Background
Proteases or peptidases constitute the largest group of enzymes in bio-industry with a long array of uses. They play an inevitable role in industrial biotechnology, especially in the manufacture of detergents, foods and pharmaceuticals. They differ in properties such as substrate specificity, active site and catalytic mechanism, pH and temperature optima, and stability profiles. Studies relating to such properties are imperative for the successful application of these enzymes in their respective industries (Sumantha et al., 2005). Proteolytic enzymes are classified on the basis of pH as acidic, neutral or alkaline. Alkaline proteases encompass commercially important proteases; they are further categorized as serine proteases, aspartic proteases, cysteine proteases or metallo proteases - depending on their specific catalytic mechanism. Proteases have a large variety of applications, mainly in the detergents, leather processing, silver recovery, medicine, food processing, feed making, and chemical industries, as well as waste treatment (Jisha et al., 2013a). Proteases and amylases are the predominant detergent enzymes, being blended for removing proteins from clothes soiled with blood, milk, sweat, grass, etc., and enzyme-blended detergents are found far more effective than non-enzyme detergents. Proteases hydrolyze proteins and break them down into more soluble polypeptides or free amino acids. Combined effect of surfactants and enzymes facilitate fast removal of bad stains intercalated in the fiber meshes; therefore, detergents with enzymes are far more effectively than non-enzyme detergents. Detergent enzymes must be cost-effective and safe to use, and should display optimal activities at flexible salt concentration, temperature, and pH.

Bacillus thuringiensis Berliner (Bt) is a ubiquitous Gram-positive and sporulating bacterium producing insecticidal crystal proteins (the δ-endotoxin) juxtaposed to the endospores in the bacterium (sporangium) during the stationary phase of its growth cycle (Jisha et al., 2013b). B. thuringiensis subsp. kurstaki (Btk) is shown to produce an extracellular, metal chelator-sensitive protease during the early stages of sporulation (Li and Yousten 1975), while Hotha and Banik (1997) showed that Bt strain H14 produced an alkaline protease in an aqueous two-phase system comprising polyethylene glycol and potassium phosphate. In fact, because of overwhelming focus on Bt-toxin, exploitation of the potentials of Bt for the production of extracellular protease with an industrial perspective was found totally neglected.

Conventionally, commercial production of Bt toxin has been achieved by submerged or liquid fermentation (SmF), or by batch or fed-batch process (Vu et al., 2010), but advantages of solid-state fermentation (SSF) for the production of both primary and secondary metabolites of microbial origin have well been appreciated by many investigators (Benjamin and Pandey 1998; Jisha et al., 2013a). Compared to SmF, SSF received more attention recently, as it uses simpler fermentation medium, requires smaller space, easier to aerate, higher productivity, lower waste water out-put, lower energy requirement, and less contamination (Benjamin et al., 2013). Combination of these strategies can yield higher titers of proteases in the fermentation medium. The product so obtained can be recovered in highly concentrated form, as against the dilute form obtained by SmF. However, only very little is known about the enzymes produced by Bt, which demonstrate industrial potentials.

Our group already reported the efficacy of SSF for the production of δ-endotoxin by Btk on potato flour (Smitha et al., 2013a, Smitha et al., 2015a) or soybean flour (Jisha et al., 2014), and concomitant production of amylase as a by-product with the enhanced production of δ-endotoxin (Smitha et al., 2013b). Based upon this, the present study is focused on the purification and characterization of a detergent stable extracellular alkaline protease produced by Btk upon its growth on soybean flour supplemented solid medium.

Results
The LB supplemented with 30% (w/v) soybean flour showed the maximum production of protease at 12 h incubation; hence, the crude protein (supernatant) obtained from this modified LB medium was used for the purification and characterization of protease.
 
Purification of extracellular protease
The active protease fractions obtained by (NH₄)₂SO₄ fractionation  (60-80% fraction), Viva spin column (below 45kDa fraction) partitioning, and Sephadex G-100 gel filtration unequivocally showed that it contained two proteases with MW 43 and 32 kDa, as judged by SDS-PAGE profile (Figure 1); with 12.8 folds purification and 0.3% yield upon  Sephadex G-100 gel filtration (Table 1).  The distinct peaks of the elution profile (fractions between 8 and 17 of G-100 gel filtration chromatography) also confirmed the existence of 2 fractions (Figure 2).

Initial activity of protease
Initial assay conditions for protease were: 10 mg/mL casein (substrate), 7.6 pH (phosphate buffer) at 37 ℃, and incubated for 20 min. At this condition, the protease active fraction obtained from sephadex G-100 gel filtration showed 2097 U/mLeqv, which was designated as initial activity (for the calculation of fold increase). The unit of activity was expressed in terms of actual LB medium (i.e., U/mLeqv) used for preparing the LB supplemented with 30% (w/v) soybean flour. Theprotease purified from the supernatant obtained from this modified LB medium was used for further studies. The initial activity of protease is the product of activities of Sephadex G-100 gel filtration fraction minus that of LB control.

Characteristics of protease
Effects of pH, temperature, substrate and metal ion concentrations were tested to fix the optimum activity of the purified protease (active fraction obtained by Sephadex G-100 gel filtration).

Effect of pH
For estimating the pH optimum, the concentration of substrate (10 mg/mL casein), temperature (37oC) and incubation period (20 min) were fixed as used for determining initial activity. At these conditions, the protease showed better activity in the range 8.5 to 11.5, with optimum activity at pH 9 (7684.4 U/mLeqv), which was 3.7 folds increase over the initial activity. Interestingly, another peak of protease activity was also obtained at pH 11 with 3.1 folds increase (6455 U/mLeqv) over the initial activity (Figure 3A). Thus, the protease showed broad range stability on the alkaline side. However, for further characterization studies, pH 9 was fixed.

Effect of temperature
For estimating temperature optimum, the concentration of substrate (10 mg/mL casein), pH (pH9) and incubation period (20 min) were fixed as used for determining initial activity. At these conditions, the protease showed better activity (7923 U/mLeqv) at 70 ℃ (Fig. 3B), which was 3.8 folds increase over the initial activity, with comparable activities at 65℃ and 75℃. Protease was more or less stable up to 80℃. At pH 11, the protease showed the maximum activity (7633 U/mLeqv) at 75℃ (Figure 3B); thus, the existence of two temperature optima further indicates that there exist two proteases.

Effect of substrate concentration
Effect of substrate concentration (casein, 0.5, 1, 5, 10, 15, 20 mg/mL) was measured at 70℃ pH 9 and 20 min incubation, i.e., 0.05 to 2%. The maximum activity was 7992 U/mLeqv noticed with 15 mg/mL casein at an incubation for 30 min; which was 3.8 folds increase over the initial activity (Figure 3C).

Effect of metal ions on protease activity
Effect of metal ions (salts) on protease activity was studied under the assay conditions: 15 mg/mL (casein) substrate at 70℃, pH 9 with incubation for 30 min.  The protease activity was enhanced with the addition of Mn²⁺, Ca²⁺ or Mg²⁺, and the maximum activity (11732 U/mLeqv) was obtained in the presence of 2 mM Mn²⁺, which was 5.6 folds increase over the initial activity (Figure 3D).

Thus, the optimized condition for the activity of protease from Btk was: 15 mg/mL (1.5%) casein as substrate, 2 mM Mn²⁺, pH 9.0 and 70℃ temperature for 30 min incubation.

Enzyme kinetics
The Km and Vmax values for Btk protease were calculated using the data obtained for different substrate concentration. The Km and Vmax values were found to be 0.90 mg/mL and 879.3 U/mg, respectively (Figure 4).

Protease inhibition and response to detergents Protease inhibition was studied by adding EDTA, the chelating agent and protein de folding agent β-mercaptoethanol in the reaction mixture. The optimized reaction condition was employed for this experiment. Of the complex compounds, presence of 1mM EDTA or β-mercaptoethanol in the reaction mixture decreased the protease activity to 6% (1972 U/mL eqv) and 15% (1741 U/mLeqv), respectively; in comparison to initial activity (Figure 5A).

At the optimized reaction conditions, the presence of detergents in the reaction system variously affected the protease activity. Protease activity was not disturbed when incubated with different concentrations (0.1, 0.2, 0.4, 0.8 or 1%) of surfactants like SDS and Triton X-100. Presence of SDS or Triton X-100 slightly affected the protease activity; i.e., in the presence of 0.2% SDS or Triton X-100, 12% reduction in activity was noticed, which was in comparison to the activity at optimized conditions (Figure 5B).

Unlike SDS, and Triton X-100, protease showed good stability in the presence of commercial detergents tested (Ariel, Tide, Surf Excel or Sunlight); i.e., the maximum stability showed in the presence of Ariel with activity 4867 U/mLeqv and was over 2.3 folds increase over the initial activity (Figure 5B); in other words 40% of the maximum activity obtained at the optimized conditions. The fold increase is summarized in Figure 6.

Discussion
Employing biphasic fermentation strategy, our group already demonstrated the amylase produced by Btk as a by-product during the process of the production of δ-endotoxin (Smitha et al., 2013a; Smitha et al., 2013b; Jisha et al., 2014; Smitha et al., 2015b). Based upon this biphasic fermentation strategy, a detergent- and thermo-tolerant protease is demonstrated in the present study.

Characterization of enzyme is an important step toward developing a better understanding on the functioning of the enzyme (Yadav et al., 2010). Alkaline protease in the culture supernatant could be purified by the conventional procedures involving fractionation by ammonium sulphate, molecular weight cut-off membrane filtration, and molecular sieving using sephadex G-100 molecular sieving. Precipitation by ammonium sulphate is the conventionally used method for the purification of protein from the crude extract (Bell et al., 1983). In the present study, the specific activity of the protease increased from 138 U/mg (crude protease) to 1766 U/mg, after final step of purification (sephadex G-100), i.e., 12.79 folds purification and 0.3% overall yield. These results indicate the effectiveness of purification method. Various percentage yields and purification folds were reported for proteases from various species of Bacillus: Bacillus sp. K25 with 40% yield and 10.08 folds purification (Mathew 1999) ; Bacillus sp. PS719 with 39% yield and 18.5 folds purification (Hutadilok-Towatana et al., 1999); B. subtilis with 7.5% yield and 21 folds purification (Adinarayana et al., 2003); and Bacillus strain HS08 with 5.1% yield and 4.25 folds purification (Guangrong et al., 2006).  Sephadex G-100 gel filtration gives rise to comparatively pure fractions of the enzyme with a significant increase in its specific activity. A variety of MWs for proteases from Bacillus species was reported: 30.9 kDa protease from a thermophilic Bacillus strain HS08 (Guangrong et al., 2006); 27.0 kDa from B. megaterium ; 75.0 kDa from Bacillus sp. S17110 (Seong and Choi 2007);34.0 kDa from B. thuringiensis (Kunitate et al., 1989); 38.0 kDa from B. cereus KCTC 3674 (Kim et al., 2001); 15.0 kDa from B. subtilis PE-11(Adinarayan and Ellaiah 2002); 34.0 kDa from B. cereus BG1(Ghorbel-frikha et al., 2005); 66.2 kDa, 31.0 kDa and 20.1 kDa from B. licheniformis strains BLP1, BLP2 and BLP3, respectively (Cheng et al., 2006).Thus, the MW of proteases secreted by Bacillus spp. range is between 20 and 75 kDa, while the MW of proteases secreted by Btk were of medium size. Most of the commercially available proteases are also active in the pH range of 8 and 12 (Gupta et al., 2002).

The results obtained in this study indicated that the protease from Btk is fit for use in laundry and dishwashing detergents, which indicates the commercial significance of the protease; because the pH optimum in the present study was 9-11. Similar results were obtained with protease from B. stearothermophilus (Dhandapani and Vijayaragavan 1994), Bacillus sp. strain MPTK 712 (Kumar et al., 2012), Bacillus sp. (Agarwal et al., 2012), and Bt (Kunitate 1989). Alkaline proteases of the genus Bacillus showed an optimum activity and a good stability at higher (alkaline) pH values (Margesin et al., 1992). The optimum pH range of alkaline proteases from Bacillus lies generally between pH 9 and 11, with a few exceptions of higher pH optima like 11.5 (Fujiwara and Yamamoto 1987), 11 and 12 (Kumar and Takagi 1999), 12 and 13 (Takami et al., 1989; Ferraro et al., 1996). In general, all currently used detergent-compatible proteases are alkaline in nature, and optimally acting at high pH (8-12).

Two temperature optima were determined for protease from Btk, i.e., 70 and 75 ℃; the temperature in washing machines (laundry, dishwasher, etc.) is normally less than this temperature. Normally, the optimum temperatures of reported alkaline proteases from Bacillus range from 50 to 70 ℃ (Zhang et al., 2010). Bt exhibited the optimum activity at a broad temperature range with the maximum (70-75 ℃) at alkaline pH (Kunitate et al., 1989). In addition, their thermal stability is the most important parameter regarding their utility in different sectors like detergent industry.

In addition, protease requires divalent cations like Ca²⁺ and Mn²⁺ or combination of these cations for the maximum activity (Kumar and Takagi 1999). Most of the previous studies showed that the role of sodium, calcium and manganese ions in increasing the activity (Adinarayana et al., 2003; Nascimento and Martins 2004), which was similar to the present results. In addition, these cations would enhance the stability of protease produced by Bacillus spp. (Durham et al., 1987). These metal ions may protect the enzyme from thermal denaturation and maintain its active conformation at high temperature by enabling proper folding.

The protease from Btk was characterized further for its Km and Vmax using casein as a substrate, which was 0.9 mg/mL and 879.3 U/mg, respectively. The Km value represents the dissociation constant (affinity for substrate) of the enzyme-substrate (ES) complex. Low values of Km indicate that the ES complex is held together tightly and dissociates rarely before the substrate is converted to product, and Vmax indicates the speed of its catalysis. Using casein as a substrate, (Kaur et al., 1998) reported a Km of 3.7 mg/mL for the protease from B. polymyxa, while Thangam and Rajkumar (2002) reported a Km and Vmax of 1.66 mg/mL and 526 U/mg/min, respectively for the alkaline protease from Alcaligenes faecalis. A Km value of 0.4 toward casein (mg/mL) was reported for the alkaline proteases from B. alcalophilus var. halodurans (Rao et al., 1998). The Km and Vmax were determined to be 0.5 mg/mL and 230 U/mg/min, respectively for the protease from B. sphaericus strain, in which the optimum reaction condition was at pH 10 and 55℃ (Aboul-Soud et al., 2011). The Km and Vmax of protease produced by a haloalkaliphilic Bacillus sp. were found to be 2 mg/mL and 289.8 μg/min, respectively. In another haloalkaliphilic Bacillus sp. AH-6, the respective Km and Vmax were 2.5 mg/mL and 625 U/min (Dodia et al., 2008). The Km (0.7614 mg/mL) and Vmax (2582 U/min) of B. subtilis DKMNR was determined at 70℃ (Kezia et al., 2011b).

The effect of inhibitors on protease activity was examined after the protease was pre-incubated with the inhibitor for 1 h at 70℃. The presence of the chelating agent EDTA and β-mercaptoethanol in the reaction mixture decreased the protease activity, which may be due to the change in the protein conformation induced by these agents. Some proteases are inhibited by metal chelating agents like EDTA, which indicate their metal ion dependency for activity (Steele et al., 1992).

Stability patterns of protease in the presence of surfactants and commercial detergents also disclosed its promising commercial utility in detergent formulations. Detergent-stable proteases have been studied by several groups with varying levels of activity in the presence of different detergents (Kuddus and Ramteke 2009). Most of the manufacturers recommend the use of detergents in the range 0.1 to 0.2% (w/v) for washing purposes, but in the present study, the detergent concentration was 0.7%. Even at this high concentration of detergent, over 40% of activity was retained, which shows the efficiency of the protease from Btk in detergent industry. Detergent stability of an alkaline protease is an important property for its industrial use, as they are currently supplemented in detergent formulations for better washing efficiency.

Briefly, the protease characterized from Btk (both fractions) is best active at alkaline pH and higher temperatures, which makes it suitable for use in detergency and treatment of effluent rich in protease.

Materials and Methods
Source organism and medium
Bacillus thuringiensis subspecies kurstaki (Btk) procured from the Institute of Microbial Technology, Chandigarh, India (strain: BA 83B; MTCC No. 868) was used in this study, which was maintained on the Luria-Bertani (LB)-agar medium. Five µl seed culture (12 h old) was inoculated in 1 mL pre-sterilized modified LB medium, which contained about 6.5 × 107 cfu. The modified LB medium was made by supplementing 30 mg soybean flour (i.e., 30% w/v) per 1 mL LB; and normal LB was used as the control.  The medium was incubated at 37℃ with constant agitation (150 rpm, and initial pH 7.0) in a temperature controlled shaker (Orbitek, India). After 12 h fermentation, the culture was centrifuged (at slow speed 1000 × g for 10 min, 4℃) to harvest the supernatant, as we described previously (Smitha et al., 2013b). The crude supernatant obtained as above was centrifuged at 9,440 × g for 10 min and 5℃ (Plastocrafts/Remi, India), and the clear supernatant obtained so was used a crude protease for further purification.

Purification of protease
The extracellular alkaline protease was purified by the method of Kunitate et al., 1989. The procedure consisted of ammonium sulphate (NH₄)₂SO₄ fractionation, spin column purification by molecular weight (MW) cut-off, and gel permeation chromatography. The purity of the protease was judged by SDS-PAGE.

Ammonium sulphate fractionation
To the supernatant obtained as above, molecular biology grade (NH₄)₂SO₄ was added slowly (by continuous stirring in a cold room) up to 80% saturation (0-20, 20-40, 40-60 and 60-80%). The precipitate formed in each step was collected after centrifugation (9440 × g for 10 min, 4℃), which was dialyzed separately against 0.1M phosphate buffer (pH 7.6) for 24 h at 5℃ with continuous stirring with two buffer changes. Protein concentration and protease activity in the dialyzate were determined at every step.

Spin column purification
The (NH₄)₂SO₄ fraction which showed the highest protease activity was subjected to spin column (Vivaspin 6, Sweden) purification; the polyethersulfone semi- permeable membrane contained in it facilitated the MW cut-off of 45 kDa (lower fraction), and this fraction was used for further purification.

Gel permeation chromatography
After concentration by dialysis, the spin column fraction of the protein was subjected to Sephadex G-100 (Sigma Aldrich, USA) gel permeation chromatography (Riviera, India), which was performed in a cold room.

Electrophoresis
Employing SDS–PAGE, the purity of protease was checked at every stage of purification. SDS-PAGE was performed on a vertical mini 12 % gel (8×7 cm) slab system (BioTech, India); the running voltage for stacking was 50 V and 70 V for resolving (Smitha et al., 2013b). Gel was stained using 0.1% Coomassie Brilliant Blue (CBB) R-250 in 50% methanol and 10% glacial acetic acid. Broad range protein MW marker (Genei, Bangalore) was sued to judge the unknown MW of the protein in the sample.

Native gel electrophoresis
Non-denaturing (native) PAGE was carried out according to the modified method of (Kazan et al., 2005). Electrophoresis was performed on 10% (w/v) gel containing 1% skim milk at 50 V and 100 V, respectively for stacking and resolving gels. After thorough wash in sterile ddH2O, the gel was incubated for 3 h at 37 ℃ in 0.1 M Glycine-NaOH buffer (pH 10.0) for the proteolysis of skim milk protein impregnated into the gel. Subsequently, the gel was stained with 0.2% CBB solution, and destained with 50% methanol and 10% glacial acetic acid mixture. A clear zone on the gel would indicate the presence of alkaline protease activity.

Calculation of Km and Vmax
The Km and Vmax values were calculated using the effect of casein on enzyme activity using the software Hyper 32 and Graph pad prism. Protease activity (U/mL or U/mLeqv) =     Where, ΔE = absorbance at 540 nm, Vf  = final volume of reaction mixture including DNS, Vs = crude supernatant (mL) containing cellulase used, Δt = incubation time for of hydrolysis, ∑ = extinction coefficient of glucose (0.0026), d = diameter of cuvette.

Characterization of protease
The protease active fraction obtained from Sephadex G-100 chromatography was used for various assays. Effects of pH, temperature, substrate concentration, different metal ions, inhibitors, surfactants and detergents on the activity of protease activity were studied.

Effect of pH on the protease activity and stability
Effect of pH on protease activity was tested using buffers of different pH ranging from pH 7 to 12. Stability of the enzyme at various pH values was studied by pre-incubating the enzyme in buffers of different pH (7-12) for 1 h; buffers used were 0.2 M sodium phosphate (pH 7-8), 0.2 M glycine-NaOH (pH 9-10) and 0.2 M NaOH-Na2HPO4 (pH 1-12). Casein (10 mg/mL) was used as substrate, and incubated for 20 min incubation at 37℃, i.e., with changing pH (substrate concentration and temperature fixed).

Effect of temperature on the enzyme activity and stability
Effect of temperature on the enzyme activity was determined by incubating the reaction mixture at different temperatures (60-80℃). To determine the temperature stability, the enzyme was pre-incubated at different temperatures (60-80℃) for 1 h, and the activity was assayed under standard assay conditions (10 mg/mL casein as substrate in phosphate buffer (pH 9) for 20 min incubation, i.e., with changing temperature (substrate concentration and pH fixed).

Effect of substrate concentration
Protease was incubated (from 5 min to 60 min at 70℃) in phosphate buffer (pH 9) with different concentrations (0.5, 1, 5, 10, 15, 20 mg/mL) of casein, i.e., with changing substrate concentration (pH and temperature fixed).
 
Effect of metallic salts
Protease was pre-incubated for 1 h with various concentrations (1, 2 and 2.5 mM) of different metallic salts (Hg²⁺, Zn²⁺,  Ca²⁺, Cu²⁺, Fe²⁺,  Mg²⁺, Mn²⁺,  Na²⁺ and K+), then the activity was measured under the reaction conditions; 15 mg/mL casein as substrate for 30 min at pH 9 and 70℃ (i.e., the optimized conditions).

Inhibitors on protease activity
Protease was pre-incubated for 1 h with varying concentrations (1-30 mM) of different protease inhibitors like ethylene-diamenetetraacetic acid (EDTA), β-mercaptoethanol, and then assayed for protease activity at standardized conditions.
 
Effect of surfactants and commercial detergents
Efficacy of protease from Btk as an additive in detergent was determined by testing its stability with surfactants, and also in commercial detergents. The protease was incubated with different concentrations of surfactants like SDS (0.1, 0.4, 0.8, and 1%), Triton X-100 (0.1, 0.4, 0.8, and 1%) for 30 min, and the activity of the protease was measured by the standard assay procedure. Stability of the protease in the presence of commercial detergents was also tested by incubating (1 h) it with measured quantity of the protease with different commercial detergents  such as Ariel, Tide, Surf excel or Sunlight. The activity was measured by standard assay procedure and compared with the control. To inactivate the enzyme in the commercial detergent, aqueous solution of detergent was initially heated at 100 ℃ for 60 min. The reaction conditions for the protease from Btk were: 15 mg/mL casein as substrate for 30 min at pH 9 and 70℃ (i.e., the optimized condition), and 0.7% inactivated detergent (to stimulate washing condition).

Statistics
For accuracy, all experiments were repeated 3 times. Microsoft Excel 2007 was used for calculating the protease activity and standard error. Adobe Photoshop CS Version 8 was used to set the figures. The Km and Vmax values were calculated using the effect of casein on enzyme activity using the software Hyper 32 and Graph pad prism.

Conclusions
Proteases associated with toxicity are reported from Bt by various authors; but this is the first report on a protease from Bt showing its utility in industry. Normally, the supernatant in the culture medium is discarded after extracting the δ-endotoxin and endospore. By this biphasic fermentation strategy, the supernatant in the medium was collected during the early phase of the growth of Btk, from which the protease was purified. Removal of free solution from the medium not only enhanced the production of δ-endotoxin as already demonstrated, but the harvest of valuable protease as by-product as well.

Author’s Contribitions
SB designed and prepared the manuscript, VNJ did the experiments, SS and PP set the reference and figures, RBS collected the literature.

Acknowledgements
JVN is grateful to the University Grants Commission, Government of India for granting Rajiv Gandhi National Research Fellowship. The support rendered by National Institute of Technology, Calicut for taking the SEM images is thankfully acknowledged.

Conflict of Interest
The authors declare that there exist no competing financial or other interests.

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