The samples of soil were collected from slaughter house, local dairy and domestic waste disposal site. Soil samples (brown color, loose texture, organic matter rich, course, pH 6 - 8) were inoculated @ 2.0 g in 100 ml of nutrient broth (pH 8 - 10) and enrichment was carried out for 48 h at 37˚C under shaking @ 200 rpm (Innova, New Brunswick, USA). Then the enriched broth was appropriately diluted and spread plated on skim-milk-agar (SKMA) which consisted of (each, % w/v): skimmed milk powder (5), peptone (0.1), NaCl (0.5) and agar (2) at pH 8 - 10. The plates were incubated at 37˚C for 48 h and then examined for halo around the colonies as the sign of protease producing ability of the organism.

The bacterial isolates which showed significant proteolytic activity on SKMA plates under high alkaline conditions were selected and subjected to submerged fermentation for protease production. Protease production medium (PPM) consisted of: (each, % w/v) glucose (0.5), peptone (0.5), yeast extract (0.5), K₂HPO₄ (0.4), Na₂HPO₄ (0.1), CaCl₂ (0.01), Na₂CO₃ (0.6) at pH 9. The culture was grown overnight under shaking at 37˚C in PPM and then inoculated into the same medium (PPM) @ 10% (v/v) and fermentation was conducted under shaking at 37˚C for 3 days. Appropriate volume of the fermented broth was centrifuged, periodically, and the supernatant obtained was considered as crude enzyme and was used for assaying the proteolytic activity.

For studying the effect of various carbon source on protease production, glucose of the PPM was replaced with different carbon sources (@ 0.5%, w/v) which were either refined ones (lactose and sucrose) or crude substrates of agricultural origin viz. wheat bran, rice bran or rice husk. Similarly, peptone and yeast extract of the PPM were replaced with either complex nitrogen sources (soybean meal, gelatin), or simple ones (ammonium sulphate, urea) @ 1%, w/v, for examining the effect of nitrogen sources on protease production. Relative activity was determined by considering control as the standard reference.

Protease activity was determined as described previously [4]. Enzyme assay mixture consisting of 1 ml of substrate (1% casein), 0.9 ml of Tris-HCl buffer (50 mM, pH 8) and 0.1 ml of appropriately diluted crude or partially purified enzyme, was incubated at 45˚C for 30 min. The reaction was stopped by adding 4 ml of TCA solution (10%, w/v), and the contents were allowed to stand on ice for 15 min. Then the contents were centrifuged at 10,000g for 10 min and 1 ml of supernatant was taken out in separate test tube and 5 ml of sodium carbonate (0.4 M) and 0.5 ml of Folin-Ciocalteu (FC) reagent was added into it. The absorbance of the solution was measured at 660 nm by spectrophotometer (Perkin-Elmer, Spectrophotometer, λ 35). A standard curve was developed by taking the various amounts of pure tyrosine (10 - 100 µmoles) along with 5 ml of sodium carbonate (0.4 M) and 0.5 ml of FC reagent. One unit of protease activity is defined as the amount of enzyme that releases 1 µmole of tyrosine per min at 45˚C under assay conditions.

Protein assay in the enzyme preparation was carried out by Lowry’s method [14] using bovine serum albumin V (BSA) as standard.

The enzyme produced by fermentation in production medium with glucose as carbon source and urea as nitrogen source was subjected to ammonium sulphate precipitation at various saturation levels of 20% - 100%. The precipitated fractions were dialyzed and analysed for protease activity and protein content. The fraction which showed the highest activity was used for further analysis.

The effect of temperature on the activity of protease was determined by carrying out the enzyme assay at different temperatures viz. 30˚C to 100˚C. The thermostability of enzyme was determined by pre-incubating the enzyme at different temperatures ranging from 30˚C to 100˚C for 30 min (pH 8) and then determining the residual activity.

For determining the effect of pH on protease activity, different buffers (50 mM) used in enzyme assay mixture were: phosphate buffer (pH 7 - 8), Tris-HCl buffer (pH 8 - 9), carbonate-bicarbonate buffer pH 10 - 11, and KClNaOH buffer (pH 12 - 13). For examining the pH stability of the protease the enzyme was pre-incubated with the buffer of appropriate pH for 30 min and then residual activity was assayed. Relative activity was determined by considering maximum activity as the standard reference.

For determining the effect some divalent ions viz. Mg²⁺, Co²⁺, Hg²⁺, Zn²⁺, Pb²⁺, Ca²⁺ and Cu²⁺ on the activity of protease, either of the ion was included in the enzyme assay mixture at final concentration of 10 mM, and activity was assayed. Relative activity was determined by considering control as the standard reference.

All the analytical experiments were conducted in triplicates and results expressed are the mean of three different experiments.

The bacterial isolates which showed considerable zone of hydrolysis on SKMA plates were subjected to submerged fermentation for examining their ability of producing enzyme under alkaline pH (8 - 10). Two isolates D-2 and D-6 (from dairy plant soil sample) showed the maximum proteolytic activity (Figure 1) on skim-milkagar plates as well as under submerged fermentation. Preliminary examination of proteases produced by the isolates D-2 and D-6 showed substantial alkalistability and thermostability. The isolate D-6 was selected for detailed investigation as it yielded higher enzyme titre as compared to the isolate D-2. The bacterial isolate was examined microscopically and found to be Gram-positive spore-former. Detailed identification of the organism

was done using Biolog System (Central Instrumentation Facility, University of Delhi South Campus (UDSC) and the bacterium was identified as Bacillus pumilus and designated as B. pumilus D-6. Protease production has been studied from bacterial isolates by various researchers [4]. B. pumilus CBS, an isolate from Tunisian soil produced protease which has compatibility with laundry detergents, and powerful dehairing, feather-degradation and blood/chocolate stains removal capabilities [1]. B. pseudofirmus was isolated from coastal region of Gujarat which produced halo-alkaline protease [12]. Hmidet et al. [15] isolated several Bacillus spp. from various natural sources (activated sludge reactor, soil near the detergent industry, polluted water from the local slaughter house and from marine water) which co-produced alkaline proteases and thermostable α-amylase. Organic solvent stable protease was reported from B. pumilus 115b isolate from contaminated soils of a wood factory [6]. B. subtilis P13, an isolate from Vajreshwari hot springs was reported to produce an extracellular serine protease that catalyses dehairing of hides as well as feather degradation [16].

Analysis of time-profile vs enzyme production showed that B. pumilus D-6 produced maximum protease titre after 48 h of fermentation, after this the protease activity remained almost constant. Incubation period for maximum protease production has been reported to vary from 24 - 48 h to a few days or weeks depending upon the type of microorganism and the cultural conditions employed [4]. B. pumilus CBS, B. pseudofirmus, and B. subtilis P13 showed maximum protease production after 24 h [1,12,16]. Qadar et al. [17] and Kumar et al. [18] reported 48 h as the optimum time for protease production from Bacillus sp. PCSIR EA-3, and from B. subtilis MTCC 9102, respectively.

Glucose of the PPM was replaced with either refined carbon source (lactose or sucrose) or crude carbon source (rice bran, wheat bran or rice husk) and fermentation was conducted. It was found that lactose and sucrose supported significant level of protease production (68 and 65.3 U/ml respectively) and interestingly even the crude substrates like rice bran, wheat bran or rice husk also gave comparable protease production (61.3, 56.6, 54.6 U/ml respectively) as depicted in Figure 2. This is quite remarkable observation as one of major bottleneck for bulk production and application of enzymes in different industries is their high cost of production. In the present study the organism not only utilized successfully the complex agricultural residues but induced considerably high enzyme production. Lazim et al. [19] reported that mixture of wheat bran and chopped dates serve good

substrate for alkaline protease production from Streptomyces sp. CN902. Although Streptomyces sp. DP2 produced maximum protease when fructose was used as carbon source, however, wheat bran also induced substantial protease production [4]. Vishalakhi et al. [20] reported alkaline protease production from Streptomyces gulbargensis using wheat bran as substrate. B. subtilis MTCC9102 and B. cereus SV1 showed substantial protease production when cultivated on shrimp wastes powder or horn meal as sole carbon sources, respectively [18, 21].

Nitrogen is one of the most essential nutrients as it is the ultimate precursor for protein synthesis, besides it also plays important role in regulating the pH of the medium, and hence may be crucial in maintaining the activity and stability of the enzyme. Synthesis of alkaline proteases by microorganisms is strongly stimulated by the presence of proteins or peptides in culture medium [22]. Utilization of low price nitrogen source must be an efficient criterion for economic production of industrial enzymes. Each of the nitrogen sources employed in the PPM lead to substantially enhanced protease production as compared to that of control (Figure 3). Urea was found to be the best and showed 80% more protease activity as compared to that in control, while ammonium sulphate enhanced enzyme production by 40%, and gelatin and soybean meal did so to the tune of 26 and 24%, respectively. We have previously reported [4] that mustard cake and soybean meal are the most-effective nitrogen sources for protease production by Streptomyces ambofaciens DP2. In most of the studies of protease or other enzyme production, soybean meal has been found to be the best nitrogen source [22]. However, a combination of yeast extract and casein [23] has been reported to

be good nitrogen source. Yeast extract as a nitrogen source has been reported to increase the protease production from Streptomyces sp. CN902 [19]. Peptone was found to be optimum nitrogen source for protease production from a Bacillus subtilis MTCC 9102 [18]. Pillai et al. [16] got enhanced protease production from B. subtilis P13 by supplementation of LB medium with various crude nitrogen sources (skimmed milk, green gram powder, bengal gram powder, milled chicken feathers and soybean meal).

Ammonium sulphate precipitation of the enzyme at different saturation levels showed that maximum activity was present in 40% - 60% salt precipitated fraction. Specific activity of ammonium sulphate precipitated fraction was 19.1 while that of crude preparation was 1.3 indicating that protease is purified by 14.69-fold.

Activity assay of partially purified protease at different temperatures showed that enzyme exerted maximum activity at 50˚C. Substantially high activity was observed at 60˚C (75.9%). But activity-reduction occurred at 70˚C (69%), 80˚C (76%), 90˚C (81.1%) and at 100˚C (83%) as shown in Figure 4. Nevertheless the most remarkable observation about B. pumilus D-6 protease was that enzyme showed certain basal level of activity even at very high temperature (90˚C - 100˚C). Thermostability analysis of B. pumilus D-6 protease at various temperatures for 30 min showed that enzyme was thoroughly stable at 30˚C - 40˚C (residual activity 100%) with slight to moderate decrease at 50˚C (9.1%) and 60˚C (31%). However, at still higher temperatures (70˚C - 100˚C) enzyme lost about half of its activity but still it retained 54% - 55% of

the activity at such high temperatures (Figure 5).

Optimum temperature for different bacterial proteases has been reported to be 50˚C - 75˚C [22]. Thermophilic Geobacillus sp. YMTC 1049 extracellular protease had an optimum temperature of 85˚C and retained activity for 10.0 h at 65˚C [24]. Alkaline protease from Pyrococcus sp. had highest activity at 110˚C [25]. Calcium chloride has been reported to have role in enhancing thermostability of protease [23]. B. cereus SV1 protease showed optimum activity at 60˚C and thermal stability at <55˚C [21]. Alkaline protease from Bacillus lehensis showed activity over 30˚C - 60˚C with optimum at 50˚C [26]. Rai et al. [2] purified alkaline protease from Paenibacillus tezpurensis sp. nov. AS-S24-II by 1.7-fold. The purified protease displayed optimum activity at pH 9.5 and 45 - 50˚C temperature range and exhibited a significant stability and compatibility with surfactants and most of the tested commercial laundry detergents at room temperature. In contrast, Bacillus sp. 17N-1 protease exhibited activity at 14˚C - 33˚C with optimum at 25˚C [7].

For studying the effect of pH on the activity of protease the assay was conducted at varying pH using appropriate buffers. It was observed that the enzyme has astonishingly high ability to work under extremely alkaline pH, and showed 91% - 100% activity over pH range of 7 - 11 (Figure 6) and around 80% of the activity at even higher pH 12 - 13. Results are amazing as the enzyme is capable of working at extremely high pH. Stabilityanalysis of the protease at different alkaline pH (Figure 7) showed that enzyme was thoroughly stable at pH 7 - 9 (residual activity, 94% - 100%), with slight decrease at pH 10 (residual activity 86%). However, further increase in pH caused reduction in the enzyme activity. The residual activity was 64.3%, 54% and 45.6% at pH 10, 11 and 12, respectively. B. cereus SV1 protease showed optimum activity at pH 8.0, pH stability in the range of 6 - 9.5 [21]. Protease from halo-alkaliphilic Bacillus sp. 17N-1 showed activity at pH 6.5 to 8.5 with an optimum at pH 7 [7].

Recombinant Bacillus lehensis protease expressed activity over broad range of pH 8 - 12 [26]. Alkaline protease of Aspergillus clavatus showed activity over a pH range of 6 - 11 with maximum at pH 9.5 [10].

Various ions were included in the enzyme assay reaction mixture at final concentration of 10mM. Ca²⁺ enhanced the activity maximally (82.35%) followed by Co²⁺ and Zn²⁺ (41.17% each) and Cu²⁺ (17%). Mg²⁺ did not cause any effect on the activity (Figure 8). The most striking feature was that enzyme activity remained unfazed even in presence of inhibitors like Pb²⁺ and Hg²⁺ which are considered universal inhibitors of enzyme activity. More than a quarter of all known enzymes require the presence of metal atoms for full catalytic activity [27]. Metal ions can influence the activity of enzymes by multiple ways: they may accept or donate electrons to activate electrophiles or nucleophiles; they themselves act as electrophiles; they may mask effect of nucleophiles to prevent unwanted side reactions; they may bring together enzyme and substrate by means of co-ordinate bonds and may hold the reacting groups in the required three dimensional orientation; they may simply stabilize the catalytically active conformation of the enzyme [27]. Protease activity of Streptomyces sp. DP2 largely remained uninfluenced by the presence of CuSO₄; however, MnCl₂ and PMSF did cause a slight reduction (5% - 6%) in activity [4]. Similar to our results, Bacillus sp. PCSIR EA-3 protease was strongly activated by Ca²⁺ [17]. Ca²⁺ enhanced thermostability of alkaline protease from B. pumilus although other ions like Mg²⁺, Mn²⁺, Co²⁺ , Cu²⁺, Fe²⁺ and Al³⁺ did not show any affect [5].