Biochemical characterization and potential for textile dye degradation of blue laccase from Aspergillus ochraceus NCIM-1146

Biotechnology and Bioprocess Engineering 15: 696-703 (2010)

DOI 10.1007/s12257-009-3126-9

Biochemical Characterization and Potential for Textile Dye

Degradation of Blue Laccase from Aspergillus ochraceus NCIM-1146

Amar A. Telke, Avinash A. Kadam, Sujit S. Jagtap, Jyoti P. Jadhav, and Sanjay P. Govindwar

Received: 23 November 2009 / Revised: 12 December 2009 / Accepted: 4 January 2010

© The Korean Society for Biotechnology and Bioengineering and Springer 2010

Abstract In our study, we produced intracellular blue

laccase by growing the filamentous fungus Aspergillus

ochraceus NCIM-1146 in potato dextrose broth. The

enzyme was then purified 22-fold to a specific activity

of 4.81 U/mg using anion-exchange and size exclusion

chromatography. The molecular weight of purified laccase

was estimated as 68 kDa using sodium dodecyl sulfate

polyacrylamide gel electrophoresis. The enzyme showed

maximum substrate specificity toward 2,2'-Azinobis, 3-

ethylbenzothiazoline-6-sulfonic acid than any other sub-

strate. The optimum pH and temperature for laccase activity

were 4.0 and 60ºC, respectively. The purified enzyme was

stable up to 50ºC, and high laccase activity was maintained

at pH 5.0 ~ 7.0. Laccase activity was strongly inhibited by

sodium azide, EDTA, dithiothreitol, and L-cysteine. Puri-

fied laccase decolorized various textile dyes within 4 h in

the absence of redox mediators. HPLC and FTIR analysis

confirmed degradation of methyl orange. The metabolite

formed after decolorization of methyl orange was charac-

terized as p-N,N'-dimethylamine phenyldiazine using GC-

MS.

Keywords: Aspergillus ochraceus NCIM-1146, laccase,

textile dyes, decolorization, redox mediators

1. Introduction

The decolorization of textile dyes can be performed with

various microorganisms, including fungi, actinomycetes,

algae, and bacteria. Fungi and bacteria especially are wide-

ly used for the biodegradation of textile dyes. Biodegrada-

tion ability is associated with the production of oxido-

reductive enzymes such as lignin peroxidase and laccase

[1]. Most of the research concerning bioremediation is

centered on a single fungal species, Phanerochaete chryso-

sporium, which is known to metabolize a wide range of

xenobiotic compounds [2]. Ascomycetes such as Asper-

gillus niger, Aspergillus fumigatus, Aspergillus oryzae, and

Aspergillus olliaceus strain 212C are capable of decolori-

zing a wide range of structurally different dyes and are

more effective than Phanerochaete chrysosporium [3-6].

Our earlier reports showed that the decolorization of textile

dyes using Aspergillus ochraceus NCIM-1146 is associated

with laccase [7].

Laccases are the most abundant members of the multi-

copper protein family, which also includes tyrosinases,

monoxygenases, and dioxygenases [8]. Phylogenetically,

these enzymes have developed from small sized prokar-

yotic azurins to eukaryotic plasma proteins, such as cerulo-

plasmin [9]. Laccases contain four histidine-rich copper-

binding domains, which coordinate copper atoms types 1-

3 that differ in their environment and spectroscopic

properties [10]. Laccases are categorized as blue laccase

(laccase containing four copper sites) and laccase lacking

type-1 copper site [11]. Laccases are the model enzymes

for multi-copper oxidases and participate in the cross-

linking of monomers, degradation of polymers and ring

cleavage of aromatic compounds [12]. Further, they possess

great biotechnological potential because of their wide

reaction capabilities and broad substrate specificities.

Promising applications for laccases include biosensors for

drug analysis and phenols in tea, polymer synthesis, textile-

dye bleaching, bioremediation, and pulp bleaching [13-16].

The majority of previous studies have focused on laccase-

Amar A. Telke, Avinash A. Kadam, Sujit S. Jagtap, Jyoti P. Jadhav, SanjayP. Govindwar*Department of Biochemistry, Shivaji University, Kolhapur 416004, IndiaTel: +91-231-2609152; Fax: +91-231-2691533E-mail: [email protected]

RESEARCH PAPER

Biochemical Characterization and Potential for Textile Dye Degradation of Blue Laccase from Aspergillus ochraceus NCIM-1146 697

producing Trametes versicolor, Pleurotus ostreatus, and

Phanerochaete chrysosporium [17]. However, there is only

limited research on intracellular laccase produced by Asper-

gillus species such as Aspergillus nidulans [18,19]. In the

present work, we report the biochemical properties of blue

laccase from Aspergillus ochraceus NCIM-1146 as well as

its mechanism of textile dye degradation.

2. Materials and Methods

2.1. Dyestuff and chemicals

2,2'-Azinobis, 3-ethylbenzothiazoline-6-sulfonic acid (ABTS),

o-tolidine, hydroquinone, pyrogallol, guaiacol, L-DOPA,

and commassie brilliant blue R-250 were obtained from

SRL Chemicals, India. Veratryl alcohol, p-cresol, o-dianisi-

dine, L-tyrosine, sodium azide, L-cysteine, EDTA, dithi-

othreitol, Methyl orange, peptone, yeast extract and agar

powder were obtained from Hi-Media laboratory, India.

Protein markers were obtained from Bangalore Genei,

India. Textile dyes were obtained from local industry at

Ichalkaranji, India. All chemicals were of the highest purity

and of analytical grade.

2.2. Microorganism and culture conditions

A. ochraceus NCIM-1146 was obtained from the National

Center for Industrial Microorganisms, Pune, India. The

stock culture was maintained on potato-dextrose agar slants

at 4ºC.

For the enzyme production, two fungal discs (8 mm

diameter) of 4-day-old culture were inoculated into 250

mL Erlenmeyer flasks containing 100 mL of PDB (potato-

dextrose broth) medium with 200 g/L of peeled potatoes

and 5.0 g/L of yeast extract, followed by incubation for 96

h at 30ºC with shaking.

2.3. Preparation of crude enzyme

The fungal mycelium was collected by filtering the 96 h

growth culture of A. ochraceus NCIM-1146. The mycelium

was suspended in 50 mM potassium phosphate buffer (pH

7.0) and homogenized using a mortar and pestle and later

a homogenizer. The homogenized sample was centrifuged

at 9,000 rpm for 15 min under cold conditions. The super-

natant obtained after centrifugation was used as a source of

crude enzyme.

2.4. Enzyme activity

Laccase activity was measured in the reaction mixture

(2.0 mL) containing 100 µM ABTS and 20 mM sodium

acetate buffer (pH 4.0). The reaction was started by the

addition of 0.2 mL of enzyme solution [20]. The substrate

oxidation was monitored at 420 nm (ε420 = 0.036/µM/cm).

One unit of enzyme activity was defined as the amount of

enzyme required to produce 1 µM of oxidized product per

min. The protein concentration of the crude sample was

determined by the Lowry method using bovine serum

albumin as a standard protein [21]. The protein concent-

ration was monitored based on the absorbance at 280 nm

after anion-exchange and size exclusion chromatography.

2.5. Purification of laccase

DEAE-anion exchange chromatography was carried out

using an automated Econo purification system (Bio-rad).

The crude enzyme was directly applied to a DEAE-cellu-

lose anion exchange column (cylindrical glass column

15 cm in height and 1 cm in diameter) equilibrated with

20 mM potassium phosphate buffer (pH 8.0) at a flow rate

of 1 mL/min. The retained proteins were eluted with a

linear NaCl gradient former (0 ~ 0.4 M). Size exclusion

chromatography was carried out using a cylindrical glass

column packed with Biogel P100 (50 cm height and 1 cm

diameter). The column was equilibrated with 20 mM pota-

ssium phosphate buffer (pH 7.0).

2.6. Gel electrophoresis, spectrum, and copper content

Native and sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE) were carried out on 5%

stacking and 11% resolving gels using a Genei vertical

electrophoresis system (Bangalore Genei, Pvt. Ltd., India).

Protein bands were stained with commassie brilliant blue

R-250. The molecular mass of purified laccase was deter-

mined by calculating the relative mobility of standard pro-

tein markers, such as phosphorylase b (97.40 kDa), bovine

serum albumin (66.0 kDa), ovalbumin (43.0 kDa), carbonic

anhydrase (29.0 kDa), soybean trypsin inhibitor (20.10

kDa), and lysozyme (14.30 kDa). Activity staining was

carried out by incubating the gel after native PAGE at room

temperature in 20 mM sodium acetate buffer (pH 6.5) with

1 mM L-DOPA [22].

Purified laccase (10 µM) in 20 mM sodium acetate buffer

(pH 4.0) was subjected for wavelength scan (200 ~ 800

nm) by a UV-visible spectrophotometer (U-2800, Hitachi,

Japan). The copper content was determined using an atomic

absorption spectrophotometer (Perkin Elmer, model no.

4100).

2.7. Effect of pH and temperature on laccase activity

and stability

The enzyme obtained after size exclusion chromatography

was used for the biochemical characterizations. The effect

of pH on laccase activity was determined within a pH

range of 2.5 ~ 8.5 using ABTS as a substrate at room

temperature (30ºC). The optimum temperature for purified

laccase was examined over the temperature range of 20 ~

698 Biotechnology and Bioprocess Engineering 15: 696-703 (2010)

80°C with ABTS as a substrate and at optimal pH.

The effect of temperature and pH on laccase stability

was studied by incubating 3.61 U of laccase at various

temperatures between 20 and 60ºC at pH 7.0 or at various

pHs between 3.0 and 9.0 at 25ºC. Sodium citrate and

sodium acetate buffers were used for maintaining the pH

between 3.0 and 6.0, potassium phosphate buffer for main-

taining the pH between 7.0 and 8.0, and sodium carbonate-

sodium bicarbonate buffer for pH 9.0 ~ 10.0. Aliquots

were transferred after specific intervals into a cuvette con-

taining 100 µM ABTS and 20 mM sodium acetate buffer

(pH 4.0) in order to determine residual laccase activity.

2.8. Substrate specificity and kinetics of laccase

Substrate specificity was studied using nonphenolic and

phenolic compounds, such as veratryl alcohol, o-tolidine,

ABTS, pyrogallol, guaiacol, hydroquinone, tyrosine, p-

cresol, o-dianisidine, and L-DOPA, as substrates. The rate

of substrate oxidation was determined by measuring the

absorbance increase with the molar extinction coefficient

(εm) obtained from the literature [11-23]. One unit of

enzyme activity was defined as the amount of enzyme

required to increase 1.0 ABS unit/min.

2.9. Effect of metal salts, salinity, and inhibitor on laccase

activity

We studied the effect of different metal salts (1 mM; MgCl2,

CaCl2, MnSO4, ZnSO4, HgCl2, FeSO4, and CuSO4), salinity

(1 to 500 mM) and inhibitors (1 and 5 mM; EDTA,

dithiothreitol, L-cysteine, and sodium azide) on laccase

activity. The enzyme (3.05 U) was incubated at various

concentrations of metal salts, NaCl and inhibitor for

15 min. Laccase activity was determined using ABTS as a

substrate.

2.10. Decolorization of textile dyes using pure laccase

The reaction mixture (5 mL) for the decolorization of

textile dyes contained 50 mg/L of textile dyes, 20 mM

sodium acetate buffer (pH 4.0), and 10 µM of pure enzyme

solution. The reaction mixture was incubated at 40ºC with

shaking. Heat inactivated enzyme was used in the control

experiment. All experiments were run in triplicate and the

average value was calculated.

2.11. Extraction and analysis of metabolites formed

after decolorization

The decolorization was monitored at λmax of respective dye

using a Hitachi U-2800 spectrophotometer. In order to

elucidate the mechanism of dye decolorization, we analy-

zed the degradation metabolites of pure azo dye Methyl

orange using HPLC, FTIR, and GC-MS. The metabolites

formed after decolorization of Methyl orange were extract-

ed three times with 10 mL of ethyl acetate with vigorous

shaking. The combined organic phase was filtered over

Na2SO4 on filter paper and concentrated in a rotary vacuum

evaporator. HPLC analysis was carried out (Waters model

no. 2690) on a C18 column (symmetry, 4.6 × 250 mm)

using the isocratic method with a 10 min run time. The

mobile phase used was methanol at a flow rate of 0.75 mL/

min with a UV detector at 280 nm. FTIR analysis was

performed in the mid IR region of 400 ~ 4,000/cm with

16 scan speed. The pellets were prepared using spectro-

scopically pure KBr (5:95) and fixed in a sample holder.

GC-MS analysis was carried out using a QP 5000 mass

spectrophotometer (Shimadzu model no. U-2800). The

ionization voltage was 70 eV. Gas chromatography was

conducted in temperature programming mode with a Re-

steck column (0.25 mm × 30 mm; XTI-5). The initial column

temperature was 40ºC for 4 min, which was then increased

linearly at 10ºC per min up to 270ºC and held for 4 min.

The temperature of the injection port was 275ºC. GC-MS

interface was maintained at 300ºC. The helium was used as

carrier gas at a flow rate of 1 mL/min with a 30 min run

time. The compounds were identified on the basis of mass

spectra using the NIST library of GC-MS (version 1.10

beta Shimadzu).

3. Results and Discussion

3.1. Purification of laccase

The majority of fungi produce both intracellular and extra-

cellular laccases. The localization of laccases seems to be

associated with the physiological functions of the enzymes

Table 1. Summary of purification of laccase from A. ochraceous NCIM-1146

Purification stepsTotal activity

(U)Total protein

(mg)Specific activity

(U/mg)Purification

foldYield(%)

Crude enzyme 55 255 0.215 − 100

DEAE-anion exchanger chromatography 28 8.98 3.10 15 50

Size exclusion chromatography 8.0 1.66 4.81 22 15

U- units.DEAE-diethylaminoethyl.


[24]. Optimal laccase production was observed after 96 h

growth of A. ochraceus NCIM-1146. The intracellular lac-

case was purified using DEAE-cellulose anion exchange

and size exclusion chromatography. The enzyme was eluted

at a NaCl concentration of 0.25 M from the DEAE-cellu-

lose anion exchange column. The procedure yielded 1.66

mg of pure protein, and the recovery of total laccase

activity was 15% with 22-fold purification (Table 1).

3.2. Gel electrophoresis, spectrum, and copper content

The purified laccase appeared as a single protein band both

on SDS-PAGE and non-denaturing PAGE (Fig. 1). The

molecular weight of purified laccase (68 kDa) was con-

sistent with the molecular weight of eukaryotic laccases

(60 to 75 kDa) [25]. Purified laccase contained 3.9 atoms

of copper. Typical blue laccase normally contains four

atoms of copper, which are distributed into three types of

copper sites [17]. UV-visible absorption spectrum of the

purified A. ochraceus laccase showed a shoulder at approxi-

mately 340 nm, which corresponds to a type-3 binuclear

copper, as well as a peak at 610 nm, corresponding to a

type-1 copper site (Fig. 2). The enzyme appears blue in

color due to the presence of copper.

3.3. Effect of pH and temperature on laccase activity

and stability

The optimum pH and temperature for the purified laccase

was 4.0 and 60ºC, respectively, using ABTS as a substrate;

this is similar to many fungal laccases [17-19]. Within 5 h

of incubation, A. ochraceus NCIM-1146 laccase complete-

ly lost its activity below pH 5.0 and above 60ºC (Figs. 3A

and 3B). The enzyme was more stable in the pH range of

5.0 ~ 7.0 and up to 50ºC.

3.4. Substrate specificity and kinetics of laccase

A. ochraceus NCIM-1146 laccase oxidized several com-

pounds, including methyl and methoxy group substituted

phenolic and nonphenolic compounds, including o-toli-

dine, ABTS, hydroquinone, o-dianisidine, gauaicol, pyro-

gallol, and L-DOPA (Table 2). Introduction of OH, OCH3

or CH3 groups into the aromatic system allows the com-

pound to be more easily oxidized by laccase [11]. The Km,

Vmax, and Kcat values for A. ochraceus NCIM-1146 laccase

were 100 µM, 6.66 U, and 111/sec, respectively. A. ochraceus

NCIM-1146 laccase did not oxidize veratryl alcohol in a

reaction mixture containing buffer, substrate, and enzyme.

Oxidation of veratryl alcohol was observed when the

reaction mixture was supplemented with suitable electron

transfer mediator (ABTS and HBT).

3.5. Effect of metal salts, salinity, and inhibitor on laccase

activity

The metal salts HgCl2 and FeSO4 completely inhibited

laccase activity. The oxidation of ABTS using pure laccase

was completely inhibited by sodium chloride at a concent-

ration of 400 mM. The ABTS oxidation rate of purified

laccase was decreased by CaCl2, MgCl2, MnSO4, and

ZnSO4 by 18, 62, 55, and 62%, respectively, compared to

control. EDTA, dithiothreitol, L-cysteine and sodium azide

were found to strongly inhibit laccase activity (Table 3),

Fig. 1. Nondenaturing PAGE and SDS-PAGE of proteins obtainedafter purification of laccase. Activity staining was carried outusing L-DOPA as a substrate, and protein staining using com-massie brilliant blue as a staining dye. The lane (A) represents theactivity staining band of purified laccase in nondenaturing PAGE;lane (B and C) represents the protein staining band of purifiedlaccase and molecular markers on SDS PAGE, respectively.

Fig. 2. UV-Visible spectroscopic analysis of A. ochraceus NCIM-1146 laccase.


similar to previously reported results [26].

3.6. Decolorization of textile dyes and analysis of obtained

metabolites

Earlier reports have demonstrated the expression of intra-

cellular laccase during the development of Aspergillus

nidulans fruitbody [18,19]. Previously, the involvement of

laccase in the decolorization of textile dyes by Aspergillus

ochraceus was shown [20]. A. ochraceus NCIM-1146 lac-

case decolorized textile dyes in the absence of redox

mediators with a decolorization efficiency from 56 to 90%

(Table 4). Trametes versicolor laccase showed 27 and 40%

decolorization in the presence of HBT after 5 and 24 h of

treatment, respectively, whereas only 7 and 15% was

observed in the absence of mediator [27]. The textile dyes

reactive navy blue HER, reactive yellow 84-A and methyl

orange showed absorbance peaks at 610, 460, and 470 nm

respectively (Fig. 4). The fact that the decolorized sample

did not show corresponding absorbance peaks suggests

decolorization of textile dyes (Fig. 4). The difference in the

Fig. 3. The pH (A) and temperature (B) stability of purified

laccase. Enzyme activity after 0 (◆), 1 (■), 2 (▲), 3 (◇), 4 (□),and 5 h (△). Data points represent the mean of three independentreplicates, standard error of the mean (SE) is indicated by errorbars.

Table 2. The oxidation of phenolic and nonphenolic substrates bypurified laccase from A. ochraceous NCIM-1146

Substratesλmax

(nm)Specific activity*

ABTS 420 0.505

o-tolidine 366 0.498

2,6-dimethoxyphenol 468 0.260

Veratryl alcohol 310 ND

Hydroquinone 248 0.333

L-DOPA 475 0.223

o-Dianisidine 460 0.111

Guaiacol 465 0.238

Pyrogallol 450 0.215

p-Cresol 400 ND

Tyrosine 278 ND

*U/mg of protein/min. ND: Not detected.

Table 3. Influence of inhibitors on the laccase activity

InhibitorsRelative activity (%)

1 mM 5 mM

Control 100 100

Sodium azide 18 09

EDTA ND ND

Dithiothreitol 09 ND

L-Cystein 09 ND

ND: Not detected.

Table 4. Decolorization potential of A. ochraceus NCIM-1146 laccase

Dyes Dye class C.I. No. CAS No. λmax (nm) Decolorization (%)*

Reactive navy blue HER Vinyl sulfone NA 77905-32-5 610 90

Reactive golden yellow HER Sulfonated monoazo NA 61951-85-7 460 90

Methyl orange Sulfonated monoazo 13025 547-58-0 470 56

*The decolorization was measured after 4 h of incubation.


FTIR spectra of Methyl orange and metabolites obtained

after decolorization suggests that biodegradation occurred.

The FTIR spectrum of Methyl orange showed peaks at

569/cm for S-O stretching vibrations, 702/cm for C-H

stretching vibrations, 844/cm for C-N bending vibrations,

1031/cm for S-O stretching vibrations, and 1168/cm for N-

CH3 stretching vibrations. The group frequency region

showed specific peaks for functional groups, peaks at

1367/cm for N=N stretching vibrations, 1518/cm for N-

CH3 bending vibrations, 1602/cm for C-H bending vibra-

tions, 2914/cm for C-H stretching vibrations in -CH3 group,

and 3481/cm for O-H stretching vibrations (Fig. 5). On the

other hand, the FTIR spectrum of metabolites obtained

after decolorization of methyl orange showed peaks at

1032/cm for N-H stretching vibrations, 1117/cm for C-O

stretching vibrations, 1464/cm for C=C stretching vibra-

tions, 1610/cm for C-C stretching vibrations, 174/cm for

C=O stretching vibrations, 2854/cm for -CH3 stretching

vibrations, 2920/cm for -CH2 stretching vibrations, and

3429/cm for N-H stretching vibrations (Fig. 5). The absence

of a peak at 1367/cm suggests that the azo bond was

cleaved. HPLC analysis of methyl orange showed a peak at

Fig. 4. Spectroscopic analysis for decolorization of reactive navy blue HER (A) reactive golden yellow HER (B) and methyl orange (C).

Spectrum of initial sample (◆) and spectrum of decolorized sample (▲). Data points represent the mean of three independent replicates,standard error of the mean (SE) is indicated by error bars.

Fig. 5. FTIR spectrum of methyl orange and metabolites obtained after decolorization.


2.26 min, whereas metabolites obtained after decolorization

showed peaks at 3.39, 3.49, and 3.84 min. The difference

in retention time between methyl orange and metabolites

formed after decolorization implies the biodegradation of

methyl orange into different metabolites. The metabolite

formed after the degradation of methyl orange was identi-

fied as p-N,N'-dimethylamine phenyldiazine using GC-MS

(Table 5).

The first step in the decolorization of azo dyes using

laccase is the formation of an electron-deficient reaction

center (carbocation). Carbocation creates a highly reactive

intermediate, which is often subject to nucleophillic attack

by nucleophiles such as -OH, -SO3 or halogen ions, result-

ing into asymmetric cleavage of the azo bond [11]. The

biodegradation of methyl orange involves asymmetric cleav-

age of the azo bond, resulting in formation of a p-N,N'-

dimethylamine phenyldiazine intermediate and a p-hydr-

oxybenzene sulfonic acid intermediate (Fig. 6).

4. Conclusion

Compared to the considerable research conducted on azo

dyes, the information exists on its biodegradation mechanism

is limited. In this paper, we describe the mechanism for azo

dye degradation by intracellular blue laccase of A. ochraceus

NCIM-1146, which has potential for environmental appli-

cations.

Acknowledgements

The authors are thankful to the University Grants Commi-

ssion, New Delhi for the financial assistance. Authors also

thank to Common Facility Center, Shivaji University,

Kolhapur, India for GC-MS facility.

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Biochemical characterization and potential for textile dye degradation of blue laccase from Aspergillus ochraceus NCIM-1146

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