Isolation , identification and screening of potential xylanolytic enzyme from litter degrading fungi

Consortia of litter degrading fungal species were developed from different baiting substrates collected in and around Western ghat forest ecosystem, Coimbatore, Tamilnadu, India. Fifty-three litter degrading fungal species were isolated by nylon litterbag technique. The production of endo-β-1,4-xylanase (1,4-βD-xylan xylanohydrolase, E.C. 3.2.1.8), β-D-xylosidase (1,4-β-xylan xylanohydrolase, E.C. 3.2.1.37) and protease was studied using oat spelt xylan as carbon source. Results showed that all fifty-three fungal species isolated from various litter samples produced fairly good xylanolytic enzyme activity. The xylanase and β-D-xylosidase activity ranges from 4.41 to 132.20 U and 48.72 to 1510.32 U, respectively. Growth was determined in terms of mycelial dry weight, which ranged between 0.209 and 1.047 mg/ml. The protease enzyme activity was from 19.7 to 60.8 U. This is the first report concerning xylanolytic enzyme production by the litter degrading fungi, isolated from litter samples.

Many microorganisms are known to produce different *Corresponding author.E-mail: palaniswamy_m@yahoo.com.types of xylanases; the nature of these enzymes varies between different organisms.Many bacterial and fungal species produce the full complement of enzymes necessary to enable them to utilize xylan as carbon source (Hann and Zyl, 2003).Production of both these enzymes has already been reported in a number of microorganisms, including bacteria (Rani and Krishna Nand, 1996), fungus (Sunna and Antranikian, 1997;Jorgensen et al., 2005), yeasts (Leathers, 1986) and actinomycetes (Nascimento et al., 2003).In the fungal kingdom, a majority of both xylanase and β-D-xylosidase enzymes producing organisms belong to the genus Aspergillus, many of which have been well characterized (Ghosh et al., 1993).Filamentous fungi are interesting procedures of this enzyme from an industrial point of view due to extracellular release of xylanases, higher yield compared to bacteria and yeast and production of several auxiliary enzymes that are necessary for debranching of the substituted xylanase (Haltrith et al., 1996).The attention on the applications of xylan degrading enzymes has led to discover many new enzymes with novel characteristics from various microorganisms (Wang et al., 2003).
In the present

Study site
Mycoflora were isolated from the Siruvani forest of the Western ghat, Coimbatore, Tamilnadu, India (April 2004-March 2005).The Siruvani forest is located at the foothills of Western ghat extension at 76° 73'N and 10° 58'E and above 500 M a.s.l.The climate in this area is typically monsoonic, characterized by three seasons, viz., warm and moist rainy season (July-October), cold winter season (November-February) and dry and hot summer season (March-June).

Isolation of mycoflora
Litter mycoflora were isolated by nylon net litterbag technique (Palaniswamy, 1997).Paddy straw, Coir pith, Eucalyptus globulus and Tectona grandis leaves were air dried and used as baits.Each of the air dried material (10 g) was placed in a nylon net bag (1 mm mesh; 20 x 20 cm size) with colored beads (to identify the materials) and buried in the aerable forest soil at 10 cm depth.For each sample, six replicates were maintained.Samples were collected at regular monthly intervals in presterilised polyethylene bags.The litter samples were powdered and suspended in sterile distilled water (0.1 g/50 ml).This suspension (1.0 ml) was plated onto the Petri plate containing rose-bengal-streptomycin-agar medium.
All fifty-three fungal species were identified and deposited at the Research and Development Culture Collection Center, Karpagam Arts and Science College.The fungal species were sub cultured on rose-bengal-streptomycin-agar slants and maintained in a refrigerated condition.The fungal species were identified by morphological and physiological analysis.

Preparation of spore inocula
Conidial spores were harvested from 7 to 10 day old agar slants by suspending spores in water and filtering through gauze into Erlenmeyer flask.

Fermentation
One percent spore suspension (10 6 spores/ml) were inoculated into 250 ml Erlenmeyer flask containing 50 ml of minimal salt medium (Carter and Bull, 1969) composed (g/l) urea 1.4, magnesium sulphate 0.25, calcium chloride 0.05, zinc sulphate 0.02, manganese sulphate 0.02, copper sulphate 0.005, ferrous sulphate 0.1, sodium sulphate 1.0, ethylene diamine tetra acetate 0.6, sodium dihydrogen orthophosphate 1.56, peptone 7.5, yeast extract 2.5, glucose 5.0 and xylan 10.0.Flasks were incubated for 7 days at 30°C in an orbitory shaker at 150 rpm.After the incubation period, the cultures were filtered over a Whatman No.1 filter paper.The residue was used for growth determination.The filtrate was centrifuged at 10,000 rpm for 20 min at 4°C and the clear supernatant was used as enzyme source.Palaniswamy et al. 1979 Biomass Mycelial biomass was collected on a pre-weighed Whatman filter paper, dried to a constant weight at 60°C and reweighed.The difference in weight denoted the mycelial growth of fungus.

Soluble protein
Soluble proteins were analyzed according to the method of Lowry et al. (1951) after preliminary precipitation with three volumes of 10% TCA.Bovine serum albumin was used as standard.

Mycelial protein
The mycelial protein content was measured in the dry mycelium by Lowry et al. (1951) method as described by Philips and Gordon, 1989.
In addition to xylanase, the organisms produced protease enzyme.The presence of protease may affect the stability of xylanase during extraction and storage (Gessesse and Mamo, 1999).Therefore, the use of appropriate protease inhibitors during extraction or selection of protease-deficient mutant may solve the effect of protease.Agosin et al. (1987) have shown that the protease secreted by the white rot fungus Dichomitus squalens was responsible for the decrease in xylanase activity.Cultures of Aspergillus awamori and Aspergillus phoenicis was found to contain a protease which was capable of inactivating the xylanase enzymes even at 4 o C (Smith et al., 1991).It was therefore considered essential that the protease titers of the selected organisms be as low as possible.Protease enzyme activity was maximum for Aspergillus oryzae (60.8 U) and low for Aspergillus glaucus (19.7 U).Similar findings were reported with Bacillus sp.(Gessesse and Mamo, 1999;Balakrishnan et al., 1997;Avcioglu et al., 2005) and Pichia stipitis (Srikrishna et al., 1997).The results were also in good agreement with production of xylanase enzyme obtained from various bacteria, actinomycetes, yeast, fungi which was studied extensively by many workers (Milagres et al., 1993;Rani and Nand, 1996;Beg et al., 2000;Passoth and Hahn-Hageradl, 2000;Wang et al., 2003;Nacimento et al., 2003;Lama et al., 2004;Avcioglu et al., 2005;Jorgensen et al., 2005;Shah and Madammar, 2005).
In conclusion, this is the first report of the production of xylanolytic and proteolytic enzyme by litter degrading fungi using shake flask batch culture method.Further studies on optimization of xylanase production by using natural lignocellulosic substrates, evaluation of Industrially important thermostable xylanase, neutral xylanase enzyme for food processing industries and using various techniques to produce low titre or free of proteolytic enzyme activity is in progress.

Table 1 .
adopted litterbag technique for developing promising xylanolytic fungal consortia using the various litter samples, Paddy straw, Coir pith, Tectona grandis and Eucalyptus globulus collected in and around study area described in materials and methods.In order to select new potential fungal species, fifty-three fungal species were screened for the production of both xylanase (E.C.Screening of litter degrading fungi for xylanolytic enzyme production