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R.Sivakumar et al.

/ International Journal of Engineering Science and Technology
Vol. 2(12), 2010, 7133-7141

Isolation, Screening and Optimization of
Production Medium for Thermostable
Laccase Production from Ganoderma sp
R. Sivakumar*
Bharathiar University
Coimbatore- 641046, Tamil Nadu, INDIA
*First Author: Shiva_grdbt@yahoo.co.in

R. Rajendran,
PG & Research Department of Microbiology, PSG College of Arts & Science,
Coimbatore-641 014, INDIA.
Second Author: rrajendranmicro@yahoo.co.in

C. Balakumar
PG & Research Department of Microbiology, PSG College of Arts & Science,
Coimbatore-641 014, INDIA.
Third Author: cbalakumar@rocketmail.com

M. Tamilvendan
Centre for Advance studies in Botany, University of Madras, Guindy campus,
Chennai-600 025, INDIA.
Fourth Author: tamilvendan@gmail.com

ABSTRACT

The present work focuses on isolating, screening and optimizing the process parameters to achieve the
maximum production of extracellular laccases by Ganoderma sp obtained from natural habitat. Guiacol and ABTS
(2, 2’- azinobis 3-ethyl-benzothiazoline-6-sulphonate) were used as substrates for screening the laccase activity.
Culture condition such as period of incubation, pH, temperature and various nutrition parameters such as carbon
source, nitrogen source, copper sulphate concentration and solvents were optimized. The optimization studies
revealed that the laccase yield was highest when operated at the following conditions: 10 days of incubation, pH 6.0,
2% starch as carbon source, 0.2% yeast extract as nitrogen source, 4% iso-propanol as solvent and 27µM of copper
sulphate in the production medium.
Key words: Laccase, Ganoderma sp, Guiacol, ABTS
1. INTRODUCTION

Laccases (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) are the most extensively studied group of
enzymes among oxidases. They belong to the family of blue multicopper oxidases, which catalyze the one-electron
oxidation of four reducing-substrate molecules concomitant with the four-electron reduction of molecular oxygen to
water1. Laccase was first described from the sap of the Japanese laquer tree Rhus vernicifera. Laccase was described
by Yoshida2, and was characterized as a metal containing oxidase by Bertrand3.
Laccase has broad substrate specificity towards aromatic compounds containing hydroxyl and amine
groups. These enzymes were known to catalyze the oxidation of a wide range of phenolic compounds and aromatic
amines. White rot fungi are the best-known laccase producers4 also there exist certain bacteria5 and actinomycetes6
that are known to produce laccases.

ISSN: 0975-5462 7133

FeSO4.1 M sodium acetate buffer (pH 5. CuSO4 (0.5). Production of Laccase Five agar disk taken from the active borders of PDA cultures were transferred to Erlenmeyer flasks (250 mL) containing 100 mL of the following liquid medium (in g. The reaction was monitored by measuring the change in A436 (€ = 2. 2. The use of inducers to enhance laccase production has been widely practiced in fungi especially in the white rots where laccase induction by aromatic compounds is well established10. 7133-7141 The genus Ganoderma includes medicinal species that belong to group of white rot fungi due to their ability to produce extracellular ligninolytic enzymes: laccase (Lac). After 14 days of incubation laccase activity and protein concentration were determined12. Na2HPO4. MnSO4 (0.05). it has been a matter of concern to find environmentally sound and economically feasible media constituents for laccase production. This present investigation we isolated the white rot fungi from natural habitat such as tamarind wood and screened for laccase activity on substrate such as guaicol and ABTS. MATERIALS AND METHOD 2. The selected strain was identified at the School of Basic Medical Science. One unit enzyme activity was defined as the amount of enzyme that oxidizes 1  mole of ABTS per minute at 270C.5). MnSO4.3. The activities were expressed in U/ml.5. Isolation of the fungal strain The fungus used in this study was isolated from natural habitat such as decaying tamarind wood in the vicinity of Kodaikanal.01). / International Journal of Engineering Science and Technology Vol. Mumbai. MgSO4 (0. ZnSO4 (0.01). Materials The reagent grade chemicals potato dextrose agar. temperature. According to Hammel8.5.L-1): starch (20). Plates containing lignolytic enzyme substrates were inoculated with a mycelial disk and incubated at 270C for five days. Czepak Dox agar. 2(12).5. two peroxidases: Mn dependent peroxidase (MnP). Chennai as Ganoderma sp. FeSO4 (0. (ii) inhibition of melanin formation and consequent increase in phenolic monomers. 2. and (iii) activation of an oxidative stress which could be indirectly responsible for laccase induction9.001). Oxidation zone around the mycelial colony indicates the presence of lignolytic enzymes. Mansur7 showed that the use of fructose instead of glucose resulted in a 100-fold increase in the specific Laccase activity of Basidiomycetes. Guiacol. H2PO4.0). CaCl2. Starch.9×104 cm−1 M−1) for 3 minutes. Further the production medium was optimized for various parameter and results are discussed in this paper. 2010. Analytical methods 2. versatile peroxidase (VP) and modify and degrade lignin.0) and 0. The substrate ABTS (1mM) was amended with the basal medium in order to screen the presence of laccase. ZnSO4. pH.1. The nutritive substances employed in the culture medium constitute significantly to the total production costs. Taramani. Tamil nadu. and etc. yeast extract (2.1. MgSO4. Owing to its vivid biotechnological applications. Hence. R.2. on Czapek-Dox agar11. 2. ABTS and CBB G-250 were purchased commercially from SIGMA.002).4. The ever-increasing demand for this enzyme requires the production process to be economical. Several hypotheses on the role of solvents in laccase production have been formulated: (i) increase in membrane permeability and promotion of protein secretion. ISSN: 0975-5462 7134 . Screening of fungi for laccase production The isolated fungal cultures were screened for lignolytic enzyme laccase. H2PO4 (1. Identifying inexpensive raw materials for enzyme production could be viewed as a solution to make the entire process cost effective and further enhancement using inducers may add to the benefit. Yeast extract.Sivakumar et al.1 ml of enzyme. The culture was periodically subcultured and maintained on potato dextrose agar plates grown at 270C and stored at 40C. The carbon sources in the medium play an important role in ligninolytic enzyme production. 2. Extracellular laccase activity Extracellular laccase activity was measured spectrophotometrically13 with ABTS as substrate. media composition. CuSO4 were procured from Hi-Media. Na2HPO4 (0. studies on laccase producing organisms have been intensified in the recent years and the optimization of laccase production from different microorganisms is being carried out by several groups. The pH of the medium was adjusted to 5. USA and used for analytical purpose. presence of inducers. The reaction mixture contains 900 µl of 1 mM ABTS in 0. CaCl2 (0. Laccase expression in fungi is influenced by culture conditions such as nature and concentration of carbon and nitrogen sources. India.001). the ligninolytic enzymes are produced during the secondary metabolism under conditions of limited nitrogen. 2.

the cross-section of the fruiting body slice showed clear. ammonium sulphate. 2.5. Flasks were incubated at 270C for a period of 14 days. The carbon sources were amended at the concentration of 2% in the production medium. The culture was harvested at every 2 day interval. ISSN: 0975-5462 7135 . the following concentrations of copper sulphate 10µM. The production medium was optimized for the following parameters for a period of 14 days.1.5. beef extract. Taramani. Effect of nitrogen sources on laccase production In order to find the suitable nitrogen source for the maximum production of laccase by Ganoderma sp the following organic and inorganic nitrogen sources namely sodium nitrate. iso propanol and 2-methyl 1-propanol on the production of laccase from Ganoderma sp was studied. Flasks were incubated at 270C for a period of 14 days. 27µm.1. bright colored tubular channels. methanol. Samples were taken at an interval of 3 days and analyzed for enzyme activity. R. 1). 2. peptone.2. 3. 40µM and 50µM were used. 20µl. 50ml of mineral salt medium was prepared and autoclaved. glucose. RESULTS AND DISSUSION 3. Thermostability Test In order to find whether the enzyme is thermostable or not. 2(12). After the medium was cooled to bearable warmth. 2.6. 5.2. This was used to determine protein content and enzyme activity. and 6.7. and ammonium chloride were amended at the concentrations 0. To these five discs (6mm in diameter) of 5 day old culture was inoculated and incubated at 270C for a period of 14 days.5. The five mycelial discs (6 mm diameter) of 5- day-old culture were transferred to Erlenmeyer flasks (250 ml) containing 100 ml of production medium. Flasks were incubated at 270C for a period of 14 days. there were spores evenly distributed. 2. Effect of carbon sources on laccase production The effect of different carbon sources like manitol. propanol. Five mycelial discs (6 mm diameter) of 5-day old culture were transferred to Erlenmeyer flasks (250 ml) containing 100 ml of production medium amended with different concentration of copper sulphate. 27µl. Estimation of protein The protein conncetration was determined14 using the Bio-Rad Protein Assay Reagent (Bio-Rad) and using bovine serum albumin (BSA) as standard. Flasks were incubated at 270C for a period of 14 days.6.Sivakumar et al. 7133-7141 2. Isolation of white rot fungi The white rot fungus was isolated from natural habitat and was cultured on Potato dextrose agar. In the channels. Effect of solvent on laccase production The effects of different solvents like ethanol.2%. Five well was cut and enzyme was loaded at different concentration (10µl.3. Effect of pH on laccase production The effect of pH on laccase production was carried out by incubating the culture flasks containing 100 ml of production medium inoculated with five mycelial discs (6 mm) at different pH such as 4.6.6. 2. The selected strain was identified at the School of Basic Medical Science. 2. 5.0. maltose. lactose. For each experiment five mycelial discs (6mm diameter) were used as inoculum in 100 mL medium.6. 20µM. One plate was incubated at room temperature and other was incubated at 600C.5. 6. Under the microscope (Fig. / International Journal of Engineering Science and Technology Vol.5. 2.6.6.6. The five mycelial discs (6 mm diameter) of 5-day-old culture were transferred to Erlenmeyer flasks (250 ml) containing 100 ml of production medium. These experiments were conducted for a period of 14 days. 2010. guiacol (1mM) was amended under aseptic condition into medium then poured in to two sterile Petri dishes and allowed to solidify. Optimization of culture conditions for laccase production by Ganoderma sp. Chennai as Ganoderma sp. This is a characteristic feature of white rot fungi. 2. Effect of various concentration of copper sulphate on laccase production In order to find out the suitable concentration of copper sulphate for the maximum production of laccase from Ganoderma sp. 40µl). and sucrose on the production of laccase from Ganoderma sp was studied. The sources were amended at the concentration of 4% in the production medium.4. Time course study In order to find the optimal time of incubation for the maximum laccase production 100ml production medium was prepared in Erlenmeyer flask (250 ml) and autoclaved.0. The mycelial discs (6mm diameter) of 5-day-old culture were transferred to Erlenmeyer flasks (250ml) containing 100 ml of production medium.

Screening of white rot fungi for laccase production White rot fungus was screened for the production of laccase on czapek dox medium amended with different laccase substrates such as guiacol and ABTS. Microscopic observation of the fruiting body. Fig. The finding that guiacol is used as marker for extracellular oxidative enzymes.1.2. ABTS and guiacol are considered unique laccase substrates in the absence of hydrogen peroxidase. 2010. 3. Screening of Ganoderma sp for laccase production of Czapek dox medium (a) Guiacol (1mM) (b) ABTS (1mM) In the present study. 3. 7133-7141 Fig. 1. The capability of degrading lignin is due to the extracellular non-specific and non-stereo selective enzyme systems.3. The oxidation of laccase substrates was observed. ISSN: 0975-5462 7136 . 3. Ganoderma sp was able to oxidase phenolic and non-phenolic compounds. / International Journal of Engineering Science and Technology Vol. The results are shown in fig 2. The maximum laccase production was observed at 10th day culture 0. therefore it is confirmed that the enzyme is a true laccase. Because the key components of the whit-rot lignin degrading system are extracellular. The extracellular enzyme system involved in lignin degradation is composed of lignin peroxidase.3. 2.59 U/ml whereas. the fungi can degrade insoluble chemicals such as lignin and many of the hazardous environmental pollutants. 2(12). Time course study The time course of laccase production by Ganoderma sp was monitored. 16. White-rot fungi are heterogeneous group of organism and have the capacity to degrade lignin as well as other wood components. supports the above result17. Optimization of culture conditions for laccase production by Ganoderma sp. laccases and manganese dependent peroxidases15. R.Sivakumar et al. maximum protein 190 µg/ml released at 14th day culture (Fig 3).

falls between 4.3.25 50 0. ISSN: 0975-5462 7137 . 0.6 Enzyme activity 150 Protein content 0.18 U/ml with a highest extracellular protein 140 µg/ml (Fig 5). In the present study.15 30 (µg/ml) (U/ml) 0. 0.0 was found suitable for the maximum growth and laccase production by Ganoderma sp.24 U/ml at pH 6.2. Effect of different carbon sources on extracellular protein and laccase activity Six different carbon sources such as Mannitol.Sivakumar et al. 3.2 40 Enzyme activity Protein content 0. Lactose and Starch were tested at 2% for laccase production in Ganoderma sp.020. Effect of different pH on laccase production. Dhaliwal19 reported that Pleurotus florida produced high amount of laccase (4.4 100 0. pH 6. 3. R. starch supported a maximum laccase activity of 0. as reported in many white rot fungi. lignolytic enzymes production and xenobiotics degradation. 7133-7141 Fig. The optimum pH of laccase production.60 U/ml) in malt extract broth after 12th days under stationary conditions.5 and laccase activity of 0.5-6. Glucose. 2(12). Maltose.3.5 6 6.3 0. / International Journal of Engineering Science and Technology Vol.1 0 0 2 4 6 8 10 12 14 Days of incubation Enzyme activity Protein content In the present study with Ganoderma sp the maximum laccase activity was recorded on 10th day of incubation. Time Course Study to determine the maximum laccase production.1 20 0.2 50 0. The fungus was able to release a maximum protein content of 40 µg/ml at pH 5. 2010. Niku-Paavola18 observed maximum laccase production in Phlebia radiata on day 3-5 and it was rapidly disappeared before the increasing activities of Manganese peroxidase and lignin peroxidase. Among the carbon sources.7 200 0. Fig.05 10 0 0 4.0 (Fig 4).3.5 Diffe re nt pH Enzyme activity Protein content The pH of the culture medium is critical to the growth. Sucrose. Effect of different pH on extracellular protein and laccase activity It was evident that the pH significantly influenced the extracellular protein content and laccase activity in Ganoderma sp. 4.5 (µg/ml) (U/ml) 0.5 5 5. 3.

0. Effect of different nitrogen sources on laccase production.05 50 0 0 Mannitol Glucose Sucrose Lactose Maltose Starch Differe nt Carbon source s (2%) Enzyme activity Protein content The production of laccase enzyme enhanced using various carbon sources such as mannitol. 2(12).15 Protein content 150 (µg/ml) (U/ml) 0. Fig.05 50 0 0 Sodium Ammonium Peptone Beef Yeast nitrate chloride extract extract Diffe re nt Nitroge nt source s (0. Propanol.2%) Enzyme activity Protein content The most widely used nitrogen sources for fungal lignolytic enzyme production are ammonium salts such as tartrate or chloride22.7 U/ml and maximum extra cellular protein content of 70 µg/ml (Fig 7).3. starch and sucrose. ISSN: 0975-5462 7138 . 3.4.propanol. 3.18 U/ml and maximum extra cellular protein content of 140 µg/ml (Fig 6). Beef extract. Basidiomycete PM123. 7133-7141 Fig. Effect of different solvent on laccase production Five different solvent sources like Ethanol. lactose. / International Journal of Engineering Science and Technology Vol.5. Among them. Effect of different nitrogen sources on extracellular protein and Laccase activity Five different nitrogen sources like Yeast extract. R. namely. 0. Effect of different carbon sources on laccase production. isopropanol supported the maximum laccase production of 0.3.1 100 0. Ammonium nitrate favored high laccase production in many white rot fungi. 6. Ammonium chloride and Sodium nitrate were tested for extracellular protein and laccase production in Ganoderma sp.2 200 Enzyme activity Protein content 0. Lentinula edodes24.Sivakumar et al.2 200 Enzyme activity 0. Isopropanol were tested for extracellular protein and laccase production in Ganoderma sp. Yeast extract supported the maximum laccase production of 0. 2-Methyl 1. 5. glucose. maltose. Methanol. Kapdan and Kargi21 reported that cultivation of Coriolus versicolor at 10 g/l glucose resulted in a better fungal growth. 2010. Peptone.15 150 (µg/ml) (U/ml) 0. Among them.1 100 0. Among the six different carbon sources starch supported good growth and laccase production.

40µM and 50µM were tested for extracellular protein and laccase production in Ganoderma sp.Methyl Methanol Isopropanol 1. 14 200 12 Enzyme activity 150 Protein content 10 (µg/ml) 8 (U/ml) 100 6 4 50 2 0 0 10 20 30 40 50 Diffe re nt C oppe r sulphate conce ntration (µM) Enzyme activity Protein content Niladevi and Prema26 obtained maximum laccase activity when copper sulphate was used at a concentration of 1mM. Effect of different solvent on laccase production. ISSN: 0975-5462 7139 . Moreover. Thermostability Test The plate incubated at 600C showed maximum activity confirming that the enzyme is thermostable. 0.4. 3.9 U/mL) also proved to be a promising inducer for laccase production by Streptomyces psammoticus as similar to that in most of the fungi 27. Fig. 30µM. cheaper and environmentally safer. ethanol is an abundant agro-industrial by-product.35 U/ml and maximum extra cellular protein content of 160 µg/ml (Fig 8). Effect of various concentration of copper sulphate on laccase production.2 20 0 0 Propanol Ethanol 2. 2(12). 20µM. / International Journal of Engineering Science and Technology Vol.Sivakumar et al. 2010. 3.8 80 Enzyme activity Protein content 0. 7133-7141 Fig. Among them.4 40 0. 30µM supported the maximum laccase production of 0.6 60 (µg/ml) (U/ml) 0. Effect of various concentration of copper sulphate on laccase production Five different concentrations like 10µM.8. R. Copper sulphate (7.3.7. The results are interpreted in table 1.Propanol Diffe re nt solve nts (4%) Enzyme activity Protein content The positive effect of methanol on laccase activity in liquid culture of Trametes versicolor was also demonstrated25.6.

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