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360 Journal of Basic Microbiology 2010, 50, 360 367

2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Research Paper

Production of laccase fromPleurotus florida

usingagro-wastes and efficient decolorization of Reactive blue 198

P. Sathishkumar1, K. Murugesan

2and T. Palvannan

1

1Bioprocess and Genetic Engineering Lab, Department of Biochemistry, Periyar University, Salem,Tamil Nadu, India

2School of Environmental Science and Engineering, Pohang University of Science and Technology,San, Hyojadong, Namgu, Pohang, Republic of Korea

Pleurotus florida NCIM 1243 produced laccase as the dominant lignolytic enzyme during the dye

decolorization. Banana peel was the best substrate for extracellular laccase production under

solid state fermentation when compared to mandarin peel and cantaloupe peel. The maximum

activity of laccase (5.4 U/g) was detected on the 10 day. The ratio of banana peel : mandarin

peel : cantaloupe peel (5:2:3) showed increased production of laccase (6.8 U/g). P. florida pro-

duced two extracellular laccase isoenzymes (L1 and L2). The half life of laccase at 60 C was 2 h

and at 4 h it retained 25% residual activity. P. florida laccase showed high thermostability and

an interesting difference was noticed in the behavior of laccase isoenzymes at different

temperature. The L1 isoenzyme of laccase showed remarked thermostability at 60 C in the

native PAGE when compared to L2 isoenzyme. The optimum pH, temperature and enzyme

concentration for maximum decolorization was found to be 4.5, 60 C and 1.2 U/ml, respec-

tively. Partially purified laccase enzyme showed excellent decolorization activity to Reactive

blue 198. The maximum decolorization (96%) was observed at lower dye concentrations

(50100 ppm) which decreased markedly when the dye concentration was increased beyond

150 ppm. The thermostable laccase ofP. florida could be effectively used to decolorize the

synthetic dyes in the textile effluent and other biotechnological applications.

Keywords: Laccase /Pleurotus florida /Thermostability / Solid state fermentation / Dye decolorization /

Reactive blue 198

Received: December 02, 2009; accepted: April 07, 2010

DOI 10.1002/jobm.200900407

Introduction*

Synthetic dyes are widely used in the textile, paper,

cosmetics, leather, dyeing, color photography, pharma-

ceutical and food industries [1]. In textile industries,during dyeing process, about 1030% or more of the

dyes used are released into water bodies [2], causing

serious environmental problem in many parts of the

world. Although some of the dyes are not themselves

toxic, after release into the aquatic environment their

degradation products are often carcinogenic [3, 4]. De-

colorization of dyes by physical or chemical methods

Correspondence: Dr. T. Palvannan M.Sc., Ph.D., Bioprocess andGenetic Engineering Lab, Department of Biochemistry, Periyar Univer-sity, Salem 636 011, Tamil Nadu, IndiaE-mail: [email protected]: +91 427 2345766, 2345520

Fax: +91 427 2345124

including adsorption and precipitation methods, che-

mical degradation or photodegradation has practical

disadvantages. Further, they are also expensive, com-

mercially unattractive, time-consuming and mostly

ineffective [5]. On the other hand, dye decolorizationusing oxidative enzymes has received great attention in

recent years due to its efficient application [6].

White rot fungi have been shown to be able to decol-

orize synthetic dyes due to their lignolytic enzymes

such as lignin peroxidase, manganese peroxidase and

laccase [710]. Generally, the white rot fungi produce

either one of the above or all the three types of the

above lignolytic enzymes. Most of the white rot fungal

strains produce laccase as the main enzyme during dye

decolorization process.

Laccase (p-diphenol: oxygen oxidoreductase, EC

1.10.3.2) is a widespread group of multi-copper enzy-

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Journal of Basic Microbiology 2010, 50, 360367 Production of laccase from Pleurotus florida using agro-wastes 361


mes which can catalyzes the oxidation of a variety of

organic compounds, with concomitant reduction of

molecular oxygen to water. The potential applications

of laccases have been shown in various biotechnological

process including pulping, detergents, textile dyes,

biosensors, food industries, detoxification of pollution

and enzymatic conversion of chemical intermediates [6,

1115]. One of the advantages associated with laccases

is that they do not require H2O2 for substrate oxidation

unlike peroxidases and moreover they have broad sub-

strate specificity [16]. Thus, laccases of fungi have at-

tracted considerable attentions for academic and indus-

trial applications. Up to date, more than 100 laccases

have been isolated from fungal cultures and their bio-

chemical properties as well as their catalytic characters

have been characterized.Submerged fermentation does not mimic the natural

living conditions of white rot fungi. Solid state fermen-

tation (SSF), defined as the fermentation of solids in the

absence of free water, has the advantage of supporting

the growth and metabolism of microorganisms under

moisture conditions [17]. Production of enzymes by SSF

on agro-wastes has gained much attention in biotech-

nology due to its higher productivities and low produc-

tion costs [18]. The use of such wastes, besides providing

alternative substrates, helps to solve environmental prob-

lems, which are caused by their disposal. In addition,

most of these wastes contain lignin or/and cellulose andhemicellulose, which act as mediators of the lignolytic

activities. Furthermore, most of them are rich in sugars,

which make the whole process much more economical.

All these facts make them very suitable as raw materials

for the production of secondary metabolites of indus-

trial significance by microorganisms [19]. Laccase pro-

duced from SSF system using agro-wastes has been

proved as an effective synthetic dye decolorizer [20, 21].

Pleurotus florida is a white rot fungi which has great

biotechnological importance and its application on

industrial effluent is well known [2224]. In this study,

we used banana (Musa cavendishii), mandarin (Citrus re-ticulata) and cantaloupe (Cucumis melo) peels as the sub-

strate to produce extracellular lignolytic enzyme from

P. florida by the SSF. The extracellular enzyme was

evaluated for its decolorization capability against syn-

thetic dye, Reactive blue 198.

Materials and methods

Microorganism and chemicals

The white rot fungus, Pleurotus florida NCIM 1243 em-

ployed in this study was purchased from National Col-

lection of Industrial Microorganism, National Chemical

Laboratory (NCL), Pune, India.

ABTS (2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic

acid) was purchased from Sigma-Aldrich. Dye used in

this study [Reactive blue 198 (RB 198)] was procured

from commercial textile dyeing industry at Erode, Ta-

milnadu, India and all other chemicals were of analyti-

cal grade.

Dye decolorization ability on agar plate

Dye degradation ability ofP. floridawas screened in low

nitrogen basal medium [25] amended with RB 198 dye

at concentration of 100 ppm in the presence of 1.5%

agar plate. Mycelia was placed on the centre of dye agar

plate and incubated at 30 C under dark condition.

Plates were regularly monitored for dye decolorizationactivities through the change of color from blue to

colorless for every 24 h.

Agricultural wastes

Chopped banana (Musa cavendishii), mandarin (Citrus reti-

culata) and cantaloupe (Cucumis melo) peels (particle size

7.5 mm 7.5 mm) were procured from local market,

and used as substrate for laccase production by P. florida

under SSF condition. Substrates were pre-treated as

follows: 10 g of each substrate which was freshly

collected were first soaked for an hour in 30 ml of

83.17 mM KOH at room temperature to neutralise or-ganic acids [26]. Then, they were thoroughly washed

with distilled water and dried at 50 C temperature.

Enzyme production and preparation

Laccase production was carried out in SSF using three

different agro-wastes such as banana peel (BP), manda-

rin peel (MP) and cantaloupe peel (CP) as the substrate.

Ten grams of moistened peels were transferred sepa-

rately to conical flask (250 ml) and autoclaved at 121 C

for 15 min. Five mycelial discs were transferred to each

flask supplemented with 100 M CuSO4 to induce lac-

case production and incubated at room temperature.Cultures were harvested at every 2 d interval for the

quantification of extracellular laccase enzyme.

Extracellular enzyme from SSF was extracted by

soaking the culture with 100 mM sodium acetate buffer

pH 5.0 at 4 C overnight. The culture supernatant was

filtered and centrifuged at 10,000 g for 15 min to

remove the fine particles. The supernatant was concen-

trated by Millipore Amicon ultrafiltration stirred cell

through a 0.22 m (10 kDa) filter under vacuum, until a

20-fold concentration was achieved. The enzyme was

precipitated with ammonium sulfate to a final concen-

tration of 80% (w/v). After standing in the ammonium

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362 P. Sathishkumar et al. Journal of Basic Microbiology 2010, 50, 360 367


sulfate solution for 5 h at 0 C, the precipitate was col-

lected by centrifugation at 10,000 g for 30 min and

resuspended with 100 mM of sodium acetate buffer

(pH 5.0) and dialysed against the same buffer for 12 h.

The resulting solution was used for the decolorization

studies.

Enzyme assay and protein estimation

Laccase activity was measured using ABTS as the sub-

strate at 30 C [27]. The assay mixture contained 50 mM

sodium acetate buffer (pH 5.0), 1 mM ABTS and diluted

laccase source. One activity unit was defined as the

amount of enzyme that oxidised 1 mol ABTS per min-ute. The absorbance increase of assay mixture was

monitored at 420 nm (420 = 36.0 mM1 cm1) in a UV

Vis spectrophotometer (Perkin-Elmer Lambda 25, Ger-many). The enzyme activities were expressed as unit

(U/ml or U/g).

Protein was estimated by the method of Lowry et al.

[28] using bovine serum albumin (BSA) as a standard.

Decolorization of RB 198 by P. floridalaccase

Dye decolorization efficiency of P. florida laccase en-

zyme obtained from SSF, was accessed using RB 198.

Reaction mixture contained of RB 198 dye, partially

purified laccase and 100 mM of buffer in a total volume

of 1 ml. The reaction mixture was incubated for dye

decolorization and the decolorization was measuredby monitoring the decrease in absorbance maximum

(592 nm) of RB 198 dye in a UVVis spectrophotometer

and expressed in terms of percentage. In parallel, con-

trol samples were maintained with heat inactivated

partially purified laccase.

Effect of different parameters on dye decolorization

The effect of pH ranging from 3 to 9 on the enzymatic

decolorization was monitored with fixed concentration

of dye (100 ppm) and enzyme concentration (2 U/ml) at

30 C. The pH of reaction mixture was adjusted by

citrate-acetate-phosphate (37) and TrisHCl (89). Tofind out the effect of temperature on the enzymatic

decolorization, the reaction mixture was incubated

with fixed concentration of dye (100 ppm), enzyme

(2 U/ml) and pH (4.5). The temperature range was be-

tween 20 to 80 C with 20 C increment. In order to

check the effect of enzyme quantity on dye decoloriza-

tion with fixed concentration of dye (100 ppm) and pH

(4.5) at 60 C, reaction was started with different quan-

tity of enzyme from 0.2 to 2 U/ml with the increment of

0.2 U/ml. The effect of dye concentration was tested

using 50, 100, 150, 200 and 250 ppm concentrations

with fixed concentration of enzyme (1.2 U/ml) and pH

4.5 at 60 C. For all these experiments, the decoloriza-

tion was monitored after 10 min incubation as descri-

bed above.

Thermostability of laccase

The thermostability of laccase was studied by incubat-

ing the enzyme at various temperatures from 20 to

80 C with the increment of 20 C. The residual laccase

activity was measured at different time intervals using

ABTS as substrate under standard assay condition.

Native PAGE

In order to identify the number of laccase isoenzymes

produced by P. florida and their thermostability, the

enzyme was subjected to non-denaturing polyacryla-

mide gel electrophoresis (10%). For zymogram analysis,the gel was incubated in 100 mM sodium acetate buffer

with 10 mM guaiacol for the laccase activity detection.

Results and discussion

Screening of dye decolorization

Decolorization ability ofP. floridawas screened by agar

plate amended with RB 198 dye. The mycelial growth

covered the agar plate completely on 6 d with 70%

decolorization zone. Further incubation lead to com-

plete decolorization on the 10 d (Fig. 1b). The decolori-zation of RB 198 proceeded through a color change

from blue to light pinkish blue and later colorless. Af-

ter 10 d of incubation, the plate was evaluated for the

type of lignolytic enzymes involved in dye decoloriza-

tion. In plate incorporated with 1 mM MnSO4, a very

slight brownish flake was observed indicating the in-

significant production of manganese peroxidase (Fig. 1c).

Another plate was screened for the laccase production

by incubating the plate with 1 mM ABTS in 50 mM

sodium acetate buffer (pH 5.0). An intense oxidation of

ABTS was observed by the formation of bluish green

color, indicating that the decolorization process wasmainly due to the production of laccase from P. florida

(Fig. 1d). The same result was observed during the de-

colorization of Remazol Brilliant Blue R (RBBR) by white

rot fungi Ganoderma lucidum [10].

Laccase production in solid state fermentation

Laccase production from P. florida in SSF using agricul-

tural wastes such as banana peel, mandarin peel and

cantaloupe peel are presented in Fig. 2 and Table 1.

Mycelial growth on the substrates was observed after

36 h incubation and the fungal mycelium completely

colonized the substrate within 6 days. The production

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Figure 1. Screening for dye decolorization by P. floridaNCIM 1243and its lignolytic enzymes on 100 ppm of RB 198 dye amendedagar plate. (a) Control; (b) after decolorization (10 days incubated);(c) Formation of manganese flakes (brown color) in MnSO

4

amended plate indicates manganese peroxidase production. (d)Oxidation of 1 mM ABTS in 100 mM sodium acetate buffer (pH 5.0)indicates laccase production.

of laccase was monitored for every second day. The

maximum laccase activities ofP. florida was found to

be 5.4 U/g (banana peel), 3.1 U/g (mandarin peel) and

4.0 U/g (cantaloupe peel) on 10 d incubation. Previously,

Osma et al. [29] reported that banana skin was novel

substrate for the growth and laccase production fromTrametes pubescens under SSF. This was attributed to

high carbohydrate content that might be easily metabo-

lized by the microorganism [30]. The same result was

observed in our study using P. floridawhich suggested

that banana peel can be used as cheap source for lac-

case production. Further experiments were carried out

with different ratio of BP:MP:CP for the laccase pro-

duction. The results indicated that a ratio of 5:2:3

(BP:MP:CP) increased production of laccase (6.8 U/g)

from P. florida (Table 1). This may be due to the syner-

getic action of the components present in all three

agro-wastes.

Figure 2. Production of laccase in SSF by P. floridaNCIM 1243.

Table 1. Effect of mandarin peel and cantaloupe peel supp-lementation to the banana peel in laccase production fromP. floridaNCIM 1243.

Ratio of banana peel : mandarin peelcantaloupe peel (w/w)

Laccase activity(U/g)

Banana peel (control) 5.4Mandarin peel (control) 3.1Cantaloupe peel (control) 4.05:5:0 4.75:4:1 5.65:3:2 6.15:2:3 6.85:1:4 6.25:0:5 5.6

Effect of different parameters on dye decolorization

by P. floridalaccase

P. florida extracellular laccase prepared from mixed

substrateculture (BP:MP:CP 5 :2 :3) was employed for

dye decolorization studies. The influence of pH on dye

decolorization was determined at different pH ranging

from 3 to 9 (Fig. 3). The results showed that the laccasehighly decolorize the dye in the range of pH 4 to 6. The

optimum pH for maximum decolorization (96%) was

pH 4.5. The decolorization activity decreased at pH 7

and no activity was observed at alkaline pH. Our result

shows that P. florida laccase favoring acidic range for

higher decolorization of RB 198 dye. This observation

was similar to the previous reports obtained by Mu-

rugesan et al. [10] and Kandelbauer et al. [31].

The effect of temperature on RB 198 dye decoloriza-

tion is shown in Fig. 4a. Temperature is one of the fac-

tors involved in dye decolorization. The result clearly

show that the optimum temperature for maximum de-

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Figure 3. Effect of pH on decolorization of RB 198 by P. floridaNCIM 1243 laccase.

colorization was 60 C, beyond that decolorization de-

creased sharply. In the case of dye samples at 30 C,

29% of decolorization was decreased when compared to

60 C. Murugesan et al. [10] reported that the white rot

fungi G. lucidum laccase had an optimum temperature

for RBBR decolorization at 60 C, which was achieved

by means of 1 h incubation. This was similar to our

result. However, P. florida laccase decolorize the dyemore rapidly (within 10 min). When incubated below

60 C, the percentage of decolorization was increased

with increase in time (data not shown). Some of the

white rot fungal laccases have been described as ther-

mostable, even though the most laccases are not active

at higher than 50 C [3235]. For example P. ostreatus

laccase showed a half life of 30 min at 60 C [36] and

P. eryngii laccase retained 10% residual activity at 60 C

after 30 min incubation [37]. However P. florida laccase

showed a half life of 2 h at 60 C and at 4 h it retained

around 25% residual activity (Fig. 4b) which indicates

its better thermostability than other Pleurotus specieslaccases. This could be due to the different ecological

origination of strains and entirely different culture

conditions. Further, in the above studies liquid culture

with chemically defined medium was used for laccase

production whereas in our study SSF with banana peel

was used. The laccase activity decreased rapidly at 80 C

and the complete inactivation occurred at 1 h incuba-

tion.

The effect of enzyme concentration on dye decolori-

zation (50 ppm) was studied to find out the amount of

enzyme essential for maximum decolorization. The

optimum concentration of laccase required for maxi-

Figure 4. a) Effect of temperature on decolorization of RB 198 byP. floridaNCIM 1243 laccase; b) Thermostability of P. floridaNCIM1243 laccase at various temperature.

mum decolorization was 1.2 U/ml (Fig. 5). However the

decolorization increased with increase in enzyme quan-tity. Previously, Soares et al. [38] had also reported that

increasing concentration of laccase increased the rate

of dye decolorization. Similarly, Pant and Adholeya [39]

reported that the concentrated laccase from P. florida

EM1303 obtained by ultrafiltration efficiently decolor-

ized undiluted distillery effluent. This further proves

the P. florida laccase utility in treating various effluents.

The effect of dye concentration on decolorization was

tested with different initial dye concentration from 50

to 250 ppm with constant amount of enzyme (1.2 U/ml).

The maximum decolorization (94%) was observed at

lower dye concentrations (50100 ppm) which decreas-

a)

b)

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Figure 5. Effect of enzyme quantity on RB 198 dye decolorization.

ed markedly when the dye concentration was increased

beyond 150 ppm (Table 2). The results revealed that the

decolorization decreased with increasing dye concen-

tration. However on further increasing the time of

incubation, a maximum decolorization was observed

(data not shown). It indicates that the tested dye con-

centrations up to 250 ppm are not inhibitory to the

enzyme whereas only the percentage of decolorization

was decreased with increasing dye concentration. At

high dye concentration the efficiency of enzyme wasreduced but did not completely diminish. This opti-

mized conditions, RB 198 was decolorized by partially

purified P. florida laccase and the absorbance spectra

were recorded Fig. 6. RB 198 decolorization was carried

in a UVVis spectrophotometer with the above opti-

mized condition and the results indicate the complete

decolorization of dye evident from the spectral scan

done between 480 and 680 nm. Interestingly, our P.

florida laccase rapidly decolorize the dye without addi-

tion of any mediator.

Laccase isoenzymesMany of the white rot fungi produce more than one

laccase isoenzyme. P. florida NCIM 1243 crude laccase

Table 2. Decolorization of RB 198 by P. florida NCIM 1243laccase at different dye concentration.

Decolorization of RB 198 (%)Dye concentration (ppm)

10 min 120 min

50 96 99100 93 99150 79 97200 43 93250 22 84

Figure 6. UVvis absorbance spectra of RB 198 decolorization byP. floridaNCIM 1243 laccase under 60 C and pH 4.5.

was subjected to zymogram analysis for the identifica-

tion of laccase isoenzymes. The results revealed that

two laccase isoenzymes (L1 and L2) were produced by

this strain (Fig. 7a). Of these two isoenzymes, L1 was

more dominant than L2. Similar pattern of laccase

isoenzymes was observed by Das et al. [32] in a previous

study using P. florida. In order to identify thermostabil-

ity of these isoenzymes, crude laccase was incubated at

different temperature for 1 h and analysed the stability

for guaiacol oxidation on native-PAGE (Fig. 7b). L1isoenzyme was more stable at 60 C than L2 isoenzyme

indicating that L1 isoenzyme of laccase is mainly re-

sponsible for dye decolorization at 60 C. At 80 C both

isoenzymes lost their activity within 1 h.

Figure 7. (a) Zymogram analysis of P. floridaNCIM 1243 laccaseisoenzymes (L1 and L2) on native PAGE by guaiacol oxidation. (b)Thermostability of P. florida NCIM 1243 laccase isoenzymes after1 h incubation at different temperature. Lane 1, 2 and 3 incubated

at 80 C, 60 C and 40 C, respectively.

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Conclusion

To our knowledge, this is the first report on thermosta-

ble laccase fromP. florida and its efficient dye decoloriz-

ing activity. Production and preparation of laccase from

this fungus using agro-wastes is easy and economical.

As the laccase in the present study shows good thermo-

stability at high temperatures it could be effectively

used to decolorize the synthetic dyes in textile effluent.

Acknowledgements

The corresponding author (Principal Investigator

Project No.: BT/PR8973/GBD/27/57/2006 Dt 14/08/07) is

grateful to Government of India, Ministry of Science &

Technology, Department of Biotechnology for financialsupport.

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