymes present. The enzymes involved in the degradation of
À
Á
Initial concentration of MICE ðas proteinÞ ꢁ Final concentration of MICE
Immobilization capacity mg gꢁ1
¼
(1)
ꢀ
ꢁ
moisture content
100
Mass of FNAC 1 ꢁ
NPG were determined using the respective assay procedures:
catechol dioxygenase,33 dehydrogenase,34 phenol hydroxylase,35
alkaline phosphatase,36 pyruvate decarboxylase,37 nitrate
reductase38 and pyruvate kinase.39 The concentration of intra-
cellular enzymes were expressed in terms of protein content,
following Lowry method,40 employing bovine serum albumin as
the standard. The intracellular enzymes were quantied and
used as a mixture of intracellular enzymes for the degradation
of NPG.
2.10.1. Nonlinear kinetic model for the immobilization of
MICE on FNAC. In order to investigate the kinetic rate constants
for the immobilization of MICE on FNAC, the pseudo rst order43
and pseudo second order44 kinetic models were employed,
following the equations (eqn (2) and (3)), respectively.
qt ¼ qe(1 ꢁ expꢁk t
)
(2)
(3)
1
2
k2qe t
qt ¼
1 þ k2qet
where qe and qt are the amounts of MICE immobilized (mg gꢁ1
of FNAC) at equilibrium and at time (t) respectively, k1 and k2
are the rst and second order rate constants.
2.7. Molecular weight determination of puried
intracellular enzymes
2.10.2. Thermodynamic studies on immobilization of
MICE on FNAC. The classical Van't Hoff equation was used to
determine the thermodynamic properties such as change in
Gibbs free energy (DGꢀ), entropy (DSꢀ) and enthalpy (DHꢀ) for
the immobilization of MICE on FNAC.
The molecular weight of enzymes was determined by using
sodium dodecyl sulphate-polyacrylamide gel electrophoresis
(SDS-PAGE) according to the method followed by Laemmli,41 on
a 5% stacking gel and 12% resolving gel. The protein marker
ranging from 5 to 240 KDa was used as a standard marker for
determination of molecular weight.
DGꢀ ¼ DHꢀ ꢁ TDSꢀ
(4)
2.8. Aminoacid composition of MICE by HPLC
ꢀ
2.11. Instrumental techniques for evaluation of
immobilization of MICE on FNAC
The MICE was hydrolyzed at 100 C for 24 h with 6 N HCl and
neutralized with 1 N NaOH. The amino acid composition was
analyzed using Agilent 1100 HPLC amino acid analyzer, and the
data analysis was performed by using HP chem station.
2.11.1. Surface morphology analysis of FNAC and MICE-
FNAC using scanning electron microscopy (SEM). The surface
morphology of FNAC and MICE-FNAC was determined using
Leo–Jeol Scanning Electron Microscope with magnication of
2500–10 000ꢂ. The FNAC samples were coated with gold by a
gold sputtering device for the clear visibility of the surface
2.9. Preparation of functionalized nanoporous activated
carbon (FNAC)
Nanoporous activated carbon (NAC) was prepared from rice morphology.
husk and the functionalization of the NAC was carried out as
2.11.2. Fourier transform-infrared spectroscopic studies.
explained by Ramani et al.42 The washed FNAC was dried at An infrared spectrophotometer (Perkin-Elmer) was used for the
110 ꢀC for 6 h and it was sieved to different sizes as 100, 300, 400 investigation of the surface functional groups. The samples
and 600 mm.
with KBr (spectroscopic grade) pellets were prepared with the
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dimensions diameter, 10–13 mm and thickness, 1 mm. The free CuSO4$5H2O, (NH4)6Mo7O24$4H2O & VCl3 and incubated at
MICE, FNAC and MICE-FNAC were scanned in the spectral 40 ꢀC for 1 h to determine the stimulatory or inhibitory effects of
range of 4000–400 cmꢁ1
.
metal ions on degradation of NPG by MICE-FNAC. The NPG
2.11.3. XRD pattern analysis. The XRD pattern of free MICE, with 15 mL of 0.1 M phosphate buffer (pH 7.0) solution alone
FNAC and MICE-FNAC was determined with high resolution served as control.
GUINER powder X-ray diffractometer (SEIFERT, Germany).
2.11.4. Thermal characterization of MICE and MICE-FNAC.
Required quantity (8–10 mg) of lyophilized MICE, FNAC or
2.14. Instrumental techniques for the NPG degradation by
MICE-FNAC
MICE-FNAC samples were loaded in platinum TGA pan and
The NPG upon degradation by MICE-FNAC was supported by
thermo gravimetric analysis was made under pure nitrogen
the instrumental techniques including high-pressure liquid
chromatography (HPLC) and uorescence spectroscopy. The
nal end product of enzymatic breakdown of NPG was also
conrmed using Fourier transform infrared spectroscopy
(FTIR), nuclear magnetic resonance spectroscopy (NMR) and
gas chromatography-mass spectroscopy (GC-MS).
2.14.1. Fluorescence spectroscopy. NPG is uorescence
inactive and the uorescence spectrum was recorded using a
uorescence spectrophotometer in the wavelength range 200–
800 nm (Cary Eclipse; Agilent Technologies, Middleburg,
Netherlands). The uorescence spectrum of NPG degraded by
MICE-FNAC was also recorded. The enzymatic reaction
mixtures were pre-scanned using the uorescence spectropho-
tometer to study the excitation and emission characteristics of
both NPG and the degraded products.
ꢀ
ꢀ
atmosphere, from 0 C to 800 C at a temperature gradient of
10ꢀC minꢁ1. The thermogram was routinely recorded as dupli-
cates using TGA Universal V4.4A (TA instruments).
DSC analysis was carried out by loading the required quan-
tity (8–10 mg) of lyophilized MICE, FNAC or MICE-FNAC
samples was loaded in aluminum DSC pan and thermo gravi-
metric analysisꢀwas carried out under reduced nitrogen atmo-
ꢀ
sphere from 0 C to 200 C at a temperature gradient of 10ꢀC
minꢁ1. Scans were routinely recorded as duplicates using DSC
Q200 (V23.10 Build 79, TA instruments).
2.12. Kinetic studies on the degradation of NPG by MICE-
FNAC
The degradation study was carried out with the immobilized MICE
on FNAC in a batch reactor. The degradation experiment was
conducted at different variables such as the contact time (5–210
2.14.2. FT-IR for spectroscopic conrmation of degrada-
tion of NPG using MICE-FNAC. The functional groups present
in the initial NPG and the MICE-FNAC degraded NPG were
identied using FT-IR spectrophotometer (Perkin-Elmer). The
samples were lyophilized and pellets were prepared with spec-
troscopic grade KBr of dimensions diameter, 10 mm and
thickness, 1 mm. The spectrum was analyzed in the spectral
ꢀ
min), pH (1–10), temperature (20ꢀ to 80 C), agitation speed (30–
150 rpm) and initial concentration of NPG (5–25 mM). The effect of
contact time was determined by mixing 10 mM of NPG with 1 g of
MICE-FNAC in 10 mL phosphate buffer of pH 7 and they were
ꢀ
incubated at 35 C under mechanical agitation of 150 rpm. The
range of 400–4000 cmꢁ1
.
samples were withdrawn at different time intervals (5 to 210 min)
and UV-visible spectra were recorded using a UV-visible spectro-
photometer (Cary varion; Agilent Technologies, Middleburg,
Netherlands).
2.14.3. High performance liquid chromatography (HPLC).
The degraded compounds of NPG were quantied using HPLC
with a PDA detector at l294 nm (Shimadzu). The sample injec-
tion volume of 100 mL was separated using cartridge containing
C18 column. The mobile phase used was methanol and water in
The optimum pH was determined by incubating the reaction
mixture at various pH ranging from 1 to 10 at room temperature
in the following buffers: 0.1 M KCl/HCl buffer (pH 1.0, 2.0), 0.1
M acetate buffer (pH 3.0, 4.0 and 5.0), 0.1 M phosphate buffer
(pH 6.0, 7.0, and 8.0), 0.1 M Tris buffer (pH 9 and 10) at
optimum time, 60 min & temperature, 35 ꢀC under mechanical
agitation at 150 rpm. The optimum temperature was deter-
mined by incubating the reaction mixture at different temper-
atures ranging from 20 to 80 ꢀC at optimum time, 60 min; pH, 7
under mechanical agitation at 150 rpm. The optimum initial
concentration of NPG was determined by incubating the reac-
tion mixture with varied concentration of NPG (5–25 mM) with 1
g of MICE-FNAC at optimized contact time, pH and tempera-
ture. The agitation speed (30–150 rpm) was optimized for the
degradation of NPG using MICE-FNAC at optimized time, pH,
temperature and initial concentration of NPG.
the ratio 95 : 5 and was applied at a ow rate of 1 mL minꢁ1
.
2.14.4. Nuclear magnetic resonance (NMR) spectroscopy.
The sample preparation for NMR analysis was carried out by
dissolving 5 to 30 mg of lyophilized samples in 650 mL of
deuterated DMSO (Brucker NMR FT 500 mHz). The degraded
products of NPG were identied using 1H and 13C NMR
analyses.
2.14.5. GC-MS analysis. GC-MS analysis of NPG and the
NPG degraded by MICE-FNAC was carried out in Agilent 7000
QQQ (USA). Mass spectra were analysed in the range of 60–650
atom mass units (amu) at the rate of 90 scans minꢁ1 for a run
time of 40 min.
3. Results and discussion
2.13. Effect of metal ions on the degradation of NPG using
3.1. Isolation and identication of bacteria by phylogenetic
MICE-FNAC
analysis
NPG (20 mM) was added to the solution of volume 15 mL con- The soil site selected for isolation of bacteria was acclimatized
taining 1 mM of KCl, CaCl2$2H2O, ZnCl2, MgCl2$6H2O, with NPG for about 45 days. The acclimatized soil sample was
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Fig. 1 Rooted phylogenetic tree showing the relationship of marine isolate S. marcescens to other Serratia sp. (values shown in the parenthesis
are accession number).
inoculated into minimal media (M9, HiMedia) containing NPG as contained polar amino acids by 60.0%, non polar amino acids
the sole carbon source. Aer 3 days of incubation, the above by 27.4% and aromatic amino acids by 13%. The aminoacids
sample was used as an inoculum for the fresh medium contain- such as aspartic acid, glutamic acid, serine and histidine are
ing NPG. The process was repeated for several times in order to very important in functioning of the protein as they constitute
isolate a microorganism for degradation of NPG with high effi- the catalytic triad of the various intracellular enzymes.46
ciency. The guanine–cytosine richness in the bacterial genome,
an important factor in characterizing a species, was about 20–
75%.45 The isolated microorganism was found to be highly stable
3.5. Immobilization of MICE on FNAC
with 53.46% guanine–cytosine content. The microorganism was
isolated for the production of intracellular enzymes. The 16S
rDNA sequencing data conrmed that the isolated organism was
Serratia marcescens (Fig. 1). S. marcescens is a red pigmented
bacterium that grows at pH, 7 and temperature, 35 ꢀC.
The effect of time on the immobilization of MICE was carried
out to determine the equilibrium values. The activity of MICE
and protein content were measured at different time intervals
(30 to 210 min) during the process of immobilization with 2 mg
of lyophilized MICE at pH 7 and at 35 ꢀC (Fig. 3a). It was found
that, the immobilization was rapid up to 90 min and then there
was a slight increase in immobilization until the equilibrium
(150 min) was attained. Initially, the number of adsorption sites
available was higher and the driving force for the mass transfer
of MICE was greater. As the immobilization time was increased,
the number of bare active sites became less and the MICE
molecules might become clustered inside the FNAC particles,
thus impairing the diffusion of MICE. The initial protein
content of MICE was found to be 1065.8 mg and the immobi-
lization capacity of FNAC was 643 mg gꢁ1 of the matrix.
The experiments were carried out at were carried out at a
wide range of pH values between pH 1 and 10 for MICE of initial
protein content of 1065.8 mg and temperature 35 ꢀC in order to
determine the optimum pH for the immobilization of the MICE
onto FNAC. The immobilization of MICE on FNAC was found to
be the maximum at pH 7 with immobilization capacity of 643
mg gꢁ1 as shown in Fig. 3b. Immobilization outside this pH
range was decreased, may be attributed to the factors that the
enzyme may become denatured or lose its tertiary structure,
and, therefore, lose its ability to function as a catalyst for the
enzymatic reactions.
3.2. Extraction and purication of MICE
The MICE extracted from S. marcescens were puried and run on
SDS-PAGE to determine their molecular weight. The SDS-PAGE
analysis showed that there were several bands of enzymes
present with different molecular weight. The MICE was found to
contain the following enzymes: catechol dioxygenase (1.141 U
mgꢁ1), phenol hydroxylase (1.255 U mgꢁ1), dehydrogenase
(0.081 U mgꢁ1), alkaline phosphatase (1.92 U mgꢁ1), pyruvate
decarboxylase (2.761 U mgꢁ1), pyruvate kinase (2.296 U mgꢁ1
and nitrate reductase (2.15 U mgꢁ1).
)
3.3. Identication of MICE
The molecular weights of various enzymes present in the mixed
intracellular enzymes were identied by SDS-PAGE (Fig. 2). The
molecular weight of extracted enzymes were as follows: 1,2-
catechol dioxygenase, 38 kDa; phenol hydroxylase, 28 kDa;
dehydrogenase, 109 kDa; alkaline phosphatase, 70 kDa; pyru-
vate decarboxylase, 71 kDa; pyruvate kinase, 63 kDa; and nitrate
reductase, 156 kDa.
The optimum temperature for the immobilization of MICE
onto FNAC was determined by varying the temperature from 20
3.4. Aminoacid composition of MICE
The amino acid composition of MICE was determined using to 60 ꢀC at an initial MICE activity (1065.8 mg) at their optimum
HPLC as shown in Table 1. It was found that the MICE pH 7. The immobilization of MICE on FNAC was found to be the
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The optimum particle size of FNAC for the immobilization of
MICE was determined by varying the particle size as 100, 300,
400 & 600 mm at the optimum conditions pH, 7; temperature,
40 ꢀC and MICE concentration, 4 mg (2131.6 mg of protein). The
immobilization capacity of FNAC was increased with decrease
in the particle size of FNAC. The optimum particle size of FNAC
was found to be 300 mm with the immobilization capacity of
1645 mg gꢁ1 of FNAC at the optimum conditions (Fig. 3e).
The optimum mass of FNAC for the immobilization of MICE
onto FNAC was determined by varying the mass (0.5 to 2.5 g) at
the optimum conditions pH, 7; temperature, 40 ꢀC; MICE
concentration, 4 mg (2131.6 mg of protein) and FNAC particle
size, 300 mm. The optimum mass of FNAC was found to be 1 g
with the immobilization capacity of 1645 mg gꢁ1 of FNAC at the
optimum conditions (Fig. 3f).
Thus, the optimum conditions required for the maximum
immobilization of MICE on FNAC was found to be time, 150
min; pH, 7; temperature, 40 ꢀC; MICE concentration, 4 mg
(2131.6 mg of protein); FNAC particle size, 300 mm and mass of
FNAC, 1 g with the immobilized MICE capacity of 1645 mg gꢁ1
.
3.5.1. Adsorption isotherms. Adsorption data are repre-
sented by isotherms and they were used in determining the
immobilization of MICE on FNAC. In the present study, the
Langmuir and Freundlich isotherm models were deliberated for
this purpose. The Langmuir isotherms were developed based on
an assumption that monolayer adsorption onto a surface con-
taining a nite number of adsorption sites with uniform ener-
gies of adsorption. It is represented as follows (eqn (5)).49
Fig.
2 SDS-PAGE of purified MICE from S. marcescens lane 1
molecular weight of marker, lane 2 purified MICE.
Table 1 Aminoacid composition of MICE from S. marcescens
Amino acids
Level (nmol mLꢁ1
)
Aspartic acid
Glutamic acid
Serine
Histidine
Glycine
Threonine
Arginine
Alanine
Tyrosine
Methionine
Valine
Phenylalanine
Isoleucine
Leucine
Lysine
Cysteine
11
91
18
85
248
80
26
235
18
74
79
25
135
29
150
97
KLCe
qe ¼
(5)
1 þ bCe
qe (mg gꢁ1) and Ce (mg Lꢁ1) are the amount of MICE adsorbed
per unit weight of FNAC and equilibrium liquid phase
concentration respectively. KL (L gꢁ1) and b (L mgꢁ1) are the
Langmuir adsorption constants (Table 2). The essential features
of the Langmuir isotherm can be expressed in terms of a
dimensionless constant called separation factor, RL (also called
equilibrium parameter), dened by eqn (6).
1
RL ¼
(6)
1 þ KLCo
Co (mg Lꢁ1) is the initial MICE concentration. The value of RL
indicates the shape of the isotherms to be either unfavourable (RL
> 1), linear (RL ¼ 1), favorable (0 < RL < 1) or irreversible (RL ¼ 0).
The Freundlich isotherm can be applied to non-ideal
adsorption onto heterogeneous surfaces as well as multilayer
sorption and is expressed by eqn (7).
maximum at 40 ꢀC. At 40 ꢀC temperature, the maximum loading
achieved was 705 mg gꢁ1 of FNAC (Fig. 3c). The reaction rate
decreased beyond the optimum temperature owing to dena-
turation of enzymes and thereby lost its ability to function as a
catalyst in the enzymatic reaction.
1
n
qe ¼ KFC
(7)
‘KF’ (mg gꢁ1) (mL mgꢁ1) is the Freundlich constant related to
adsorption capacity of FNAC and ‘n’ is the Freundlich exponent
(Table 2).
The adsorption isotherms indicated that immobilization of
MICE on FNAC obeyed Freundlich isotherm model based on the
regression coefficient (R2). The value of RL, the separation factor
fell in the range of less than zero, indicating that the immobi-
lization of MICE on FNAC was irreversible. This conrmed the
The immobilization capacity of FNAC was found to increase
with increase in the initial MICE concentration, however, the
percentage immobilization decreased with increase in MICE
concentration. The optimum MICE concentration was found to
be 4 mg (2131.6 mg of protein) with the immobilization capacity
of 1287 mg gꢁ1 of FNAC at the optimum pH and temperature
(Fig. 3d). The high immobilization capacity of FNAC may be due
to more number of anchoring sites in the matrix.
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Fig. 3 Effect of (a) time, (b) pH, (c) temperature, (d) initial concentration of MICE, (e) particle size of FNAC and (f) mass of FNAC on the
immobilization of MICE on FNAC.
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Table 2 Isotherm parameters for the immobilization of MICE on FNAC
Langmuir constants
Freundlich constants
KL (L gꢁ1
)
3.294
5.868
KF (mg gꢁ1) (L mgꢁ1
)
0.513
3.12
0.9349
b (L mgꢁ1
)
n
R2
RL
0.8627
R2
2.8 ꢂ 10ꢁ4
immobilization of MICE, through the formation of a strong
bonding with adsorption sites of FNAC.
3.5.2. Nonlinear kinetic model for the immobilization of
MICE on FNAC. The validity of kinetic order for the immobili-
zation of MICE on FNAC process was based on the regression
coefficients and c2 values. The experimental data were plotted
under each model (gures not shown). The values of k1, k2 and
qe for each sorption process were determined from the slopes
and intercepts of the plots and they are presented in Table 3
along with their relevant regression coefficients (R2). Relatively
higher R2 values, least c2 values and more or less closer exper-
imental qe and calculated qe values showed a better agreement
of immobilization of MICE on FNAC with the pseudo second
order kinetic model (Fig. 4). The pseudo second order kinetic
model suggests that the rate of this adsorption process depend
both on concentration of MICE and adsorption sites of FNAC.
Fig. 4 Kinetic modelling for the immobilization of MICE on FNAC.
3.6. Thermodynamic studies for immobilization of MICE on
FNAC
The thermodynamic parameters were calculated using the eqn
(4). The change in positive entropy value (DSꢀ ¼ 0.256 kJ molꢁ1
Kꢁ1) indicates that the immobilization of MICE on FNAC was
accompanied with increased random distribution of it at stan-
dard temperature, 25 ꢀC. Moreover, the negative value of
enthalpy (DHꢀ ¼ ꢁ78.19 kJ molꢁ1) of the system indicates the
immobilization of MICE on FNAC was an exothermic process at
25 ꢀC. The negative free energy (DGꢀ ¼ ꢁ153.20 kJ molꢁ1) value
indicates the immobilization of MICE on FNAC was sponta-
ꢀ
neous at 25 C.
3.7. Evidences for the immobilization of MICE on FNAC
Fig. 5 SEM images of (a) FNAC and (b) FNAC-MICE.
3.7.1. SEM morphology of FNAC and MICE-FNAC. The SEM
images of FNAC (Fig. 5a) reveal that the surface was covered
Table 3 Comparison of pseudo first- and second-order kinetic parameters of immobilization of MICE on FNAC (initial MICE activity, 1065.8 mg;
pH, 7.0; temperature, 40 ꢀC; particle size, 300 mm; mass of FNAC, 1 g and agitation, 150 rpm)
Pseudo rst order
Pseudo second order
Temperature
(ꢀC)
qe (exp)
k1
qe (Cal)
k2
qe (Cal)
(mg gꢁ1
)
(minꢁ1
)
(mg gꢁ1
)
R2
c2
2.63
0.26
4.80
8.94
9.52
(mgꢁ1 g minꢁ1
)
(mg gꢁ1
)
R2
c2
20
30
35
40
50
60
223
456
643
705
546
210
0.0057
0.0194
0.0201
0.0203
0.0117
0.0124
248
467
701
789
623
305
0.973
0.943
0.970
0.980
0.487
0.852
3.17 ꢂ 10ꢁ5
2.42 ꢂ 10ꢁ5
1.26 ꢂ 10ꢁ5
1.20 ꢂ 10ꢁ5
1.06 ꢂ 10ꢁ5
2.46 ꢂ 10ꢁ5
230
450
652
692
554
254
0.996
0.969
0.998
0.999
0.942
0.983
0.21
0.08
0.12
0.24
0.12
7.62
29.5
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Fig. 6 FT-IR spectra of (a) MICE, (b) FNAC and (c) MICE-FNAC.
with an epitaxially grown deposit on the outer pore surface area
The FT-IR spectrum of FNAC shows the peak corresponding
and the chemical deposit was uniform in nature with regard to to the N–H stretching vibration of a secondary amine at 3416.26
the opacity. This may be regarded as an amino aldehyde cmꢁ1. The signicant increase in the intensity of the band at
chemical deposit on the nanoporous activated carbon surface. 1715.57 cmꢁ1 in FNAC corresponds to C–O stretching vibrations
The surface morphology of MICE-FNAC shows that the MICE of carbonyl or aldehyde groups. The bands at 2923.82 cmꢁ1 and
have been immobilized onto the walls of the pores of FNAC 1365.59 cmꢁ1 may be attributed to methylene stretching and
(Fig. 5b).
bending vibrations respectively. This may be due to the addition
3.7.2. Fourier transform-infrared spectroscopic studies. of ethylenediamine and glutaraldehyde during the preparation
The FT-IR spectrum of puried MICE is shown in Fig. 6a. The of FNAC. This conrms the condensation of ethylenediamine
FT-IR spectrum of MICE shows major protein bands (due to and glutaraldehyde onto nanoporous activated carbon.42
vibration of the peptide linkage) in the spectral region of 1200–
The FT-IR spectrum of MICE-FNAC (Fig. 6c) showed that the
1700 cmꢁ1. The band at 1635.49 cmꢁ1 is due to the C]O frequency at 1652.34 cmꢁ1 may be attributed to amide I of
stretching vibrations of amide I. The bands at 3434 cmꢁ1 and immobilized MICE. The peak corresponding to C–O stretching
1547.03 cmꢁ1 may be attributed to N–H stretching and C–N vibration (1715.57 cmꢁ1) in MICE-FNAC of ketone and aldehyde
bending. The bands at 633.59 cmꢁ1 and 662.85 cmꢁ1 may be groups were masked because of the strong binding of NH2
attributed to C–N stretching vibrations in MICE. The bands at group of the MICE with the aldehyde group of FNAC. The wide
2925.88 cmꢁ1 and 2855.09 cmꢁ1 may be due to the asymmet- peak observed at 3414.43 cmꢁ1 in MICE-FNAC is due to the N–H
rical and symmetrical C–H stretching of methylene groups stretching vibration of secondary amine.
respectively. The band at 1162.55 cmꢁ1 may be attributed to
3.7.3. XRD pattern of MICE, FNAC and MICE-FNAC. The
C–O stretching vibrations present in MICE.
XRD patterns of the MICE, FNAC and MICE-FNAC are shown in
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Fig. 7 XRD pattern of (a) MICE, (b) FNAC and (c) MICE-FNAC.
Fig. 7a–c respectively. The XRD pattern of MICE (Fig. 7a) loss at 114.45 ꢀC due to the removal of moisture. A major weight
ꢀ
suggests its crystalline nature with diffracting peaks of 2q angle loss occurred from 196.32 to 563.06 C due to the decomposi-
at 10.88, 16.67, 17.36, 23.76, 26.55, 28.21, 31.13, 33.08, 35.30, tion of the major components of the MICE. At the end of the
ꢀ
36.14, 39.61, 43.23, 51.02, 53.80, 63.26 and 66.04ꢀ due to pres- scan (800 C), 39.62% of the sample remained as xed residue
ence of the mixed enzymes. The XRD pattern of FNAC (Fig. 7b) indicating the thermal sensitivity of the constituents of MICE.
contains peaks corresponding to 2q values at 11.3, 12.9, 15.8,
The TGA of FNAC (Fig. 8b) showed 3.17% weight loss at
ꢀ
18.0, 20.1, 22, 25.05, 25.6, 26.6 and 30.9ꢀ due to surface func- 101.34 C due to the removal of moisture. A major weight loss
ꢀ
tionalization (Ramani et al., 2012). The immobilization of MICE occurred from 194.24 to 533.58 C and 62.05% of the sample
in FNAC (Fig. 7c) was further conrmed with XRD pattern, remained as xed residue at 800 ꢀC, indicating the thermal
which showed the peaks at 12.9ꢀ, 19.46 and 22ꢀ due to surface resistance of the constituents of FNAC.
functionalization and peaks at 2q values of 10.69, 14.11, 21.26,
23.62, 27.37, 48.52, 52.58 and 62.98ꢀ conrmed the presence of weight loss at 90.31 ꢀC due to the removal of moisture. A weight
MICE in MICE-FNAC.
loss of 9.64% was observed at 190.39 ꢀC which could be due to
The thermogram of MICE-FNAC (Fig. 8c) showed 8.05%
3.7.4. Thermogravimetric analysis of MICE, FNAC, MICE- decomposition of smaller particles in MICE. A major weight loss
FNAC. The thermogram of MICE (Fig. 8a) showed 8.91% weight occurred from 215.12 ꢀC to 376.89 ꢀC is due to the
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Fig. 8 TGA of (a) MICE, (b) FNAC and (c) MICE-FNAC and DSC of (d) MICE, (e) FNAC and (f) MICE-FNAC.
decomposition of the major components of the MICE-FNAC. At transition 144.7 J gꢁ1 and 187.2 J gꢁ1 respectively. The DSC of
the end of the scan (800 ꢀC), 62.60% of the sample remained as FNAC (Fig. 8e) shows there was a thermal transition at 118.09 ꢀC
xed residue, indicating the higher thermal resistance of the with the enthalpy of transition 203.9 J gꢁ1. The DSC of MICE-
constituents of MICE-FNAC than MICE.
FNAC (Fig. 8f) showed a sharp endothermic thermal transi-
The DSC of MICE (Fig. 8d) shows that there was an endo- tion at 111.38 ꢀC and small bumps at 171.34 ꢀC & 233.33 ꢀC with
thermic transition at 75.97 C, 208.05 C with the enthalpy of the enthalpy of transition 178.9 J gꢁ1
.
ꢀ
ꢀ
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Fig. 9 Degradation of NPG using MICE-FNAC (a) effect of time (experimental conditions: pH, 7; temperature, 30 ꢀC; initial NPG concentration,
10 mM; agitation speed, 70 rpm), (b) effect of pH (experimental conditions: time, 3 h; temperature, 30 ꢀC; initial NPG concentration, 10 mM;
agitation speed, 70 rpm), (c) effect of temperature (experimental conditions: time, 3 h; pH, 7; initial NPG concentration, 10 mM; agitation speed, 70
rpm), (d) effect of initial concentration of NPG (experimental conditions: time, 3 h; pH, 7; temperature, 40 ꢀC; agitation speed, 70 rpm) and (e)
effect of agitation speed (experimental conditions: time, 3 h; pH, 7; temperature, 40 ꢀC; initial NPG concentration, 20 mM).
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at l290 nm indicating partial degradation of NPG at 20 ꢀC
(Fig. 9c). The peak at l390 nm was completely disappeared at
ꢀ
30 C and the maximum amount of new compound at l290 nm
was found at 40 ꢀC. As the temperature increased, the intensity
of characteristic peak at l390 nm decreased progressively and the
intensity of new peak at l290 nm increased steadily.
The initial concentration of NPG (Fig. 9d) was varied and it
was found that the optimum concentration of NPG was about 20
mM to obtain the maximum concentration of new compound.
The effect of agitation speed on degradation of NPG using
MICE-FNAC was studied. It was observed that there was a small
peak at l390 nm and a new peak at l290 nm indicating partial
degradation of NPG at agitation speed of 30 rpm (Fig. 9d). As the
agitation speed increased, the peak at l390 nm was completely
eliminated but a new compound of high intensity appeared at
70 rpm.
Fig. 10 UV-visible spectra of degradation of NPG by MICE-FNAC in
the presence of metal ion enhancers.
The optimum conditions for the degradation of NPG using
MICE-FNAC was found to be time, 3 h; pH, 7; temperature, 40
ꢀC, initial concentration of NPG, 20 mM and agitation speed, 70
rpm.
These results suggest that the thermal stability of the MICE
was enhanced due to the stabilized bonding between MICE and
functional groups of FNAC. And also the results suggested that
there was not much change in their enthalpy of transition,
indicating that the nature of MICE was not being altered upon
immobilization.
3.9. Effect of metal ions
Most enzymes require the presence of metal ion activators to
express their catalytic activity. Metal ions act as cofactors for
3.8. Degradation kinetics of NPG using MICE-FNAC
The effect of various factors such as contact time (5–210 min), many enzymes to increase their catalytic activity at lower
ꢀ
pH (1–10), temperature (20ꢀ to 80 C), agitation speed (30–150 concentrations while the catalytic activity was reduced due to
rpm), initial concentration of NPG (5–25 mM) was studied on the toxic effect of metal ions at high concentrations.47 The effect
degradation of NPG using 1 g of immobilized MICE (1645 mg of various metal ions such as K+, Zn2+, Cu2+, Ca2+, Mg2+, Mo6+
gꢁ1 of FNAC). The degradation of NPG as a function of time and V3+ on the degradation of NPG using MICE-FNAC was
(Fig. 9a) shows the disappearance of characteristic peak of NPG studied in the present investigation. Zn2+, Cu2+, Ca2+ and V3+
(l390 nm) with increase in time and increase in the intensity of enhanced the catalytic effect on degradation of NPG using
the formation of new compound at l290 nm using MICE-FNAC. MICE-FNAC (Fig. 10). All other metal ions inhibited the degra-
The degradation of NPG using free MICE also showed the dation of NPG as evidenced from the absence of new compound
disappearance of characteristic peak of NPG (l390 nm) and the formation at l290 nm. The metal ions such as Zn2+, Cu2+, Ca2+
formation of new compound at l290 nm (Fig. 9a). The maximum and V3+ had stimulatory effect on the degradation of NPG were
ꢀ
degradation was attained in 3 h at pH, 7; temperature, 30 C; used in combination (Fig. 10). The intensity of new compound
initial NPG concentration, 10 mM with agitation speed of 70 formed at l290 nm was in the decreasing order as V3+ > all metal
rpm. The degradation of NPG using FNAC was used as the ions > Cu2+ > Ca2+ > Zn2+ ions.
control; where there is no formation of new compound at l290
The MICE from S. marcescens such as dehydrogenase and
nm suggesting MICE mediates the degradation of NPG.
alkaline phosphatase could be enhanced by the presence of zinc
The effect of pH on the degradation of NPG using MICE- ion.46 Copper ion enhanced the activity of dioxygenase,48 while
FNAC was studied. It was observed that MICE-FNAC was able the kinase was activated by calcium and reductase by vanadium
to degrade NPG at different pH conditions from 1–10 (Fig. 9b) ion.49 In protein metal-binding sites, the metal ion is coordi-
and the maximum degradation of NPG was observed at pH 7.0 nated by different combinations of protein side chains,
using MICE-FNAC. This also suggests that the carrier matrix, including the nitrogen of histidine, the oxygen of aspartate or
FNAC provides MICE the ability to retain the geometry from glutamate and the sulfur of cysteine; among these, histidine is
denaturation despite the wide uctuations in pH. The chemical the most commonly observed, followed by cysteine.50
resistance of MICE in its immobilized state may be correlated
with the strong binding established with FNAC.
The Zn2+ activates alkaline phosphatase by binding to the
ligands three histidine and two water molecules, while Zn2+
The effect of temperature on the degradation of NPG using activates dehydrogenase enzyme by binding with two cysteine,
MICE-FNAC was studied. The NPG degradation increases with one histidine and a water molecule. Copper ion activates
increase in temperature. It was observed that the MICE-FNAC dioxygenase by forming square planar coordination with two
could withstand wide range of temperatures. The intensity of histidine, one cysteine and a water molecule while calcium ion
formation of new compound varied but the degradation of NPG forms a octahedral coordination with two aspartate residues (or
was achieved. It was observed that the characteristic peak of NPG one aspartate and one asparagine) bound in a monodentate
at l390 nm was reduced in its intensity and a new peak appeared manner, one glutamate residue bound in a bidentate manner,
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Fig. 11 Fluorescence spectra of (a) NPG, (b) NPG degradation using MICE-FNAC in presence of vanadium (c) all metal ions (d) calcium, (e) copper
and (f) zinc ions.
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Fig. 12 FT-IR spectra of (a) NPG and (b) NPG after treatment with MICE-FNAC.
one main-chain carbonyl group and one water molecule. The showed that uorescent active compounds were formed with
sixth ligand is variable and may be water, serine, asparagine or excitation and emission peaks around l340 nm and l410 nm
aspartate.51 Vanadium ion activates reductase by binding with respectively (Fig. 11). The presence of an emission peak in the
the carboxylate groups in aspartate, glutamate and cysteine spectrum could be due to the complete destruction of NPG and
molecules. The metal ion in the active site of intracellular the formation of uorescence active end products. Thus, the
enzymes causes polarisation of hydrogen–oxygen bond, making uorescence spectra conrm the degradation of NPG by MICE-
the oxygen slightly more negative, thereby weakening the FNAC in the presence of Zn2+, Cu2+, V3+, Ca2+ ions and AM ions.
peptide enhancing the enzyme activity.52
3.10.2. FT-IR spectra of NPG and degraded NPG by MICE-
FNAC. The FT-IR spectrum of NPG (Fig. 12a) shows the char-
acteristic peaks. The bands at 1489.48 cmꢁ1 and 1646.17 cmꢁ1
are attributed to stretching vibrations of C]C in aromatic ring.
The bands at 1549.43 cmꢁ1 and 1588.17 cmꢁ1 are due to the
asymmetric stretching vibrations of N]O in nitro group. The
band at 1312.91 cmꢁ1 is attributed to symmetric N]O
stretching vibration in nitro group. The stretching of aromatic
C–H is observed at 3096.1 cmꢁ1. The C–N stretching in NPG is
conrmed at 836.67 cmꢁ1 and 699.02 cmꢁ1. The bands at
770.25 cmꢁ1, 520.92 cmꢁ1, 470.13 cmꢁ1 are due to the out of
plane bending vibrations of C–H in aromatic ring. The inplane
3.10. Instrumentation evidences for the degradation of NPG
NPG was treated with MICE-FNAC at the optimized conditions
and the degradation was studied using the following instru-
mentation techniques.
3.10.1. Fluorescence spectroscopy. The uorescence
spectra of NPG and degraded NPG using MICE-FNAC in the
presence of Zn2+, Cu2+, V3+, Ca2+ ions and all metal (AM) ions are
shown in Fig. 11. NPG is a uorescent inactive compound as
shown in Fig. 11a. The uorescence spectra of NPG degraded by
MICE-FNAC in the presence of Zn2+, Cu2+, V3+ and Ca2+ ions
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Fig. 13 HPLC chromatogram of (a) NPG and (b) NPG after degradation by MICE-FNAC.
aromatic C–H bending vibrations were conrmed at 1081.8 MICE-FNAC, which is further conrmed with H1 and 13C NMR
cmꢁ1 and 1015.6 cmꢁ1. The band at 3456.25 cmꢁ1 is attributed spectroscopy.
to the strong bonded O–H vibrations.
3.10.4. 1H & 13C nuclear magnetic resonance spectroscopy.
1
The degradation of NPG by MICE-FNAC was conrmed from The H NMR spectrum of NPG was scanned between d 0 and d
the FT-IR spectrum (Fig. 12b). The band at 1642.17 cmꢁ1 is 12 ppm. The 1H-NMR spectrum showed signals in the aromatic
attributed to asymmetric stretching of carboxylate ion in degraded region around d 6.4 and 6.6 ppm which indicates hydrogen
sample. The peak at 1450.3 cmꢁ1 is attributed to asymmetric attached to substituted benzene skeleton. The chemical shi
bending of CH3 group. The band at 3436.68 cmꢁ1 is due to the around d 4.0 ppm can be assigned to the proton in OH group
stretching of O–H group. The peak at 1084.12 cmꢁ1 may be attached to aromatic ring (Fig. 14a). The MICE-FNAC degraded
attributed to stretching of C–O group. The FT-IR spectrum NPG shows the presence of aliphatic proton at d 3.4 and d 3.8
conrmed the degradation of NPG and the presence of a keto ppm.
group concludes the degraded end product could be pyruvic acid.
The 13C NMR spectrum of NPG was scanned between d 0.0
3.10.3. High-performance liquid chromatography. The and d 200 ppm. The chemical shi around d 130–140 ppm
HPLC chromatogram of NPG and NPG aer degradation is indicates the evidence for phenyl carbon in the aromatic ring
shown in Fig. 13. The HPLC chromatogram of the initial NPG (Fig. 15a). The occurrence of chemical shis at d 118.45, d
shows a compound with retention time (Rt) at 9.6 min. Fig. 13b 118.22 and d 115.41 ppm conrm the presence of carbon atoms
shows that the retention time of the parent compound dis- attached to hydroxyl groups. The chemical shi at d 78.93 ppm
appeared and new peaks were appeared at retention time (Rt) of can be assigned to the carbon attached to nitro groups. The 13C-
8.6, 8.3 & 1.2 min. This conrms the degradation of NPG by NMR spectrum of MICE-FNAC degraded NPG sample shows
that the peaks around d 110–140 ppm were disappeared. The
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Fig. 14 1H NMR spectra of (a) NPG and (b) NPG after degradation by MICE-FNAC.
chemical shi at d 208 ppm indicates the evidence for keto peak d 180 ppm may be due to the masking effect of the solvent.
carbon. The chemical shi at d 18.71 ppm indicates the The absence of peaks at d 78.93, 118.45, d 118.22 and d 115.41
evidence for methyl carbon (Fig. 15b). The absence of carboxylic ppm conrms the degradation of NPG by MICE-FNAC.
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Fig. 15 13C NMR spectra of (a) NPG and (b) NPG after degradation by MICE-FNAC.
[M+] at m/z ¼ 173.9 with a retention time of 10.93 min was found
to be identical to the mass spectral properties of the standard
NPG (Fig. 16a). The mass spectrum data of NPG shows the
3.10.5. GC-MS spectra of NPG before and aer degradation
by MICE-FNAC. GC-MS analysis was used to identify the end
products of NPG degraded by MICE-FNAC. The molecular ion
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Fig. 16 GC-MS spectra of (a) NPG and (b) NPG after degradation by MICE-FNAC.
fragment peak of C6H5O4 (i.e. 141) on elimination of neutral NO fragmentation of the COOH group, yielded CH3CO+, corre-
molecule with a rearrangement to form the phenoxy cation (m/z sponding to a peak at m/z ¼ 43 and on elimination of the CH3
¼ 143.1). The MS data show the molecular ion at m/z ¼ 101 group, a peak corresponding to m/z ¼ 33 (Fig. 16b). The frag-
could be due to the elimination of two alcohol groups.
ments with molecular ions conrm the presence of pyruvic acid
The MICE-FNAC degraded NPG sample showed the presence as the product of degradation of NPG by MICE-FNAC.
of pyruvic acid, corresponding to a molecular ion peak at m/z ¼ The plausible pathway for the degradation of NPG by MICE-
88 with the retention time of 8.258 min. Pyruvic acid, on FNAC involves the reduction of the nitro group in NPG by
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Acknowledgements
P. Saranya is grateful to the Council of Scientic and Industrial
Research (CSIR), India. The nancial assistance under the
STRAIT (CSC0201) programme is also gratefully acknowledged.
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