Production and partial characterization of neutral protease by an indigenously isolated strain of Aspergillus tubingensis NIICC-08155

摘要

An indigenously isolated strain of Aspergillus tubingensis NIICC-08155 from soil of teak dominated vegetation of kusmi forest at Gorakhpur was subjected to neutral protease production and its partial characterization. The maximum production of neutral protease i.e. 68.50 U/ml was attained after 96h of incubation. The crude preparation of protease showed reasonable activity at temperature range of 40 to 60 °C with maximum activity at 40 oC and had a maximum enzyme activity at pH 6.4. The Km value of the enzyme was found to be 45.0 mg/ml and the molecular weight as determined by SDS-PAGE was approximately 45 kDa. The enzyme was completely inhibited by DTT and EDTA. Introduction The microbial proteases represent 60% of the worldwide sales value of the total industrial enzymes (Godfrey, 1996; Gupta et al., 2002). Extracellular proteases (generally alkaline and acid proteases) from Aspergillus have high commercial value and find applications in industries like the detergent, diagnostics, food, leather, pharmaceutical, waste management and silver recovery industries (Chiplonkar et al.,1985; Godfrey & West 1996; Rao et al., 1998; Agarwal et al., 2004). Neutral proteases also have potential applications in baking, food processing, protein modification, leather processing industry, animal feeds and pharmaceutical industries. (Monod et al., 1993; Basu et al., 2007; Merheb-Dini et al., 2009; Paranthaman et al., 2009).The members of Aspergillus are highly explored for the production many industrial enzymes (Adrio et al., 2003). There are few reports of neutral proteases from Aspergillus (Rao et al., 1998; Paranthaman et al., 2009), though substantial amount of work has been reported for alkaline and acid protease. This paper reports production and partial characterization of neutral protease secreted from an indigenous strain of Aspergillus tubingensis isolated from soil of teak dominated vegetation of kusmi forest at Gorakhpur. Material and Methods MicroorganismThe indigenously isolated Aspergillus tubingensis was isolated from kusmi forest (Ramgarh forest range, 26o45’N and 83o24’20”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. ChemicalsAll analytical reagents and media components were purchased from Hi-Media (Mumbai, India), Merk BDH (Germany) and SISCO Research Laboratories Pvt. Ltd, (Mumbai, India).Growth mediaFor 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 30oC, 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