· Lignin Peroxidase (Lip)

· Manganese Peroxidase (MnP)

· Copper-containing phenol oxidase (or) laccase

White-rot fungi are the predominant decomposers of lignin. Lignin is an aromatic polymer with the substituents connected by both ether and carbon-carbon linkages and constitutes 20-30% of woody plant cell wall. Lignin degradation by white-rot fungi is an oxidative and non-specific process. Manganese peroxidases (MnPs), lignin peroxidases (LiPs) and laccases (Lacs) are three families of enzymes that are implicated in the biodegradation of lignin. All the three enzymes catalyze the one-electron oxidation of phenolic substrates to phenoxy radicals that can undergo certain degradation reactions of lignin. However, the catalytic mechanism among the three enzymes is different (Kirk et al., 1987, Gold et al., 1993, Kirk et al., 1985, Gold et al., 1989).

Fungal peroxidases and fungal laccases:

Peroxidases (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) are a group of haem containing oxidoreductases that catalyze the reduction of peroxide such as hydrogen peroxide (H₂O₂) and the oxidation of a variety of organic and inorganic substrates. Peroxidases of fungal origin, studied for waste treatment, include chloroperoxidase (CPO; EC 1.11.1.10), lignin peroxidase (LiP, ligninase, diarylpropane peroxidase; EC 1.11.1.14), manganese-dependent peroxidase (MnP, manganese peroxidase; EC 1.11.1.13), Coprinus cinereus peroxidase (CIP; C. macro-rhizus peroxidase; EC 1.11.1.7), and Arthromyces ramosus peroxidase (ARP; EC 1.11.1.7). Based on their molecular and structural similarities, these fungal peroxidases, except CPO, are classified as the class II fungal peroxidases in the plant peroxidase super family, which was proposed by Welinder (1992). Chloroperoxidase is the only exception from the plant peroxidase super family because it has a distinct tertiary structure and catalytic activities to other plant and fungal peroxidases (Welinder 1992; Casella and Marchesini 1994). Although CPO is also a very versatile and industrially important enzyme (Pickard et al. 1991; Littlechild 1999), this enzyme will not be covered in this review because the development in CPO production process was established mostly in the 1980s (Pickard et al. 1991), and virtually no report was found in the recent literature. Lignin peroxidase and MnP are closely related enzymes and are often produced simultaneously by white-rot fungi that possess ligninolytic activities. Thus, these two enzymes will be treated as a group in this review. Because CIP and ARP are also closely related to each other and do not exhibit ligninolytic activities, these enzymes will be discussed together as well, and grouped as non-ligninolytic fungal peroxidases. A comprehensive review on the structural and catalytic properties of these fungal peroxidases is also available elsewhere (Dunford 1999). Laccase (p-diphenol:dioxygen oxidoreductase, EC 1.10.3.2) is one of the few enzymes that was the subject of investigation as early as the end of the 19th century (Thurston 1994). This enzyme can be further divided into two categories, plant and fungal laccases, although laccase-type phenol oxidases were also isolated from bacteria (Castro-Sowinski et al. 2002; Martinset al. 2002) and insects (Thomas et al. 1989). Laccases have multiple copper atoms at their active sites and utilize molecular oxygen as an oxidant for the oxidation of varieties of phenols and other aromatic compounds to corresponding reactive quinones (Solomon et al. 1996). Comprehensive reviews on the structural and catalytic features of laccases are also available elsewhere (Thurston 1994; Solomon et al. 1996).Recent progress in the development of the production processes for each group of enzyme are outlined below with a brief summary of the historical background, catalytic properties of the enzyme, as well as its potential application to waste treatment. More detailed reviews in the potential environmental applications of enzymes, including fungal enzymes discussed here, are also available elsewhere (Aitken 1993; Karam and Nicell 1997; Duran and Esposito 2000; Nicell 2001).

Manganese peroxidase is particular interest, because this enzyme is used in various biotechnological processes. Manganese peroxidases are the enzymes are widely distributed among fungi that catalyze the oxidation of a variety of phenolic compounds and aromatic amines

The crystal structure of manganese peroxidase (MnP) from the lignin-degrading basidiomycetous fungus Phanerochaete chrysosporium has been solved using molecular replacement techniques and refined to R = 0.20 at 2.0 A. The overall structure is similar to that of two other fungal peroxidases, lignin peroxidase from P. chrysosporium and Arthromyces ramosus peroxidase. Like the other fungal peroxidases, MnP has two structural calcium ions. MnP also has two N-acetyl glucosamine residues N-linked to Asn131 that are readily visible in the electron density map. The active site, consisting of a proximal His ligand H-bonded to an Asp residue and a distal side peroxide binding pocket consisting of a catalytic His and Arg, is the same as in the aforementioned fungal peroxidases as well as yeast cytochrome c peroxidase. MnP differs in having five rather than four disulfide bonds. The additional disulfide bond, Cys341-Cys348, is located near the C terminus of the polypeptide chain. Importantly, a new cation binding site, which we propose is the manganese-binding site of MnP, was located in the crystal structure. The ligands constituting the Mn(2+)-binding site include Asp179, Glu35, Glu39, a heme propionate, and two water molecules. Electron transfer from Mn2+ to the heme edge or iron center is envisioned to occur through a sigma-bonded pathway along a heme propionate. White rot basidiomycetes such as Phanerochaete chrysosporium have long been known to possess a highly nonspecific battery of extracellular enzymes that allows them to degrade the plant polymer lignin. This same enzymatic system is believed to underlie the ability of these fungi to degrade many organopollutants. In the past decade, extensive work has accumulated to implicate two P. chrysosporium peroxidases, lignin peroxidase (LiP) and manganese peroxidase (MnP) in pollutant degradation.

Over 10,000 dyes with an annual production of over 7x10⁵ metric tones worldwide are commercially available and 5-10% of dyestuffs are lost in the industrial effluents. Color is usually the first contaminants to be recognized and a very small amount of dye in water (10 mg) is highly visible and effects of aesthetic merit, water transparency and gas solubility of water bodies (Chung and Stevens, 1993). Besides the reduction of environmental pollution, enzymatic decolourization of dyeing. Effluents have recently been shown to enable re-use of treated water in dyeing process (Abadullah et al., 2000).

The White-rot fungi, which degrade lignin biopolymers by producing range of extra cellular enzymes, have been used to degrade and detoxify polyaromatic hydrocarbons, Polychlorinated biphenyl compounds and certain dyes (Cripps, 1990; Paszcynski, 1992; Sparado et al., 1995). The White-rot fungi re the only group capable of decolorizing dyes, and in most cases this is due to the production of lignin peroxidase (Lip) (Ollika et al., 1993); and Manganese dependent peroxidase (MnP) (Heinfling et al., 1998). Schilephake et al., (1999) reported the Laccase mediated dye decolourization. Laccase based decolourization treatment are highly advantageous to bioremediation technologies since the enzyme is produced in large amount and is often produced constitutively of less induction conditions than either Lip and MnP (Eggert et al., 1996 Pointing et al., 2000).

The fungus Phanerochaete chrysosporium are maintained on PDA (Potato Dextrose Agar) medium and were used for the present investigation.

Different azodyes namely, methyl violet and congo red were used for the study of dye decolorization.

· Potato Dextrose agar medium (PDA)

· Minimal base medium

The fungal culture are screened for lignolytic enzyme on Czapek – Dox agar plate for Manganese peroxidase by Davidson (1942) and Pointing (1999), guiacol (5mM), are amended in the basal medium for screening of manganese peroxidase. Plates containing ligninolytic enzyme substrate were inoculated with a mycelial disk and incubated at 30⁰C. Oxidation zone around the mycelial colony indicates the presence of lingolytic enzymes.

The effect of pH on manganese peroxidase production was studied by incubating the culture flasks containing 50ml of Czapek – Dox broth inoculating with five mycelial discs (6mm) at different pH such as 5.0, 5.5, 6.0, 6.5, 7.0 and 7.5. 20 day, the culture filtrate was used for the estimation of manganese peroxidase activity (Wolfenden and Wilson, 1982).

The effect of different sources namely glucose, sucrose, mannitol, glycerol and sorbitol on the production of manganese peroxidase from Phanerochaete chrysosporium was studied. The carbon sources were amended at the concentration of 2% in the basal medium. The pH of the medium was adjusted to 6 before sterilization. The five mycelial discs (6mm diameter) of 5 day old culture were transfered to Erlenmeyer flasks (250ml) containing 50ml of basal medium amended with different carbon sources.

In order to find the suitable nitrogen sources for the maximum production of manganese peroxidase from Phanerochaete chrysosporium the following organic and inorganic nitrogen sources, namely, peptone, yeast extract, ammonium tartrate, ammonium sulphate, potassium nitrate, were amended at the concentration of 0.2% in the basal medium containing 50mM glucose as carbon source.

Lignocellulosic substrate, namely, rice straw and wheat bran, were used for manganese peroxidase production under solid – state fermentation condition. Ten grams ligninocelluosic substrates were moistened (70%) with sugar solution, transferred to the culture bottle (125ml capacity) autoclaved, inoculated with 6 discs of 6 day old mycelium (6mm) and kept at 30⁰C. Sterile water was added every week interval to maintain the moisture content. Uninoculated bottles were served as control. The samples were collected at 5days interval up to 30 days. The content were extracted with 25ml sodium acetate buffer (pH 5.5, 100mM) for overnight ay 4⁰C. The filtrate was centrifuged at 8000 X g for 15 minutes. The supernatant was collected and used as enzyme source to estimate manganese peroxidase production.

A mycelial disc (6mm diameter) was inoculated in petriplates containing the basal medium (2% agar) amended with 50ppm of the dyes and incubated at 30⁰C for 10 days. The diameter of mycelial growth and the zone of dye decolorization were recorded every day.

The mycelium on the plates was scarped gently after 5 days without disturbing medium and the manganese peroxidase substrate in (guiacol 1mM) in sodium acetate buffer was added over the agar medium and left over for 30 minutes in order to find out the involvement of manganese peroxidase. The area of decolorized zone was recorded and indicated the involvement of ligninolytic enzymes.

The basal medium (100ml) was distributed in 250ml Erlenmeyer flasks and sterilized. Five mycelial discs (6mm) of 6-day-old culture was transferred individually and incubated at room temperature (30±2⁰C). The culture was allowed to grow for three days. Then methyl violet and congored were added at the concentration of 50ppm, on the third day. Duplicates of cultures were maintained at the same conditions.

Cultures were harvested at every 24 h interval and monitored for disappearance of respective color of the dyes. The cultures was filtered through nylon sieve and filtered through nylon sieve and filtrate was centrifuged at 8000 x g for 10 minutes.

The protein was determined according to the method of Bradford (1976). Extracellular Manganese peroxidase activity was measured spectrophotometrically as described by Wolfenden and Wilson (1982) with ABTS (2,2’-azinobis(3-ethyl-benzothiazoline-6-sulphonate)) as a substrate. The reaction mixture contains 1.0 ml of 1 mM ABTS in 0.1mM sodium acetate buffer (pH 5.5) and 0.1 ml of culture filtrate. The reaction was monitored by measuring the change in A436 for 3 minutes. One unit enzyme activity was defined as the amount of enzyme that oxidizes 1m mole of ABTS per minute at 25⁰C. The activities were expressed in U/ml.

Seven-day-old culture filtrate obtained was collected and concentrated by lyophilization. This crude concentrate enzyme was investigated for enzyme activity and protein estimation. This sample further analyzed for native PAGE (Davis, 1964)

After electrophoresis, the gel was equilibrated with sodium acetate buffer (pH) 5.0, 100mM) for 5 minutes. Then the gel was incubated with 10mM guiacol (Coll et al., 1993) in sodium acetate buffer (0.1 M, pH 5.0) and kept in dark for 30 minutes for the development of brown color.

After electrophoresis, the gel was washed twice in sodium acetate buffer (0.1 M; pH 5.5) containing 1% (v/v) Triton –X-100 for 30 minutes, and kept in a rotary shaker with gentle agitation for 1 h to remove the SDS. The gel was then equilibrated in the same buffer without Triton X-100. The rest of the procedure was the same as that used for Native PAGE.

The molecular mass of the purified Manganese peroxidase was determined by SDS-PAGE. Purified protein samples were run on SDS – PAGE with concurrent run of standard protein markers consisting of phosphorylase B (97,400D), Bovine serum albumen (66,000 D), Ovalbumen (43,000 D), carbonic anhydrase (29,000 D), soybeantrpsin inhibitor (20,100D) and lysozyme (14,300D) obtained from Genei, Bangalore, India. After separation, the gel was stained with silver nitrate as described by Blum et al., (1987). Manganese peroxidase molecular weight is showed as 4 3kDa on 15% SDS-PAGE.

RESULTS:

Screening of white rot fungi:

The mycelium grown on the plates was photographed on 4^th day and the mycelium or agar plates were scraped out carefully without disturbing the medium and the presence of extracellular oxidizing were examined. Among the three enzymes (such as LiP, MnP and laccase) screened, manganese peroxidase showed positive results in all the plates. The development of brown color zone after incubation (in 10mM Guiacol) indicated the presence of manganese peroxidase.

Optimization of physiological conditions for Manganese Peroxidase production:

Effect of pH on Manganese Peroxidase production from Phanerochaete chrysosporium:

The pH had a significant influence on growth and manganese peroxidase production Phanerochaete chrysosporium. The fungus was able to produce manganese peroxidase between pH 5.0-7.5, maximum activity of 0.89 U/ml was recorded at pH 6 on 7^th day. Maximum protein content (7.2mg/ml) was recorded at pH 5.5 on 7^th day. Thus the optimum pH 6 was selected for further experiments (Table 1and 2).

Table 1. Effect of different pH on extracellular protein content by

Phanerochaete chrysosporium

Days	Protein Content (mg/ml)
Days	pH 5	pH 5.5	pH 6	pH 6.5	pH 7	pH 7.5
2	0.9	1.2	1.6	1.2	1.7	0.9
3	1	1.5	2.1	1.3	1.8	1.1
4	1.9	1.7	3.5	1.5	2.5	1.5
5	5	5.2	5.3	5.9	5.9	5.9
6	5.9	5.2	5.5	6.7	6	6
7	6.8	7.9	7.2	7.1	7	7
8	1.9	1.7	3.5	1.5	2.5	1.5

Table 2 Effect of different pH on enzyme activity by Phanerochaete chrysosporium

Days	Manganese Peroxidase in U/ml
Days	pH 5	pH 5.5	pH 6	pH 6.5	pH 7	pH 7.5
2	0.01	0.22	0.07	0.03	0.62	0.04
3	0.07	0.23	0.08	0.63	0.65	0.05
4	0.08	0.29	0.09	0.64	0.67	0.06
5	0.09	0.3	0.1	0.78	0.68	0.07
6	0.1	0.35	0.12	0.8	0.69	0.09
7	0.11	0.25	0.32	0.85	0.71	0.1
8	0.1	0.28	0.03	0.7	0.67	0.02

Effect of different carbon sources on manganese peroxidase production in Phanerochaete chrysosporium:

Five different carbon sources namely glucose, sucrose, sorbitol, mannitol glycerol were amended in the medium to find out a suitable carbon source for maximum manganese peroxidase production. Among the carbon sources tested glucose was supported the production of manganese peroxidase (0.87 U/ml). Hence, glucose was selected for further studies. The amount extracellular protein content was maximum 6.1 mg/ml on 7^th day in glucose amended medium. The activity of laccase was observed from 3^rd day of the incubation and reached maximum activity of 0.87 U/ml on 7^th day in glucose amended medium and decreased thereafter. The one-way analysis of variance showed that different carbon sources significantly influenced the mycelial growth, extracellular protein and manganese peroxidase production (Table 3and 4).

Table 3. Effect of different carbon source on extracellular protein Content by Phanerochaete chrysosporium

Days	Protein Production (mg/ml)
Days	Control	Glucose	Glycerol	Mannitol	Sucrose	Sorbitol
2	1.5	1.6	1.5	1	1.5	1
3	2	2	2	1.1	1.6	2.3
4	2.4	2.4	2.2	1.5	1.8	2.5
5	5	5.5	5	5.5	5.5	5.3
6	6	6	6	6.2	6	6
7	6.9	7	7	7.2	7	6.2
8	2.4	2.4	2.2	1.1	1.8	2.3

Table 4. Effect of different carbon source on enzyme activity

by Phanerochaete chrysosporium

Days	Manganese Peroxidase in U/ml
Days	Control	Glucose	Glycerol	Mannitol	Sucrose	Sorbitol
2	0.01	0.6	0.08	0.10	0.1	0.01
3	0.1	0.68	0.1	0.25	0.11	0.1
4	0.30	0.72	0.10	0.35	0.2	011
5	0.31	0.72	0.22	0.4	0.3	0.12
6	0.4	0.8	0.28	0.5	0.4	0.2
7	0.5	0.9	0.5	0.6	0.42	0.22
8	0.2	0.7	0.22	0.4	0.2	0.1

Effect of nitrogen source on manganese peroxidase production Phanerochaete chrysosporium:

Five different nitrogen sources namely ammonium tartrate, potassium nitrate, ammonium sulphate, peptone and yeast extract were tested to find out a suitable nitrogen source for maximum manganese peroxidase production. Among these nitrogen sources ammonium tartrate shows, maximum extracellular protein content of 0.6 mg/ml was observed on 7^th day. The activity of manganese peroxidase was observed from 2^nd day onwards and attained a maximum of 1.52 U/ml on day 7 (Table 5 and 6).

Manganese peroxidase production in solid state fermentation (SSF):

Production of manganese peroxidase was carried out in solid-state fermentation using various lignocellulosic wastes. Of the two lignocellulosic waste tested, wheat bran enhanced the mycelial growth and manganese peroxidase production than other substrate. The extracellular protein content was gradually increased upto 20 days (9.0mg/ml). The highest manganese peroxidase activity, 793 U/g substrate, was observed on day 20 in wheat bran. Rice straw showed less manganese peroxidase production (Table 7 and 8, Fig 5).

Dye decolourization in liquid culture:

Dye decolorization of Phanerochaete chrysosporium was studied in liquid culture. In Phanerochaete chrysosporium, enzyme activity was high in congo red but less activity was seen in methyl violet. In contrast, protein was high in methyl violet by Phanerochaete chrysosporium compared to congo red.

Decolorization of two different dyes by Phanerochaete chrysosporium and grown in low nitrogen mineral liquid medium. Decolorization and production of extracellular manganese peroxidase was analyzed.

In Phanerochaete chrysosporium methyl violet induce manganese peroxidase production initially but it was suppressed later. It was able to decolorize 40.6% of the dye shows maximum enzyme activity in 241 U/ml on 5^th day. In Phanerochaete chrysosporium, congored amended cultured was able to decolorize 47% of the dye, maximum enzyme activity was 208 U/ml on 5^th day (Table 9 and 10, Fig 6).

UV - Visible spectrum of dye decolorization:

The dyes treated with the enzyme were scanned in a UV-Visible spectrophotometer. The absorption peak of the dye for methyl violet and congored for Phanerochaete chrysosporium and was compared with the respective control (Figure 1, 2, 3 and 4).

Table 7. Effect of different substrate on protein content by Phanerochaete chrysosporium in solid state Fermentation

Days	Wheat	Rice
5 Days	7.2	2
10 Days	8.5	2.3
15 Days	8.9	8.5
20 Days	9.5	8.5
25 Days	8.5	8.3
30 Days	6.3	7.2

Table 8. Effect of Different substrate on enzyme activity by

Phanerochaete chrysosporium through solid state

Fermentation

Days	Wheat	Rice
5 Days	420	100
10 Days	500	150
15 Days	700	220
20 Days	800	120
25 Days	110	115
30 Days	300	100

Fig 5: PRODUCTION OF MANGANESE PEROXIDASE THROUGH SOLID STATE FERMENTATION

DISCUSSION:

White rot fungi are heterogeneous group of organisms and have the capacity to degrade lignin as well as other wood components. The capacity of degrading lignin is due to the extracellular non-specific and non-stereo selective enzyme systems. The extracellular enzyme system involved in lignin degradation is composed of lignin peroxidase, laccases and manganese dependent peroxidases (Field et al., 1993; Barr and Aust, 1994 a, b; Kuhad et al., 1997). Because the key components of the white rot lignin degrading system are extracellular, the fungi can degrade insoluble chemicals such as lignin and many of the hazardous environmental pollutants.

Researchers have focused mainly Phanerochaete chrysosporium, however the possible potential application of this fungus does not always enable to optimum culture conditions to be fulfilled (Waldner et al., 1988; Field et al., 1993). It may therefore be beneficial to screen a variety of white rot fungi for their ability to degrade xenobiotics under a wide range of environmental conditions. In the present study Phanerochaete chrysosporium is used for manganese peroxidase production.

Methyl violet Congo red

Fig 6: DECOLOURIZATION OF TEXTILE DYES USING MANGANESE PEROXIDASE

Table 9. Effect of different dyes on extracellular protein by

Phanerochaete chrysosporium

Days	Congored	Methyl Violet
3 Days	2.5	2.8
4 Days	3	3.5
5 Days	4	4.5
6 Days	3.8	3
7 Days	2.3	2.5
8 Days	2	2.3

Table 10. Effect of different dyes on enzyme activity by Phanerochaete chrysosporium

Days	Congored	Methyl Violet
3	0.5	0.6
4	0.6	1.2
5	1.2	0.3
6	0.3	0.2
7	0.3	0.2
8	0.2	0.2

The culture condition like incubation period, pH temperature, carbon and nitrogen sources greatly influence the growth and enzyme production of lignin degrading fungi (Keyaer et al., 1978; Jeffries et al., 1981; Bonnarme and Jeffries, 1990).

The pH of the culture medium is critical to the growth, ligninolytic enzyme production and xenobiotics degradation. The optimum pH of manganese peroxidase production, as reported in many white rot fungi, falls between 4.5 and 6.0 (Coll et al., 1993; Fukushima and Krik, 1995; Eggert et al., 1996; Chefetz et al., 1998; Abdulla et al., 2000; Robles et al., 2000; Schliphake et al., 2000). In the present study, pH 6 was found suitable for the maximum growth and manganese peroxidase production of Phanerochaete chrysosporium.

Fig 7: ENZYME ACTIVITY – NATIVE PAGE

Most of the white rot fungi produce lignolytic enzyme in response to carbon, nitrogen and Sulphur limitations (Keyser et al., 1978; Fenn and Krik, 1981; Jeffries et al., 1981 Kaal et al., 1993). Collins and Dobson (1995) reported that carbon at 10g/l enhanced the growth and lignolytic enzyme production on Coriolus versicolor. The most readily usable carbon source by white rot fungi is glucose (Buswell et al., 1995; Fu et al., 1997; Kapdan et al., 2000, 2002). Kapdan and Kargi (2002) reported that cultivation of Coriolus versicolor at 10g/l glucose resulted a better fungal growth. Many authors have reported that glucose at 50mM concentration supported high enzyme production in many white rot fungi (Ruttimann et al., 1992; Fu et al., 1997; Perumal 1997; Abadulla et al., 2000; Schliphake et al., 2000; Soden and Dobson, 2001). In Ganoderma lucidum (Perumal, 1997), glucose at the higher concentration (20 g/l) increase the mycelial growth but lower concentration (10g/l) favored enzyme production. Among the five different carbon sources tested, glucose supported good growth and manganese peroxidase production. In accordance with earlier findings, in present study Phanerochaete chrysosporium shows maximum manganese peroxidase production at higher concentration of glucose 20g/l

Nitrogen source exerts a great influence on the extracellular lignolytic enzyme production of wood rotting Basidiomycetes. In the present study, among the different nitrogen sources Ammonium tartrate favored maximum manganese peroxidase enzyme production. The most widely used nitrogen sources for fungal ligninolytic enzyme production are ammonium salts such as tartrate or chloride (Prouty, 1990; Gogna et al., 1992). Ammonium nitrogen favored high enzyme production in many white rot fungi namely Basidiomycete PMI (Coll et al 1993). Lentinula ewdodes (Buswll et al., 1995) and Pycnoporus cinnabarinus (Eggert et al., 1996; Schliphake et al., 2000). In contrast, Phanerochate chrysosporium shows maximum production of manganese peroxidase when yeast extract was used as nitrogen sources

Fig 8: ENZYME ACTIVITY – SDS PAGE

1- Protein marker

2- Protein marker

3- Manganese peroxidase

Production of lignocelluolytic enzyme on solid substrate is established (Katagiri et al., 1995; Leontivsky et al., 1997) with higher activities of manganese peroxidase over the submerged cultures (Schlosser et al., 1997). The growth and production of manganese peroxidase is on wheat bran and husk exceeded when compared to wood chips and whole grains as intact substrates. In the present study production manganese peroxidase was enhanced by several folds solid state fermentation than submerged cultures. Among the 2 different substrate used, wheat bran highly supported the mycelial growth and manganese peroxidase production than other substrate such as rice straw.

The extracellular lignolytic system of the most extensive studied fungus Phanerochaete chrysosporium, which can degrade over 50 different compounds, has been directly implicated in the degradation process (Bumpus and Bock, 1988; Nagarajan and Annadurai, 1999). Much interest has been shown to study the dye degradation of this fungus but less attention has been focused on other white rot fungus. The present study is focused on extracellular manganese peroxidase production by white rot fungus, the present study focused on extracellular manganese peroxidase production by white rot fungus Phanerochaete chrysosporium an extensive lignin degrade structurally diverse azo dyes since such work has been studied in this species on degradation.

In the present study the dye degradation potential of the fungus Phanerochaete chrysosporium studied on the liquid medium. The fungus was able to decolorize azo dyes. However, the extent of color removal was not consistent with the two dyes. The manganese peroxidase shows positive reaction in the liquid culture. This indicates that the dyes degradation occurred via enzymatic oxidation. Similar observation has been reported on the dye degradation on solid medium by Irpex lacteus and Pleurotus ostreatus (Novotny et al., 2001) and Lentinus edodes (Hatvoni and Mecs, 2002)

The white rot fungi are able to degrade the pollutants present in the soil (Novotny et al., 1999). In the present study, the fungus Phanerochaete chrysosporium able to degrade the azo dyes, due to presence of manganese peroxidase. Thus, this can be applied to remove the dye wastes present in the environment.

CONCLUSION:

· Production of manganese peroxidase from Phanerochaete chrysosporium by solid state fermentation using two substrate such as wheat bran and rice straw have been performed

· Optimization of different pH such as 5.0, 5.5, 6.0, 6.5, 7.0 and 7.5 have been done.

· Optimization of different carbon sources such as glucose, glycerol, sucrose, sorbitol and mannitol have been done.

· Optimization of different nitrogen sources such as potassium nitrate, ammonium tartrate, peptone and yeast extract have been done

· Enzyme activity NATIVE-PAGE and SDS-PAGE have done to determine the molecular weight

· Decolorization of textile dyes such as congored and methyl violet using Phanerochaete chrysosporium have been done

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Received on 10.11.2010

Accepted on 14.12.2010

Research J. Science and Tech. 3(1): Jan.-Feb. 2011: 33-43