Production and partial characterization of neutral protease by an indigenously isolated strain of Aspergillus tubingensis NIICC-08155
V Morya, D Yadav
aspergillus tubingensis, indigenous isolate, neutral proteases, teak forest
V Morya, D Yadav. Production and partial characterization of neutral protease by an indigenously isolated strain of Aspergillus tubingensis NIICC-08155. The Internet Journal of Microbiology. 2009 Volume 8 Number 1.
An indigenously isolated strain of
The microbial proteases represent 60% of the worldwide sales value of the total industrial enzymes (Godfrey, 1996; Gupta
The members of
Material and Methods
The indigenously isolated
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
Media for fungal growth and spore production
Preliminary screening for protease production from
The proteases from different isolates of
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 1g 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 and characterization
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).
Results and Discussion
A total of ten indigenously isolated strains of
The optimum temperature of the neutral protease was found to be 40 oC (Figure 2) while thermal stability was up to 60 oC for 30 minutes, showing similarity with earlier reports (Monod et al., 1993; Basu et al., 2007; Sumantha et al., 2006). The optimum pH found was 6.4 though the enzyme showed reasonable activity in the pH range of 3.0 to 9.0 (Figure 3). The pH optima of most of the neutral protease reported in literature also supports this finding (Monod et al., 1993; Basu et al., 2007; Merheb-Dini et al., 2009). Optimum substrate concentration for maximum enzyme activity was determined in terms of Km using casein as substrate. The Km values were interpreted from Line Weaver Plots. The Km values (Figure 4 and 5) for neutral protease from
The influence of various metal ions and inhibitors on enzyme activity was studied (Figure 6). Among the metal ions tested, Mn2+, Cd2+, Co2+ and Mg2+ were insignificant as they do not influence the enzyme activity (Basu et al., 2007; Merheb-Dini et al., 2009 ) while Zn2+, Cu2+ and Co2+ showed significant inhibitory effect (Figure 6). The effect of inhibitors and detergents on enzyme activity of the partially purified protease was also studied (Figure 6). The enzyme retained its activity in the presence of PMSF and pepstatin while it was completely inhibited by 0.1 mM EDTA (Figure 6) as reported in literatures (Monod et al., 1993; Basu et al., 2007; Merheb-Dini et al., 2009). Enzyme activity and stability in presence of some available commercial detergents was studied with a view to exploit the enzyme in detergent industry. The enzyme retained its activity in the presence of detergents like Tween, Triton X-100 and DMSO (Figure 6). However, the enzyme activity was drastically reduced when subjected to SDS detergent. The enzyme activity was completely inhibited in the presence of 0.5 % 2-Mercaptoethanol and DTT (Figure 6).
A summary of purification steps for neutral protease from
The partially characterized neutral protease from
The authors are thankful to Head, Department of Biotechnology, D. D. U. Gorakhpur University, Gorakhpur, India for providing infrastructural facilities. One of Author V. K. Morya is thankful to CSIR, New Delhi for financial assistance as Senior Research Fellowship.