Partial Purification Of An Alkaline Protease From A New Strain Of Aspergillus Oryzae AWT 20 And Its Enhanced Stabilization In Entrapped Ca-Alginate Beads
J Sharma, A Singh, R Kumar, A Mittal
alkaline protease, aspergillus oryzae, ca-alginate, immobilization, purification
J Sharma, A Singh, R Kumar, A Mittal. Partial Purification Of An Alkaline Protease From A New Strain Of Aspergillus Oryzae AWT 20 And Its Enhanced Stabilization In Entrapped Ca-Alginate Beads. The Internet Journal of Microbiology. 2005 Volume 2 Number 2.
An alkaline protease was produced and partially purified from a new strain of
Industrial enzymes have seen a spectacular rise in their production in the last three decades. The growth of industrial enzyme market has expanded to nearly 85 enzymes, which are currently in commercial production. With the discovery of a variety of new and more active enzymes, the enzyme market has been forecasted to go upto US $ 1.7-2.0 billion by 2006 .
Proteases, which account for about 60% of total enzyme market and among the most valuable commercial enzymes, are the single largest class of enzymes occupying a pivotal position due their wide application in the industrial processes . Plants, animals and microbial sources are employed for protease production. Microbes serve as the preferred source of proteases because of their rapid growth, the limited space required for their cultivation, and the ease with which they can be genetically manipulated to generate new enzymes with altered properties . Microbial extracellular alkaline proteases are important enzymes and are mainly used in detergents to facilitate the release of proteinaceous stains such as blood, milk, egg and meat. They account for approximately 40 % of the total worldwide enzyme sale.
Though a good number of bacterial alkaline proteases such as subtilisin Carlsberg, subtilisin BPN and Savinase are commercially available, having their major application as detergents enzymes but alkaline proteases of fungal origin offer an advantage over bacterial proteases because (i) the mycelium can be easily removed from the final product by simple filtration, (ii) ability of the fungus to grow on cheaper substrate, (iii) easy immobilization of mycelium for repeated use, (iv) broad range of pH (4-11) and substrate specificity and hence low cost of production. As only few reports are available on the use of fungal proteases in detergent industry, therefore there is a growing need to exploit fungal proteases for commercial exploitation in detergent industry.
For industrial use enzyme must be produced at low cost and should be reused reproduce result with consistent efficiency. To achieve this many techniques for immobilization of enzymes on different types of supports have been developed [3,4]. The immobilization of proteases on solid supports has been widely used in many investigations [5,6]. When a protease is immobilized, enzyme autolysis is minimized. For industrial applications, immobilization of the enzyme in gel or solid supports may offer several advantages such as repeated use of the enzyme, ease of product separation and improvement of enzyme stability [5,6].
In the present study, the alkaline protease produced by
Materials and Methods
Bovine serum albumin (BSA), 1,10-phenanthroline, β-N-tosyllysine chloromethyl ketone (TLCK), β-N-tosylphenylalanine chloromethyl ketone (TPCK), phenylmethane sulphonyl fluoride (PMSF), β-casein, sodium alginate (medium viscosity), Coomassie brilliant blue G and Blue Dextran-2000 ®were supplied by Sigma chemical Co., St. Louis, MO USA. Cysteine, calcium chloride were brought from Hi-Media (Banglore, India). The chromatographic media like Sephadex G-100 and CM-Sephadex C-50 were supplied by Pharmacia (Uppsala, Sweden). Sodium dodecyl sulfate (SDS) was procured from Hi-Media Laboratories, India. Other chemicals used were of highest quality available. Refrigerated centrifuge IEC-25 and table top centrifuge Remi R8C were used for routine centrifugation. Systronics Spectrophotometer 108 was used for recording the absorbance in UV/VIS range. Protein samples were concentrated using Ultrafiltration Amicon Cell Model 8200 having YM10 membrane under compressed nitrogen pressure of 4-5 psi.
Isolation and screening of alkaline protease producing fungi
Proteases producing fungal strains were isolated from different samples such as garden soil, decaying wheat straw, spoiled cheese etc. Proteolytic fungi were screened on skim milk agar medium containing skim milk powder, 100 g; peptone, 5 g and agar, 20 g per litre at pH 8.0. Fungal isolates showing zone of clearance were picked up, purified by repeated streaking on the same medium and finally transferred to PDA slants and maintained at 4 °C. Fungi were further screened for alkaline protease production on modified Reese medium (pH 9.0) containing KH2PO4, 2.0 g; (NH4)2SO4, 1.4 g; MgSO4.7H2O,0.3 g; CaCl2,0.3 g; Urea,0.3 g; trace element solution 1.0 ml; Tween 80, 0.5 ml and supplemented with 0.30 % glucose, 0.5 % casein and 0.05 % yeast extract. Trace element solution contained MnSO4.H2O, 1.56 g; FeSO4.7H2O, 5.00 g; ZnCl2, 1.67 g; CoCl2, 2.00 g per liter . The best producing strain identified as
The amount of protein was estimated by the method of Lowry
Determination of alkaline protease activity
Activity for alkaline protease was determined spectrophotometrically by the Anson method , with a slight modification. Enzyme (0.1 ml) was incubated with 1.0 ml (0.5 % casein) and 1.9 ml 0.1 M Tris-HCl buffer, pH 9.0 at 37 °C for 30 min. and then the reaction was arrested by the addition of 2.0 ml of 5 % trichloroacetic acid (TCA). This mixture was centrifuged and the released amino acids were measured as tyrosine by Lowry method. One unit of alkaline protease activity was defined as the amount of enzyme required to liberate one µg of tyrosine per min per ml under the standard assay conditions.
Alkaline protease production
Two ml spore suspension (10 4 to 10 6 spore/ml) was added to 250 ml Erlenmeyer flask containing 100 ml Reese medium (modified) pH 9.0 supplemented with 0.25% glucose, 0.5% casein and 0.5% yeast extract and the flasks were incubated at 30 °C for 72 h in an incubator (200 rpm) on rotary shaker. The culture medium was centrifuged at 5,000 rpm to remove the fungal mycelia and medium debris; the supernatant was used as crude enzyme solution.
Purification of alkaline proteases
All the purification steps were carried out at temperatures from 0° to 4 °C unless otherwise stated.
Ammonium sulfate fractionation
The crude enzyme was first saturated upto 30% with solid (NH4)2SO4 and then centrifuged at 5,000 ×
Ion-exchange chromatography on CM Sephadex C-50
The dialyzed enzyme resulting from ammonium sulfate fractionation was loaded on CM-Sephadex C-50 column (35 × 2.5 cm) equilibrated with buffer A. After washing the column free from unadsorbed proteins, the bound proteins were eluted by applying a linear NaCl gradient (0.0-1.0
Gel filtration chromatography on Sephadex G-100 column
The concentrated pool of activity obtained above was fractionated on Sephadex G-100 column (120 × 2.0 cm) with eluent buffer B. The fractions eluted from the column were assayed for alkaline protease. The fractions (41-55) having high alkaline protease activity were concentrated and stored at 4 °C and used further to check the homogeneity of the enzyme by electrophoresis and characterization studies.
Polyacrylamide gel electrophoresis
The purity of the enzyme was established by gel electrophoresis at pH 8.4  and the molecular mass of the enzyme was determined by gel filtration on analytical Sephadex G-100 column and by SDS–PAGE .
The purified alkaline protease was immobilized in the calcium alginate beads through entrapment by the method of Banerjee
Effect of pH and temperature on the alkaline protease activity
The effect of pH and temperature on the free and immobilized enzyme was determined under standard assay conditions using casein as substrate. Alkaline protease activity was studied in the pH range from 5.0 to 11.5 for free and immobilized form of enzymes and than their activity was measured at various temperatures (10 o C to 80 °C).
Stability of immobilized alkaline protease enzyme
The stability of the enzyme in buffers at different pH values (0.1 M sodium acetate buffer pH 4.0 to pH 5.5; 0.1 M histidine-HCl buffer pH 6.0 to 7.5; 0.1 M tris-HCl buffer pH 7.5 to 9.0; 0.1 M glycine-NaOH buffer pH 9.5 to 11.5) was measured by incubating 10 beads (0.125 mg enzyme) in 1.0 ml of different solutions at 37 °C. The temperature stability (from 0 to 80 °C) was monitored by incubating the immobilized enzyme at different temperature for 10 min in 0.1 M tris-HCl buffer pH 9.0 and the residual activity was monitored under the standard assay procedure.
Effect of substrate concentration on immobilized alkaline protease
Effect of various metal ions and inhibitors on alkaline protease activity
To investigate the effect of some metal ions and inhibitors on the free form of enzyme activity, the purified enzyme solution was pre-incubated along with various metal ions and inhibitors for 15 min at 37 °C. The concentration of metal ions and inhibitors used was 1 mM and 5 mM, respectively. The residual enzyme activity was measured by adding the substrate and carrying out the enzyme assay under the optimum conditions.
Results and Discussion
Purification of alkaline protease
The purification of the alkaline protease enzyme produced by
[Casein was used as substrate to measure the activity of alkaline protease as described under ‘
The purification fold of purified alkaline protease from
Characterization of partially purified alkaline protease
The apparent molecular mass of the partially purified alkaline protease was estimated to be ~33 kDa as measured on analytical Sephadex G-100 column and SDS–PAGE (Fig. 3). The native enzyme is thought to be a monomer, which is composed of only one subunit. This result is very similar for alkaline protease purified from
Effect of some chemicals and inhibitors on alkaline protease activity
The different concentrations of each of the tested ions and inhibitors significantly affected the activity of the purified alkaline protease from
[Activity of alkaline protease was measured with casein as substrate at pH 9.0. Enzyme was pre-incubated with the respective metal ions for 10 min and then the residual enzyme activity was determined by the standard assay procedure for alkaline protease. The inhibition was taken as 0% when no metal ion was added]
[Activitiy of alkaline protease was measured with casein as susbtrate at pH 9.0. Enzyme was pre-incubated with the respective inhibitor for 10 min and then the residual enzyme activity was determined by the standard assay procedure for alkaline protease. The inhibition was taken as 0% when no inhibitor was added]
Immobilization of alkaline protease enzyme
Table 1 shows the effect of varying concentrations of sodium alginate (1.5-3.5%) on alkaline protease immobilization. The percent-entrapped activity was maximal (~48%) at 2.0% (w/v) of sodium alginate concentration. Leakage of enzyme occurred at 1.5% (w/v) sodium alginate concentrations owing to the larger pores of the less tightly crosslinked gel. But, the amount of entrapped activity is comparatively very low at 2.5-3.5% (w/v) sodium alginate concentrations. It may be because of the high viscosity of the enzyme-BSA-sodium alginate mixture that results less diffusion of high molecular weight of substrates into the alginate beads. Thus, 2.0% concentration of alginate was selected for entrapment of alkaline protease to study its altered physico-chemical behavior. The sodium alginate in the concentration range of 2-3% has been used for the immobilization of keratinase , lipase ,
Effect of pH and temperature on the enzyme activity
The optimum pH value of the immobilized alkaline protease shifted to pH 9.5 from pH 9.0, which was the optimum pH of the free enzyme (Fig 4). This may be due to the anionic nature of the alginate support used for immobilization and change in the microenvironment of the immobilized enzyme. Similar observations were reported in annase-immobilization studies . The stability of immobilized alkaline protease was compared with the free enzyme in acidic and alkaline environment (Fig 5). The free alkaline protease was stable over the pH range 7.0-9.0, whereas the immobilized alkaline protease was stable over a wider range of pH (5.5-10.0). Even at pH 10.5, the immobilized enzyme could retain ~80% activity whereas free enzyme was totally inactivated at this pH. The stability of the two forms was also compared by their incubation for 10 min at various temperatures. At 55 °C, the immobilized alkaline protease retained ~86% of its activity while free enzyme could retain ~10% activity. The thermostability of the enzyme increased very significantly after entrapment (Fig 6). This probably reflects the fact that the entrapped enzyme is not chemically modified but remains in its native form in the gel matrix. Tanriseven
Effect of substrate concentration on immobilized alkaline protease
To investigate further the catalytic activity of the immobilized alkaline protease, the Michaelis constant
In the present study, an alkaline protease has been partially purified using two chromatographies from a new strain of A.
Financial assistance received from Kurukshetra University Kurukshetra in the form of University Research Scholarship to Avtar Singh, Rajesh Kumar and Ashwani Mittal during the course of this work is gratefully acknowledged. The authors also thank Dr. Ashok Aggarwal, Dept of Botany, K.U. Kurukshetra for his help in identification of fungal strain.
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