Textile dye degradation potential of plant laccase significantly enhances upon augmentation with redox mediators

Article abstract of DOI:10.1039/c5ra12454a

Cell suspension cultures of Blumea malcolmii Hook. exhibited 98% decolorization of a textile dye Brilliant Blue R (BBR) at a concentration of 40 mg L^-1 within 24 h. A significant induction in the intracellular laccase activity (607%) was observ

Full text of DOI:10.1039/c5ra12454a

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Textile dye degradation potential of plant laccase significantly enhances upon
augmentation with redox mediators
a
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Anuradha N. Kagalkar , Rahul V. Khandare and Sanjay P. Govindwar *
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aDepartment of Biochemistry, Shivaji University, Kolhapur-416004, India
bDepartment of Biotechnology, Shivaji University, Kolhapur-416004, India
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*Address for correspondence
Prof. S. P. Govindwar
Department of Biochemistry,
Shivaji University, Kolhapur- 416 004, India
Email: spg_biochem@unishivaji.ac.in
Tel: +91-231-2609152
Fax: +91-231-2691533
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Abstract
Cell suspension cultures of Blumea malcolmii Hook. exhibited 98% decolorization of a
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textile dye Brilliant Blue R (BBR) at a concentration of 40 mg L within 24 h. A significant
induction in the intracellular laccase activity (607%) was observed during decolorization
of BBR. Twelve different redox mediators showed noteworthy degradation of BBR when
added independently to cell cultures. Nevertheless, augmentation of 2, 2’ Azino-bis 3-
ethylbenzothiazoline 6-sulfonic acid (ABTS) achieved 100% decolorization within 30 min.
Purified laccase from B. malcolmii was revealed to have a molecular weight of 40 kD.
Thirteen different phenolic and non-phenolic substrates were also oxidized by the purified
enzyme. Purified enzyme was found to degrade five structurally different textile dyes in
presence of ABTS. The enzyme took 12 h to completely remove BBR from the solution,
however, addition of ABTS tuned up the catalytic action of enzymes achieving up to 96%
decolorization within 5 min. Degradation of BBR was confirmed by high performance liquid
chromatography and gas chromatography-mass spectroscopy. Precise role of laccase in
phytodegradation of BBR was further proposed in a schematic pathway. Phytotoxicity
studies revealed decrease in toxicity of degradation metabolites of the parent dye.
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Keywords: Decolorization; phytoremediation; cell suspension cultures; Blumea malcolmii
Hook.; Brilliant Blue R; purified laccase
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1. Introduction
The extraordinary pollutant degrading abilities of plants directly rely on their
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complex mechanisms of metabolism. Plants are endowed with efficient enzymatic
machineries which are known to treat a variety of xenobiotic compounds covering heavy
metals, polyaromatic hydrocarbons, pesticides, munitions, petroleum products and crude
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oils. Out of the different types of pollutants that have been contaminating our natural
resources, dyes which are released by textile, dyestuff and dyeing industries constitute
heavily recalcitrant pollutants. Many of the textile dyes are reported to have carcinogenic
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and mutagenic effects with a potential toxicity to all life forms. Textile dyes generally have
very complex chemical structures which craft them difficult to be degraded by physico-
chemical methods. For instance, in sulfonated dyes, the organosulfonate group plays an
important role in altering the solubility and dispersion properties of the dye molecule. It
increases recalcitrance to normal environmental breakdown because of the
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thermodynamically stable carbon-sulfur bond. Plant enzymes have been shown to carry
out the breakdown the dye structures through oxidation, desulfonation, dehalogenation,
denitrification and can even cleave the aromatic structures.5-8
Phytodegradation research still lacks the knowledge regarding the basic
mechanisms with respect to enzymatic involvement for the removal of dyes and their
products formed after degradation. This sort of basic research is an essential prerequisite
that would lay the foundation for in situ application of these phytotechnologies. In the last
decade, a number of reports on plant based removal of textile dyes have focused on
enzymatic degradation. Various enzymes like lignin peroxidase, tyrosinase, azo reductase,
laccase, dichlorophenol indophenol reductase and riboflavin reductase from Blumea
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malcomii, Typhonium flagelliforme, Aster amellus, Glandularia pulchella, Petunia grandiflora,
Zinnia angustifolia, Portulaca grandiflora etc. have been shown to play a vital role in
breaking the complex structures of textile dyes to form different products.9-15 All these
studied enzymes were used from crude extracts of plant tissues, therefore the roles of
individual enzymes could not be predicted. Because of this lacuna in earlier works, the
underlying cellular and metabolic mechanisms during enzymatic degradation of dyes were
partially proposed or are unclear.
Enzymatic processes for bioremediation are characterized by high reaction rates
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and stoichiometric efficiencies. Laccases have always been endorsed as key enzymes for
the biodegradation of xenobiotics. Most of the isolated laccases are from fungal sources and
there are fewer reports on purification of the enzyme from plant sources.17 A broad
substrate specificity of laccases facilitate their application in the degradation of textile dyes.
In an earlier study, in vitro grown suspension cultures of B. malcolmii were proposed to
metabolize Malachite Green dye showing a prominent involvement of laccase during the
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degradation process. In the present work, B. malcolmii cell suspension was used for the
degradation of dyes with different structures and Brilliant Blue R (BBR) was taken further
for detailed mechanistic studies with purified laccase. Phytoremediation technologies are
generally slower when compared to other physico-chemical and even biological treatment
methods. In this study, various redox mediators were therefore explored to enhance the
degradation rate of BBR. The biochemical mechanism of metabolism of BBR by purified
laccase is also proposed.
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2. Materials and Methods
2.1. Chemicals and dyes
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2, 2’ Azino-bis 3-ethylbenzothiazoline 6-sulfonic acid (ABTS) was purchased from
Sigma Aldrich (St Louis, MO, USA). Tartaric acid, dichlorophenol indophenol (DCIP), n-
propanol, o-tolidine, hydroquinone, pyrogallol, guaiacol, catechol, DMP, o-danisidine, DAB,
2
L-DOPA, MgCl , MnSO
4 2
, CaCl , MnCl
2
, ZnSO
4
, CuSO , sodium azide, L-cysteine, EDTA, NADH
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(nicotinamide adenine dinucleotide reduced disodium salt) and coomassie brilliant blue R-
250 were obtained from Sisco Research Laboratories, India. DEAE-cellulose, 3,4-dimethoxy
benzyl alcohol (veratryl alcohol), syringic acid, catechol, N,N’-dimethyl phenylenediamine,
caffeic acid, Murashige and Skoog medium and clarigel were obtained from Hi-Media, India.
Protein markers were obtained from Bangalore Genei Pvt. Ltd. All chemicals used were of
the highest purity available and of analytical grade. Malachite Green used was from
Himedia, India and Methyl Orange was obtained from Merck Limited, Mumbai, India. Other
textile dyes and the effluent were obtained from Manpasand textile industry, Ichalkaranji,
India.
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2.2. Suspension cultures of B. malcolmii malcolmii Hook.
Cell suspension cultures of B. malcolmii were a kind gift from Prof. V. A. Bapat,
Department of Biotechnology, Shivaji University, Kolhapur, India. The suspension cultures
were maintained on Murahige and Skoog’s medium supplemented with 2,4-
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Dichlorophenoxyacetic acid (5 mg L ), glutamine (100 mg L ), sucrose (3%) and coconut
milk (20%). The pH of the medium was adjusted to 5.8±0.05 and was autoclaved at 15 psi
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and 120 C for 20 min. The cultures were maintained at 25±2 C with 16 h light and 8 h
darkness with continuous shaking at 100 rpm which facilitated a uniform dispersion of
cells. A 10 mL of the inoculum was further transferred into 250 mL Erlenmeyer flasks
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containing 40 mL of autoclaved medium. The cells were cultured regularly after every 10
days and were used for phytoremediation studies.
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2.3. Decolorization experiments with suspension cultures of B. malcolmii
To determine decolorization ability of the suspension culture of B. malcolmii, BBR at
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the concentration of 20 mg L was added into Erlenmeyer flasks containing 40 mL of the
medium. The dye containing medium was autoclaved and then inoculated with 10 mL of
inoculum of the suspension culture which was grown for 10 days. The flasks were exposed
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to continuous shaking at 100 rpm at 25 ± 2 C. Aliquots of the sample were withdrawn at
regular intervals. The samples were filtered through Whatmann filter paper. The clear
filtrate was used to determine the absorbance at the respective absorption maxima of the
dyes used. Following dyes namely BBR, Reactive Red 2, Direct Red 5B, Malachite Green and
Methyl Orange with absorbance maxima of 560, 530, 520, 620 and 540, respectively were
used in this study. Abiotic controls consisted of the dye containing medium without the
inocula and biotic controls comprised of the medium inoculated with the suspension
culture and devoid of the dye.
Decolorization percentage for the respective dyes in all the experiments was
calculated using the equation 1,
InitialꢀAbsorbance − Observedꢀabsorabance
Decolorizationꢀꢁ%ꢂ =ꢀ
Initialꢀabsorbance
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……………Equation (1)
Based on the screening experiments, BBR was selected as the model dye for all the
further experiments.
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For studying the effect of varying dye concentrations on decolorization performance
of suspension cultures of B. malcolmii, the cells were subjected to 20, 40, 80, 160 and 320
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mg L concentrations of BBR. Absorbances were noted after every 12 h. To study the effect
of increasing biomass on the removal of BBR, 5, 10, 15 and 20 ml of 10 day old inocula of
the suspension culture of B. malcolmii were added into different flasks containing 40 mg L-1
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of the dye BBR. The flasks were exposed to continuous shaking at 20 ± 2 C and the
absorbance was noted after every 6 h.
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2.4. Preparation of cell free extract
Cell suspension cultures of B. malcolmii before and after decolorization of BBR (40
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mg L ) were used for the preparation of cell free extracts. Cells were separated from the
medium by filtration through a Whatmann filter paper. The clear filtrates obtained were
used as sources of extracellular enzymes. The collected cells were suspended in ice cold 50
mM potassium phosphate buffer (pH 7.4), ground finely in a mortar and pestle and then
homogenized in a glass homogenizer with intermittent cooling and the extract was further
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centrifuged at 8000 rpm for 20 min at 4 C. The supernatant thus obtained after
centrifugation was used as a source of intracellular enzymes.
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2.5. Enzyme assays
Activities of lignin peroxidase, laccase, tyrosinase, veratryl alcohol oxidase, NADH-
DCIP reductase, azo reductase and riboflavin reductase were assayed
spectrophotometrically in cell free extract of test as well as in the control supernatant.
Lignin peroxidase activity was calculated by monitoring the formation of propanaldehyde
at 300 nm in a reaction mixture of 2.5 mL containing 100 mM n-propanol, 250 mM tartaric
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acid, 10 mM H
2
O
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containing 10% ABTS in 0.1M acetate buffer (pH 4.9), and increase in optical density was
measured at 420 nm.20 For tyrosinase activity, the final assay concentration in 3 ml
reaction mixture with 50 mM potassium phosphate (pH 7.4), 0.17 mM catechol, 0.070 mM
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L-ascorbic acid equilibrated to 30 C. The absorbance monitored at 265 nm. A 0.1 mL of the
enzyme sample was added and decrease in the absorbance was recorded for 1 min.21
Veratryl alcohol oxidase activity was determined using veratryl alcohol as a substrate. The
reaction mixture of volume 2 mL containing 4 mM veratryl alcohol, in 0.05 M citrate
phosphate buffer (pH 3.0), and 6.8 mg of enzyme was prepared and oxidation of the
substrate at room temperature was monitored by an absorbance increase at 310 nm due to
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the formation of veratraldehyde. NADH-DCIP reductase activity was measured in cell-free
extract. DCIP reduction was monitored at 620 nm, and calculated using an extinction
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coefficient 19 mM cm . The reaction mixture (5.0 mL) contained 50 mM substrate (DCIP)
in 50 mM potassium phosphate buffer (pH 7.4) and 0.1 mL enzyme. From this, 2.0 mL
reaction mixture was assayed at 620 nm by addition of freshly prepared 50 mM NADH.23
Azo reductase activity was measured in cell free extract by taking 2.0 mL reaction mixture
contained 25 mM of Methyl Red, 50 mM NADH, 1.2 mL of potassium phosphate buffer of 50
mM concentration (pH 7.4). Substrate Methyl Red reduction was observed at 430 nm at
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room temperature and determined using an extinction coefficient 23 mM cm . Riboflavin
reductase NAD(P)H:flavin oxidoreductase was measured by monitoring the decrease in
absorbance at 340 nm. Cell free extract was added to a solution (final volume 2 mL)
containing 100 mM of Tris-HCl (pH 7.4), 25 mM of NADPH and 10 mM of riboflavin.
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1.25 All enzymes assayed for test sample and biotic control sample were carried out in
triplicates.
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2.6. Phytotoxicity studies
Phytotoxicity studies with 1000 ppm concentration of BBR and its extracted
products for two common edible plants viz. Phaseolus mungo and Triticum aestivum seeds.
Ten seeds of each plant were independently taken in petridishes were watered with dye
and metabolite solutions (5 mL) for 8 d. A control set was also kept supplying distilled
water to it. Germination percentages and lengths of plumules and radicles were recorded
on 8 d.
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2.7. Mediator studies with cell suspension cultures
With a view of enhancing the decolorization potential of suspension cultures of B.
malcolmii for the dye BBR, different known redox mediators [ABTS (0.4 μM), 1-
hydroxybenzotriazole (HBT), acetosyringone (ACS), syringic acid (SA), vanillin (VAN),
hydroquinone (HQ), 2,6-dimethoxy phenol (DMP) and pyrogallol (PG) (2 mM)] were added
into the suspension culture medium in earlier mentioned section 2.2. The suspension
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culture medium was autoclaved with the dye BBR (40 mg L ) and sterilized solutions of
the different mediators were aseptically added into each of the respective flasks followed
by inoculating them with 10 day old suspension cultures and decolorization percentage
was calculated as mentioned above. The mediators were selected looking at chemical
structures and ability to undergo repeated oxidation-reduction cycles.
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2.8. Purification and characterization of laccase
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Ten day old cultures of B. malcolmii were used for purification studies. The cell
cultures were filtered with a Whatmann filter paper No. 1 under cold conditions (4 °C). The
filtrate was used as the crude source of extracellular laccase. The enzyme was dialyzed in a
dialysis bag against sodium phosphate buffer (pH 7.4) for 4 h at 4 °C to remove media
components. DEAE-anion exchange chromatography was further carried out using
automated Econo purification system (Bio-Rad). The dialyzed enzyme was directly applied
on the DEAE-cellulose anion exchange column (cylindrical glass column with 15 cm height
and 1 cm diameter) equilibrated with 0.2 M potassium phosphate buffer (pH 8.0) at a flow
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rate of 0.88 mL min . The retained proteins were eluted with a linear NaCl gradient former
(0 to 1 M).
Native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
was carried out using 5% stacking gel and 11% resolving gel using a Genei vertical
electrophoresis system (Bangalore Genei, Pvt. Ltd., India). Activity staining was carried out
using non-denaturing PAGE using L-DOPA as the substrate. Molecular mass of the purified
enzyme was determined by SDS-PAGE using high molecular mass-standards such as
phosphorylase b (98 kD), bovine serum albumin (66.0 kD), ovalbumin (43.0 kD), carbonic
anhydrase (29.0 kD), lactoglobulin (18.4 kD) and aprotinin (6.5 kD). Protein bands were
visualized using silver staining method.26
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2.9. Substrate specificity, kinetic constants and spectral character of the purified
enzyme
Substrate specificity of the purified enzyme was studied by using several phenolic
and nonphenolic compounds, such as 3,4-dimethoxy benzyl alcohol, guaiacol, 2,6-
dimethoxy phenol, L-DOPA, o-tolidine, hydroquinone, catechol, ABTS, caffeic acid,
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pyrogallol, p-cresol, syringic acid, o-dianisidine and syringaldazine. The reaction mixture
(2.0 mL) contained 1 mM substrate, 35 μM ABTS and 20 mM sodium acetate buffer. The
reaction was started by adding 0.2 mL of enzyme solution.
Kinetics of the purified enzyme was studied by observing the oxidation of increased
concentration of ABTS at optimum condition. Michaelis constant (K ) and maximum rates
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(Vmax) were determined by using ABTS in the range of concentrations 0.125-1.25 μM at pH
4.8 and 30°C. The reaction was followed in a spectrophotometer (Hitachi U-2800) and data
were plotted according to Lineweaver-Burk. Sixty μg of laccase in potassium phosphate
buffer (pH 8.0) was subjected to wavelength scan (200 to 800 nm) on UV-visible
spectrophotometer (Hitachi UV 2800).
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2.10. Effect of pH, temperature, metal salts and inhibitors on laccase activity
The effect of pH on laccase activity was determined by observing the oxidation of
ABTS within the pH range of 2.0 to 10.0 at room temperature (30 °C). Optimum
temperature for purified laccase was examined over the temperature range of 0-100°C
with ABTS as a substrate at optimal pH. Salt concentration was standardized before
application with varying concentrations (0.01-1.5 nM). The effect of different metal salts (1
4 2
mM; MgSO , CaCl , MnCl
2
, MnSO
4
, ZnSO
4
, CoCl
2
, and CuSO ) and inhibitors viz. sodium azide,
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EDTA and L-cysteine (5 mM) on the activity of purified enzyme was also studies. The
enzyme activity was determined by using ABTS as substrate. Heat inactivated enzyme was
used as a control. All the experiments were run in triplicates and average values were
calculated.
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2.11. Decolorization of the textile dyes and analyses of decolorization products using
purified laccase
Five structurally different dyes viz. BBR, Malachite Green, Reactive Red 2, Direct Red
5B and Methyl Orange were studied for their decolorization by using purified laccase. The
reaction mixture for the degradation of textile dyes using purified laccase contained the
-1
respective dyes (40 mg L ), 0.2 M sodium acetate buffer (pH 4.8) and 0.5 mL enzyme and
35 μM ABTS. The reaction mixture was incubated at 30 °C at static as well as shaking
conditions at 100 rpm.
Decolorization of all the individual dyes in culture was monitored qualitatively using
UV-visible spectrophotometer (Hitachi U-2800; Hitachi, Tokyo, Japan) at the respective
wavelengths of maximum absorption of dyes used as mentioned earlier in section 2.3,
while HPLC was used for the confirmation and analysis of phytotransformation. GC-MS
analysis was carried out for the identification of metabolites produced. The cells were
separated from the liquid medium after 48 h of their exposure to BBR by filtration through
a Whatmann filter paper No. 1 and the clear filtrate was used for the extraction of the
products formed after the degradation of BBR. In case of studies with the purified enzyme,
the reaction mixture after complete decolorization was used for the extraction of the
products formed owing to the action of the enzyme.
The dye and its products were extracted using equal volume of ethyl acetate and the
extract was then evaporated over anhydrous Na
2
SO
in vacuum and later dried.14 HPLC
4
analysis was carried out (Waters model no. 2690; Waters Corp., Milford, MA) on C18
-1
column (symmetry, 4.6 mm x 250 mm) using methanol with flow rate of 1 mL min for 10
min and UV detector at 254 nm.11 Gas Chromatography Mass Spectroscopy (GC-MS)
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analysis of the metabolites was carried out using a Shimadzu 2010 MS Engine, equipped
with integrated gas chromatograph with an HP1 column (60 m long, 0.25 mm i.d.,
-1
nonpolar). Helium was used as carrier gas at a flow rate of 1 mL min . The injector
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temperature was maintained at 280 C with oven conditions as: 80 C kept constant for 2
o
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o
-1
min-increased up to 200 C with 10 C min , raised up to 280 C with 20 C min rate. The
compounds were identified on the basis of mass spectra and using the National Institute of
Standards and Technology (NIST) library.11
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2.12. Statistical analysis
Data were analyzed by one-way analysis of variance (ANOVA) with Tukey–Kramer
multiple comparisons test. Readings were considered significant when P<0.05.
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3. Results and discussion
3.1. Decolorization of the dye Brilliant Blue R by suspension cultures of B. malcolmii
The cell cultures were found to be efficient in decolorizing a wide variety of dyes
belonging to structurally diverse groups but the maximum decolorization was observed in
case of the dye BBR (98% within 24 h and more than 90% within 12 h). BBR was therefore
selected as the model dye for further studies. UV-visible spectrophotometric analysis (400-
800 nm) of the untreated dye showed a distinct peak at 600 nm, which was completely lost
after treatment of the dye with the cell culture indicating the decolorization of BBR (Fig.
1a). B. malcolmii suspension cells have earlier been reported to degrade toxic paint
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preservatives called Troysan S-89. Nopalea cochenillifera cell cultures have also shown
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degradation of a textile dye Red HE7B at a concentration of 1000 ppm within 7 d. The
efficacy B. malcolmii cell to decolorize most of the dyes taken in this study was found to be
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similar to or even more efficient than many microbial systems. These findings with the use
of suspension cell cultures challenges the previous observations on phytoremediation of
dyes which reported a sluggish removal rate of pollutants.
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While studying the effect of various dye concentrations starting from 20 mg L to
320 mg L-1 on the decolorization of BBR mediated by cell cultures, the percentage
decolorization was found to decrease and the highest percentage decolorization values
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from 20 to 80 mg L quite insignificantly reduced the percentage decolorization values
while the dye concentrations of 160 and 320 mg L-1 showed a drastic reduction in
decolorization percentages (Fig. 1b). A drastic reduction in decolorization at 160 and 320
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mg L could be attributed to the toxicity of BBR at these concentrations. SEM images of the
cells subjected to increasing dye concentrations showed no apparent damage to cells (Fig.
1c). Since almost similar percentage decolorization values were obtained for the dye
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concentrations of 20 and 40 mg L , the concentrations selected for further studies was 40
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mg L . Inability to tolerate higher concentrations of pollutants is said to be one of the
major disadvantages of phytoremediation technologies and has limited their applications.28
However, B. malcolmii cell cultures have shown the capacity to tolerate and decolorize
higher dye concentrations. In case of decolorization of Malachite Green by Kocuria rosea,
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only 13 and 6% decolorization was observed at 70 and 100 mg L dye concentrations,
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respectively. While, B. malcolmii cell cultures showed 52 and 42% decolorization even at
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160 and 320 mg L concentrations which proved them to be more efficient than many
microbial dye degrading systems.
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3.2. Enzymatic analysis of degradation by B. malcolmii cell cultures
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B. malcolmii cell cultures have shown to be metabolically highly active tissues with a
rich source of degradative enzymes. The cell suspension cultures showed significant
inductions in intracellular and extracellular activities of laccase by 607 and 28%,
respectively during decolorization of BBR. Inductions in the intracellular activities of DCIP
reductase (8%) and veratryl alcohol oxidase (167%) were also observed during
decolorization. Interestingly, azoreductase seems to be repressed after the degradation of
dye which may have occurred due to its toxicity. Similarly, tyrosinase was also found to be
repressed. A noteworthy induction in the laccase activity recommended its importance in
the dye biodegradation process (Table 1). This enzyme was therefore targeted for further
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studies. B. malcolmii root tissues showed the absence of laccase while treating Direct Red
5B. The cell suspensions however revealed a clear activity of laccase during BBR
degradation in this report. These observations ultimately lead to purification of the enzyme
from B. malcolmii suspension cultures.
Marigold hairy roots have shown complete absence of laccase activity in control
tissues that were unexposed to the dye Reactive Red 198 while activity of the enzyme was
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detected in root tissues that were exposed to the dye. Similar observation were also made
with studies on hairy root cultures of B. juncea. Methyl Orange exposed hairy roots
revealed an enhanced activity of laccase, however, it was completely absent in the dye
unexposed roots.36 In this study, activity of laccase was found to be induced during the
decolorization of BBR which helped to speculate that laccase could be the principal enzyme
from B. malcolmii suspension cultures that imparts it to possess dye degrading properties.
Thus, to determine the exact role of laccase in decolorization of textile dyes, further
attempts were made to purify the enzyme.
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3.3. Phytotoxicity studies
1000 ppm concentration of BBR was found to cause total (100%) inhibition of seed
germination of Phaseolus mungo while 60% germination in case of Triticum aesitivum.
Germination percentage was found to be slightly higher in case of T. aestivum seeds treated
with products than those treated with the parent dye. In case of P. mungo, no difference in
germination percentage was observed in distilled water and dye metabolites. Lengths of
plumules and radicles of both T. aestivum and P. mungo were observed to be superior in the
solutions of dye metabolites than those germinated in BBR. These studies showed that the
products of BBR after treatment with B. malcolmii cell cultures were with reduced toxicity
as compared to BBR and thus could be proposed to be effective to render textile waste
waters safer for the environment (Table 2).
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3.4. Effect of redox mediators on the decolorization of BBR
Out of eight different mediators used, ABTS was observed to be the most efficient
mediators which led to complete decolorization of the dye within 30 min whereas control
set (those without any mediator) took 24 h. HBT was found to mediate almost 85%
decolorization within 30 min while in absence it required more than 12 h. DMP was found
to give 95.21% decolorization at the end of 30 min. However, it was surprising to find
lesser decolorization in subsequent hours. This might have taken place due to formation of
some other color intermediates which showed the absorbance at same wavelengths. In
case of other mediators tested viz. VAN, ACS, PG, SA and HQ, the absorbance measured at
24 h showed no significant loss of color (Fig. 2). The redox mediators showed varying
performances owing to their redox potentials. This work is the first report where redox
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mediators have been used to enhance the decolorization of dyes mediated by plant
suspension cultures thereby facilitating such a rapid dye removal.
Redox mediators speed up the reaction rate by shuttling electrons from biological
oxidation of primary electron donors or from bulk electron donors to the electron-
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accepting organic compounds. A mediator goes through many oxidation reduction cycles.
Oxidized form of the mediator further gets reduced because of the oxidation of dye
substrates. Lesser decolorization of BBR (as compared to controls) in presence of certain
mediators such as VA, ACS, PG, SA and HQ could have taken place because of the inhibition
of laccase caused by radicals generated in the process (Fig. 2). Prediction of prospective of
a redox mediator is difficult as there are a number of factors such as redox potential
between laccase and the mediator, and type and position of substituent in the mediator.
Properties of the oxidized form of mediator such as stability, inactivation and substrate
affinity also affect the redox process.31-33 The negative effect of HBT on laccase has been
studied where it has been known to attack aromatic amino acids such as tyrosine and
tryptophan.33 Inactivation of laccase by HBT during degradation of an indigo dye depended
on the presence of substrates oxidizable by HBT radical, since this factor avoids the enzyme
inactivation reaction.34 In our experiments, dyes were the oxidizable substrates by this
mediator and have a positive influence on the stability of laccase. Despite great promise,
slow removal rate of pollutants has limited the application of phytoremediation
technologies. Such enhanced decolorization by using mediators and finding natural and
less toxic mediators could help us take a step forward in overcoming the hurdles in the
application of phytoremediation technologies.
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3.5. Purification of laccase from B. malcolmii cell suspension cultures
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The DEAE-cellulose anion exchange chromatography elution profile of proteins is
shown in Fig. 3a. The enzyme was eluted with 0.4 M NaCl concentration from DEAE
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cellulose anion exchange column. The procedure yielded 0.72 mg mL of pure protein. The
recovery of laccase activity was 62.4% with a purification fold of 7.8 (Table 3).
UV-Visible spectrum of purified B. malcolmii laccase showed peaks at 280 nm and
360 nm whereas no absorbance was detected at 610 nm (Fig. 3b). Similar observation was
also made by Madhavi and Lele (2009).37 Laccase contains four copper atoms that have
been classified according to their electron paramagnetic resonance (EPR) features. They
are classified as Type 1 or blue, Type 2 or normal and Type 3 or coupled binuclear copper
site. Type 1 enzyme is associated with an intense optical absorption band near to 610 nm.
In addition, Type 3 non paramagnetic is supposed to be associated with a cooperative two-
electron acceptor, which show a strong optical absorption at about 340 nm in the oxidized
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state. These observations helped to infer that the enzyme lacks Type 1 copper which is
found to be present in blue laccases. The absorbance shown by the enzyme at 360 nm could
be a characteristic of Type 3 copper (Fig. 3b).
The purified laccase appeared as a single protein band on SDS-PAGE (Fig. 4). The
molecular weight of purified laccase was observed to be 40 kD. The molecular weight of
purified B. malcolmii laccase was found to be in correspondence with the molecular weight
of plant laccases. Fungal laccases have been reported to have a molecular weight between
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40-80 kD. The protein size of PPOs depends on plant species and varies from 39 kD in
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snapdragon to 73 kD in spinach. Laccases from spent Lentinus polychrous Lev. Mushroom
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compost was found to have the molecular weight of 32 kD. Botrytis cinerea laccase was
previously reported to be as small as 38 and 36 kD.42
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3.6. The effect of pH, temperature, metal ions and inhibitors on laccase activity
The purified laccase exhibited the oxidation of ABTS within a wide range of pH at 30
oC. The highest activity of enzyme was demonstrated at pH 3.0 (Fig. 5a). The activities of
enzyme at pH 4.0, 5.0 and 6.0 did not show any drastic changes and the values of activities
plotted against pH showed almost a plateau on the graph within this range. The pH optima
of laccase from Rhus vernicifera for ABTS as the substrate, has been found to be close to
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4.0. Laccase from fungus Ceriporiopsis subvermispora also showed an optimum pH value
of 3.0 when ABTS was used as a substrate.44
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Although the optimum temperature of purified enzyme was found to be 30 C, the
enzyme could remain active within a broad temperature range (Fig. 5b). Extracellular
laccase from Pseudomonas sp. LBC showed optimum activity at 40 C temperature.12
Characterization of laccase from tuberous roots of Amorphophallus campanulatus showed
that the enzyme underwent rapid inactivation when pre-incubated for 5 min at
temperatures greater than 40 C.45 B. malcolmii laccase thus was found to oxidize ABTS
even when the reaction mixture was pre incubated for 10 min at high temperatures.
When the effect of different metal ions on laccase activity was studied, it was found
o
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that ZnSO
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led to marginal increase in enzyme activity. The effect of metal ions on activities
of laccase from a white rot fungus Ganoderma lucidum also showed that at low
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concentrations, ZnSO
also enhanced by about 8% in the presence of CuSO
site of laccases. Previous reports also confirm the enhancement of laccase activity in the
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was found to enhance the activity of laccase. Activity of laccase was
4
. Since, copper is a component of active
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presence of CuSO . This might have happened because of the filling of Type 2 copper
binding sites with copper ions.44 The highest inhibition of laccase was observed in the
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presence MnSO
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which about 13%. Other metal salts viz. MgSO
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were similar to those obtained with Ganoderma lucidum where Mn was again found to
show maximum inhibition of laccase activity.44
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3.7. Substrate specificity and kinetics of purified laccase
Besides ABTS, the purified enzyme was found to oxidize a wide range of phenolic
and non-phenolic substrates including guaicol, o-tolidine, pyrogallol, syringaldazine, L-
DOPA, o-danisidine and 2,6-dimethoxyphenol in presence of ABTS as the redox mediator
(Table S1). The Km for B. malcolmii laccase was found to be 20 μM while Vmax was found to
be 5.04 moles L-1 (Fig. 6). Trametes hirusita laccase has been reported to have the Km of 41
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μM for ABTS. Generally, lower Km values have been observed for syringaldazine as the
substrate while comparatively higher Km values were observed for ABTS. A low Km value
for B. malcolmii laccase indicated that the enzyme possessed a very high affinity for ABTS
as the substrate.
B. malcolmii laccase showed the ability to oxidize polyphenols, methoxy substituted
phenols, diamines etc. in the presence of ABTS. The enzyme seemed to show higher activity
values for methyl and methoxy substituted substrates and diamines. Introduction of OH,
OCH3 or CH3 groups into aromatic system renders the compound easily oxidizable by
laccase. Methoxy groups can donate an electron easily to introduce one-electron
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oxidation. The highest activity was observed for o-tolidine which has methyl and amino
groups as substituents. DMP which has methoxy substituent and o-danisidine which has
additional amino substituent were also significantly oxidized. Guaicol and syringaldazine
were oxidized considerably though syringic acid and veratryl alcohol were not oxidized
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despite of having methoxy substituents and pyrogallol. A polyphenol was also oxidized
despite the absence of methyl, methoxy or amino substituent. The enzyme however failed
to oxidize substrates which were diphenols. The ability to oxidize syringaldazine is a
distinctive characteristic of laccase (Table S1). A lower redox potential of substrates or
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higher redox potential of laccase results into higher oxidation rates of substrates. Redox
mediators allow laccases to oxidize non-phenolic compounds, thereby vastly expanding the
range of substrates that can be oxidized by this enzyme.47 As the catalyzed reactions
depend on the difference of redox potential between laccase and substrate.33, 47 The organic
compound best fitting the term “redox mediator” was found to be ABTS as evident with the
outcome of this work.
When different known inhibitors at 5 mM concentrations were used to detect their
effect on laccase activity, no inhibition was observed in the presence of EDTA whereas,
complete inhibition was observed in the presence of L-cysteine and sodium azide.
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3.8. Decolorization of dyes by purified laccase
Purified laccase from suspension cultures of B. malcolmii showed the ability to
decolorize a variety of different dyes like BBR, Malachite Green, Reactive Red 2, Methyl
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Orange and Direct Red 5B at 40 mg L concentrations. The model dye BBR was found to be
completely removed by purified laccase within 12 h. This performance was tuned upon
addition of ABTS as the redox mediator. Addition of ABTS achieved a decolorization of 96%
just within 5 min at static conditions. Other dyes namely Reactive Red 2 and Direct Red 5B
were also decolorized up to 95 and 77% within 30 min of addition of the enzyme. While
Malachite Green and Methyl Orange were decolorized up to 80 and 49%, respectively
within 2 h in presence of ABTS. UV-Visible spectra of all the dyes screened showed a
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decrease in the absorbances measured at the characteristic wavelength maxima of dyes
(Table S2). Fungal laccases have been known to be responsible for the degradation of
recalcitrant compounds like phenols while plant laccases are known to be involved in
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synthetic processes such as lignin formation. There are only a few reports where purified
plant laccases have been used for the degradation of dyes. Decolorization studies with cell
suspension cultures of B. malcolmii have shown the prominent role of laccase in the
decolorization of the dye BBR. Moreover, ABTS was found to be the best mediator that gave
the most rapid degradation of the dye. These results led to the use of ABTS as the mediator
for the degradation of the different textile dyes using purified B. malcolmii laccase. The
decolorization of Remazol Brilliant Blue R by a commercial laccase formulation with a
nonionic surfactant as the redox mediator was also reported.30
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HPLC analysis was performed in order to confirm the degradation of the BBR. HPLC
profile of the untreated dye showed peaks at 1.915, 2.236 and 3.106 min while, the
products formed with whole cell cultures of B. malcolmii showed peaks at 2.899, 3.183,
3.517, 3.676, 5.824, 9.698 min and the products formed after 5 min of degradation of the
dye by purified B. malcolmii laccase showed peaks at 2.662, 2.858, 3.359, 5.505 and 9.285
min. Differences in the HPLC profiles of BBR and the metabolites formed confirm the dye
degradation. HPLC profile of the control sample (extracts of the buffer, ABTS and the
enzyme) and that of the metabolites formed after the degradation of BBR by purified
laccase showed just one peak in common (9.285 min) which indicated that all the other
peaks in the HPLC profile of the metabolites were a consequence of degradation of the dye
by B. malcolmii laccase (Fig. 7).
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To detect the products of metabolism of BBR with whole cell cultures of B.
malcolmii, GC-MS of control samples (extracts of the medium inoculated with B. malcolmii
suspension cultures and devoid of the dye) and test samples (extracts of the products
formed due to the metabolism of the dye) was done. A number of peaks were detected
which were common in test and the control samples while there was only one peak which
was eluted at the retention time of 22.598 min which was not present in the control
sample. The GC-MS analysis of the products formed with the purified enzyme also showed
a peak at the retention time of 22.598 min along with an additional peak at 18.684 min.
Moreover, the mass spectrum of products formed both in case of degradation of dye with
purified enzyme and with whole cell culture, eluted at the retention time of 22.598 min was
found to be exactly similar. The pathway predicted for degradation of BBR by whole cell
cultures involved a asymmetric cleavage of BBR followed by demethylation with laccase to
give N-ethyl-4-4-{(Z)-[(4Z)-4-(methylimino)cyclohexa-2,5-dien-1-ylidene]methyl}aniline;
(MW = 238) (Fig. 8). Strikingly similar fate of metabolism seems to be carried out by
purified laccase where only difference was the detection of another compound [3-
methylbenzenesulfonic acid (MW =172)] in the GC-MS analysis. This compound was
thought to be formed after the first step where the enzyme is suspected to bring about the
asymmetric cleavage of BBR. No other products were detected with whole cell cultures
which could be attributed to a complete mineralization of the products with the help of the
other enzymes present in the suspension cultures of B. malcolmii. The GC-MS analysis
revealed a remarkable similarity in the nature of products formed due to the decolorization
of BBR by crude enzyme source and that of purified laccase both of which show a peak at
22.598 min (Fig. 8). The present study clearly helped to determine the role of laccase in
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degradation of BBR and gave confirmatory evidences to elucidate the mechanism of action
of laccase. This also reveled that the enzyme catalyzes exactly the same reaction when
present in whole cell and when present in its purified form. Laccase thus seems to
dominate the degradation process even when there are a couple of other enzymes that can
compete with laccase for degradation of BBR.
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4. Conclusions
The present study explored the potential of B. malcolmii suspension cultures to
decolorize structurally different textile dyes along with unraveling the detailed mechanistic
basis underlying degradation of BBR dye by whole cell cultures and purified laccase. The
studies also demonstrated an interesting approach to enhance degradation process with
the use of mediators and the ABTS-laccase mediator system was found to prove to be
highly efficient. The purified enzyme also showed versatility in degrading structurally
dissimilar dyes and many other laccase substrates. A schematic pathway of degradation by
purified laccase was deciphered to understand the fate of metabolism of dye. Novel insights
towards understanding the phytoremediation mechanisms behind removal of textile dyes
with purified laccase lays the foundation for future advances. Enhancement and application
of these technologies will help in broadening the horizons of phytoremediation research.
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Acknowledgements
Authors are thankful to Prof. V.A. Bapat, Department of Biotechnology, Shivaji
University, Kolhapur for the kind gift of B. malcolmii cell culture. Thanks are also due to the
Department of Biotechnology, Government of India, New Delhi for funding under
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Interdisciplinary Program for Life Science to Shivaji University, Kolhapur, India (Grant No.
BT/PR4572/INF/22/147/2012).
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5 V. Adki, J. Jadhav and A. Bapat, Int. J. Phytorem., 2012, 14, 554–569.
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Figure captions
Fig. 1. a). UV-Visible spectra of Brilliant Blue R before and after decolorization, b) Effect of
increasing concentrations of BBR on the decolorization performance of B. malcolmii cell
cultures and c) SEM images of B. malcolmii cell cultures unexposed to and those subjected
to increasing concentrations of BBR.
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Fig. 2. The effect of different mediators on the decolorization of Brilliant Blue R measured
at 0 h ( ) and after 0.5 ( ), 4 ( ), 8 ( ) and 12 ( ) hours of decolorization.
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Fig. 3. a) DEAE cellulose anion exchange elution profile of purified enzyme showing
absorbance (■) at 280 nm and enzyme activity in U (▲) which was assayed in each fraction
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with ABTS as substrate. The samples (200 μl) from each fraction were added in reaction
mixture containing 1.7 mL acetate buffer (pH 4.8). The formation of oxidized product was
measured at 420 nm and b) UV-Visible spectrum of purified B. malcolmii laccase
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Fig. 4. SDS-PAGE of proteins obtained after purification of enzyme. The lanes A and B
represent the activity staining bands of crude and purified laccase, respectively on PAGE.
The lanes C and D represent the protein staining bands of crude and purified laccase,
respectively and the lane E represents molecular weight markers on SDS PAGE.
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Fig. 5 a) Optimum pH for purified B. malcolmii laccase, b) Optimum temperature for
purified B. malcolmii laccase and c) The effect of metal salts on the activity of purified B.
malcolmii laccase
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Fig. 6. Km of purified laccase from B. malcolmii.
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Fig. 7. HPLC profile of a) Brilliant Blue R, b) products formed after the degradation of
Brilliant Blue R by whole cell cultures of B. malcolmii malcolmii, c) the control sample after
12 h of inoculation of cells in the medium devoid of the dye, d) Products formed after the
degradation of BBR by purified B. malcolmii laccase and e) control sample containing the
buffer, ABTS and the enzyme.
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Fig. 8. Proposed pathway for the degradation of the dye BBR by whole cell cultures and
purified laccase from B. malcolmii on the basis of GC-MS data showing the detected
metabolites.
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