The indigenously isolated Aspergillus tubingensis was isolated from kusmi forest (Ramgarh forest range, 26^o45’N and 83^o24’20”E INDIA) Gorakhpur, a teak dominating forest by plating method. The strain was deposited at National Institute of Interdisciplinary Science and Technology (NIIST), Thiruvananthpuram, India with accession number NIICC-08155.

All analytical reagents and media components were purchased from Hi-Media (Mumbai, India), Merk BDH (Germany) and SISCO Research Laboratories Pvt. Ltd, (Mumbai, India).

For isolation of Aspergilli, Potato Dextrose Agar and Czapek’s Agar was used (Raper and Fennell, 1965).

Media for fungal growth and spore production

Preliminary screening for protease production from Aspergillus awamori isolates was carried out by skimmed milk agar plate assay on standard media with slight modification (Ventosa et al., 1982; Vermelho 1996). For preparation of 2 liter of media 2.0 g K2HPO₄, _{6.0 g NaNO3, 1.0 g MgSO4, 1.00 g KCl and 40.0 g Agar was dissolved in 1 liter of double distilled water, sterilized by autoclaving after adjusting pH to 7.0. Then 1 liter of autoclaved skimmed milk solution (13.3 g milk powder in 1 liter double distilled water) was mixed and subsequently poured approximately 20-25 ml in autoclaved Petri plates under laminar hood. After incubation for 96 hours at 30^oC, the inoculated plates were observed for the clear zone formed around mycelia margins, for indicating the production of protease. The plates showing clear zone were subsequently photographed using Olympus digital micrographic camera (Model C-5050, Japan).}

The proteases from different isolates of Aspergillus were produced under submerged culture condition using standard media comprising of Czapek solution with final composition of 0.1 g Yeast extract, 0.3 g Peptone, 2.0 g glucose, 3.0 g NaNO3, 1.0 g KH₂PO₄, 0.5 g MgSO₄, 0.01 g FeSO₄, 10.0 g soluble casein, and 0.001 g Thymine HCl dissolved in 1.0 lit. warm double distilled water (Vermelho et al., 1996). The pH of media was adjusted to 7.0. The prepared media was then aliquoted as 50 ml dispensed in150 ml conical flasks prior to sterilization by autoclaving. The above media was then inoculated with Aspergillus spore suspension with final concentration of 10⁶ spores/ml along with penicillin (final concentration of 0.5 mg/ml (Devi et al., 2008). The flasks were incubated at 30⁰C under stagnant condition for 96 hrs. The culture was filtered and centrifuged at 8,000 rpm for 15 min and supernatant thus obtained was used as crude enzyme extract.

The production of protease was quantified by tyrosine methods (Kunitz, 1947). The reaction mixture comprising of 1 ml of 2% casein solution, 1.0 ml of 50 mM buffer (pH 7.0) and 0.1 ml of crude enzyme was incubated for 15 min. at 30⁰C. After incubation, the reaction was stopped by adding 2ml of cold 10% TCA. After one hour, the mixture was centrifuged at 10, 000 g for 15 min. to remove the precipitate. The acid soluble material was estimated spectrophotometrically at 280 nm. Enzyme activity was calculated by measuring mg of tyrosine equivalent released and compared with the standard. One enzyme unit activity was defined as the amount of enzyme required to liberate 1g of tyrosine per minute under experimental conditions.

The protease activity subjected to temperature ranging from 20 -100ºC was monitored by standard enzyme assay while thermostability of the enzyme was determined by incubating the crude enzyme preparation at temperatures ranging from 40-100ºC for 30 minute in a constant-temperature water bath. The residual enzyme activities were then assayed after each treatment. Optimum pH for enzyme activity was determined by subjecting the crude enzyme preparation to three different types of buffers namely citrate buffer (pH range 2-5), Phosphate buffer (pH range 6-9) and Glycine NaOH (pH range 10-12). The assay conditions were similar except variability of pH from 2 to 11 as mentioned above. The Km value was calculated by Michaelis- Menten and double reciprocal graph (Lineweaver & Burk, 1934). The effect of different metal ions namely Ca²⁺, Mg²⁺, Fe²⁺, Co²⁺, Zn²⁺, Mn²⁺, Hg²⁺, Cu²⁺, chlorides and inhibitors like pepstatin, EDTA, DTT and PMSF was studied by taking metal ions in assay buffer ^{at a final concentration of 5 mM.}

Partial purification of crude enzyme preparation was achieved by standard method using ammonium sulphate fractionation (Ogundero and Osunlaja, 1986). The crude enzyme preparation were fractionated in different ranges of ammonium sulfate namely 0-30%, 30-60% and 60-90% saturation. During ammonium sulphate precipitation, the salt was added in small quantity under constant stirring to prevent increase of high local concentrations. The precipitate is then dialyzed against 1.0 mM Phosphate buffer and protein concentration was estimated by standard Lowery method. The purified fractions were then assayed individually for total soluble proteins and protease activity. The approximate molecular weight was determined by subjecting the partially purified enzyme to 12 % SDS PAGE (Laemmli, 1970).