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.

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 [₇]. The best producing strain identified as Aspergillus oryzae was isolated from spoiled cheese and was used for further studies.

The amount of protein was estimated by the method of Lowry et al [₈] with bovine serum albumin as the standard. The protein content of the immobilized enzyme was calculated by subtracting the amount of unbound protein from the protein originally added.

Activity for alkaline protease was determined spectrophotometrically by the Anson method [₉], 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.

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.

All the purification steps were carried out at temperatures from 0° to 4 °C unless otherwise stated.

The crude enzyme was first saturated upto 30% with solid (NH₄)₂SO₄ and then centrifuged at 5,000 × g for 15 min. The supernatant obtained was further saturated upto 70% with solid (NH₄)₂SO₄ and again centrifuged. The pellets obtained were dissolved in minimum volume of 0.1 M phosphate buffer, pH 6.0 (buffer A), and dialyzed against the same buffer extensively and then concentrated through Amicon Diaflo Ultra-filtration cell using YM-10 membrane.

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 M NaCl), at a flow rate of 40 ml/h. In this step, alkaline protease was eluted at 0.3 M NaCl concentration. The fractions (27-37) showing alkaline protease activity were pooled, concentrated and dialysed against 0.1 M Tris-HCl buffer, pH 9.0 containing 0.2 M NaCl and 1 mM EDTA ((buffer B).

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.

The purity of the enzyme was established by gel electrophoresis at pH 8.4 [₁₀] and the molecular mass of the enzyme was determined by gel filtration on analytical Sephadex G-100 column and by SDS–PAGE [₁₁].

The purified alkaline protease was immobilized in the calcium alginate beads through entrapment by the method of Banerjee et al [₁₂] Bovine serum albumin (5.0 mg) was added to 5.0 ml of enzyme solution containing 2.5 mg proteins. To this 1.5% of sodium alginate was added and stirred gently. The entrapment was carried out by dropping the mixture through a hypodermic needle (No.16) into 50 ml of 2.0% (w/v) CaCl₂ solution. The beads so formed were left for 1 h in calcium chloride solution and then stored in 0.1 M Tris-HCl buffer pH 9.0, at 4 °C.

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).

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.

The K _m ' (apparent) value was determined for the hydrolysis of casein by immobilized alkaline protease. A Lineweaver-Burk plot was drawn between the inverse of different concentrations of casein and the reaction velocity to determine the apparent Michaelis constant, K _m ', for the immobilized alkaline protease.

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.

The purification of the alkaline protease enzyme produced by A. oryzae was simple, economical, efficient, reliable and rapid. When crude enzyme was subjected to 30-70 % ammonium sulfate fractionation, there was 3.34-fold purity with 90.6% yield. Precipitates obtained after ammonium sulfate saturation were dissolved, dialyzed and subjected to cation exchange chromatography on CM Sephadex C-50 column, pH 6.0. The elution profiles for alkaline protease and protein from the cation exchange as shown in Fig 1 have two peaks. The second peak contains the highest specific activity (312.5 U/mg protein). The most active fractions (26 to 37) eluted 0.25 M NaCl gradient from the CM Sephadex C-50 column were pooled and loaded on Sephadex G-100 column (Fig 2), pH 9.0 leading to an apparently pure form of alkaline protease. Enzyme was purified ~29.29-fold with 35.2 % yield having specific activity 750 U mg-1 after Sephadex G-100 gel filtration chromatography (Table 1). However Ogundero & Osunlaja [₁₃] reported 26.2% recovery by using DEAE-cellulose and Sephadex G-200 columns for purification of alkaline protease from Aspergillus clavatus.

Figure 1

Figure 1: Cation-exchange chromatography on CM-Sephadex C-50. [Ammonium sulfate fraction (30-70%, 10 ml) of alkaline protease was loaded on CM-Sephadex C-50 column at pH 4.6. Protein (?---?); alkaline protease activity (?-?). The bound protein was eluted with a linear NaCl gradient (0-1.0 )]

Figure 2

Figure 2: Molecular sieve chromatography of on Sephadex G-100 column [CM-Sephadex pool of DPP IV enzyme was loaded on Sephadex G-100 column equilibrated and eluted with 0.1 Tris-HCl buffer pH 9.0 containing 1m EDTA and 0.2 NaCl. Protein (?---?); alkaline protease activity (?-?).

Figure 3

Table 1: Purification of alkaline protease from

[Casein was used as substrate to measure the activity of alkaline protease as described under ‘Materials and Methods'. The activity units are expressed one µg of tyrosine liberated per min per ml under the standard assay conditions at 37 °C].

The purification fold of purified alkaline protease from Asperigillus oryzae is ~30-fold which is almost similar to that obtained from Nocardiopsis sp and T. koningii [₁₄,₁₅] but this factor is ~3 × higher than the enzyme purified from Bacillus and Pseudomonas sp.[₁₆,₁₇].

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 Bacillus and Pseudomonas spp [₁₄,₁₅] while other workers have reported molecular mass ~48 kDa for alkaline protease from Aspergillus sps [₁₈,₁₉] .

Figure 4

Figure 3: Determination of molecular mass of purified alkaline protease by SDS-PAGE: [Lane 1, Alkaline protease enzyme (40 µg); lane 2, protein markers, (20 µg each of phosphorylase , 97.4 kDa; , Fructose 6 phosphate Kinase, 84 kDa; bovine serum albumin, 68 kDa; Glyceraldehydes 3 phosphate dehydrogenase, 36 kDa; trypsinogen, 24 kDa and ?-Lactoalbumin, 14.2 kDa )]

The different concentrations of each of the tested ions and inhibitors significantly affected the activity of the purified alkaline protease from A. oryzae. This protease was strongly inhibited by PMSF and moderately by trypsin inhibitor, TLCK and TPCK (Table 3). However, it was not inhibited by proteinase inhibitors such as EDTA, 1,10-phenanthroline (inhibitor of metalloproteinases). Neither the sulfhydryl reagents like benzamidine nor the thiol activators like β-ME and cysteine had any effect on the enzyme activity. These findings indicated that a SH-residue is not involved in the expression or regulation of the activity of alkaline protease. Among the metal ions, Hg2+ was found to e the strongest inhibitor whereas Li+ and Mn2+ inhibited the enzyme slightly. However, the enzyme activated by Ca2+ and Mg2+ by 35 % and 19 % at 1 mM concentration, respectively, indicated that the enzyme might require metal ions for its activity (Table 2). These observations indicate that metal-binding region on alkaline protease molecule is important for the regulation of the enzyme activity. These results and particularly strong inhibition by PMSF indicated that the active-centre of alkaline protease contains serine residues. Similar result of PMSF inhibition has also been reported by Hossan et al [₁₉] for protease produced by Aspergillus flavus. Alkaline proteases isolated from different fungal and bacterial sources behaved differently in the presence of metals ions. At a concentration of 1 mM, Fe ²⁺ , Co ²⁺ and Ca ²⁺ increased the activity of alkaline protease from T. koningii, whereas K ⁺ , Na ⁺ , Ag ⁺ and Pb ²⁺ inhibited its activity [₂₀]. Cu ²⁺ strongly stimulated activity of the alkaline protease produced by ABacillus spp [₂₁]. Some metal ions such as Ca ²⁺ , Mg ²⁺ and Mn ²⁺ increased and stabilized the protease activity of both the proteases (AP-1 and AP-2) of Bacillus sp. NCDC 180, and Hg2+ ion (1mM) resulted in maximum inhibition of both the enzymes AP-1 and AP-2 by 74% and 73 % respectively [₂₂]. In case of B. pumilus protease, Ca ²⁺ , Mg ²⁺ and Na ⁺ slightly increased enzyme activity whereas Cu ²⁺ and Zn ²⁺ inhibited the enzyme activity. Similarly Kunert et al [₂₃] observed the stabilization effect of Ca ⁺² ions on the activity of serine protease Alp isolated from Aspergillus fumigatus.

Figure 5

Table 2: Effect of metal ions on activity of alkaline protease from A AWT 20.

[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]

Figure 6

Table 3: Effect of various inhibitors on the activity of alkaline protease from AWT 20.

[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]

Figure 7

Table 4: Immobilization of alkaline protease in the presence of different concentration of sodium alginate in 2.0% CaCl concentrations

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 [₂₄], lipase [₂₅], Teredinobacter turnirae [₂₆] and Saccharomyces cerevisiae [₂₇] cells. There was no significant effect of varying concentration of CaCl₂ on the immobilization of alkaline protease when the sodium alginate concentration was maintained at 2.0% (w/v).

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 [₂₈]. 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 & Dogan [₂₉] have also reported the similar type of result with invertase immobilized in Ca-alginate capsules makes it a suitable candidate for detergent formulations.

Figure 8

Figure 5: Determination of pH stability of free and immobolized alkaline protease. After exposing the free and immobilized enzymes to various pH values for 10 min the residual activity were measured at pH 9.0.

Figure 9

Figure 6: Determination of temperature stability of free and immobilized alkaline protease. The enzyme were first exposed to different temperature for 10 min and then immediately cooled to 4°C The residual activity were determined at 40°C

To investigate further the catalytic activity of the immobilized alkaline protease, the Michaelis constant K _m was determined using casein as substrate. The values for the immobilized (apparent K _m ' =5.0 mg casein/ml) and free form (K _m =3.3 mg casein/ml) of alkaline protease were different. The increase in the K _m ' value caused by immobilization is due to the alginate matrix, which can offer significant resistance to the transport of substrates. The increase in K _m ' has also been reported for glucose oxidase after immobilization within calcium alginate gel [₃₀]. But the V_max value for both form of the enzyme were almost same i.e. 750 ± 2.45 µg tyrosine/min/ml.

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.