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Purification and properties of an extra cellular alkaline protease produced by Bacillus Subtilis (MTCC N0-10110).


Alkaline serine proteases play an important role in the cellular metabolic processes and they are most important group of commercial enzymes and widely used in the detergent, food, pharmaceutical and leather industries [1-5]. Alkaline proteases are one of the important industrial enzymes accounting for nearly 60% of the total world wide enzyme sales. Bacillus subtilis is the main group that is used in international enzyme industry [6]. In addition, proteases from microbial sources are preferred to the enzymes from plant and animal sources since they possess characteristics desired for the industrial applications [7]. The proteases produced from Bacillus subtilis (neutrophillic) and B. licheniformis (alakaphillic) are mainly used in detergent industry [8]. Of late, alkaline proteases have also been used in the recovery of silver from photographic plates [9] and peptide synthesis [10]. The alkaline proteases find their largest use in household laundry with a worldwide annual production of detergents of approximately 13 billion tons [10]. Hence, there is a considerable interest has been made in the production of alkaline proteases from different microbial sources. Recently, large portions of commercial alkaline proteases are available from the Bacillus species [11-14], although the potential use of several fungal sources is now being increasingly realized [3,15-17]. In the present study purification and characterization of an extra cellular alkaline protease produced by Bacillus Subtilis (KHS-1) isolated from the slaughter house soil samples of Anakapalli, Visakhapatnam district, Andhra Pradesh, India, was reported.

Materials and Methods


EDTA(Ethylene Diamine Tetra Acetic acid), PMSF (Phenyl Methane Sulphonyl fluoride), Acryl amide, N,N Methylene-Bis- acryl amide, TEMED(N,N,N',N' Tetra methyl ethylenediamine), APS (Ammonium Per Sulfate), Casein and other chemicals used were of analytical grade and purchased from Himedia Mumbai, Qualigens, India.


KHS-1 was isolated from slaughter house soil sample of Anakapalli, Visakhapatnam district, Andhra Pradesh, India. KHS-1, was identified as Bacillus Subtilis by Biochemical characterization and 16S RNA analysis. Microbial type culture collection (MTCC) number was 10110, given by IMTECH, Chandhigarh, India.

Assay of protease

Protease activity was assayed according to Anson using casein as the substrate [18]. The reaction mixture (7.0ml) contained 5.0 ml of Tris-Hcl buffer pH 8.5, 1.0 ml of 0.5% casein, and 1.0 ml of enzyme. After 30 min, of incubation at 37[degrees]C, the reaction mixture is terminated by addition of 0.5 ml of 10 % TCA and kept in ice for 10 min, and contents are filtered through watts man No-50 filter paper. To 2ml of filtrate, 5.0 ml of 0.2M Alkaline sodium carbonate and 1.0 ml of Folin & ciocalteu's phenol reagent was added and incubated at 37[degrees]C for 30 min and absorbency was measured at 660 nm in UV Spectrophotometer (Shimedzu Model).

Protein Assay

Total protein of the cell free filtrate was determined by the method of Lowry et al., Bovine serum albumin was used as a standard [19]. One unit of protease activity was defined as micrograms of tyrosine equivalents liberated by 1.0 mg of protein at 37[degrees]C in 30 min.

Production of enzyme

Protease production was carried out in medium containing (g/L) casein 2; peptone 5; NaCl 5 and pH was adjusted to 8.5. The production medium was inoculated with 50 [micro]L of the KHS1 overnight culture (1 X [10.sup.8] Cells/mL) and incubated at 37[degrees]C and at 150 rpm in shaking incubator (Remi-Ris-24, Mumbai, India) for 96 h. At the end of incubation, the whole broth was centrifuged at 10,000g for 15 min (Plastocraft Super spin--RV/FM High speed, Mumbai, India) and the clear supernatant was used as enzyme source.

Purification of alkaline protease: All the subsequent steps were carried out at 4[degrees]C. Step 1:

Ammonium Sulfate Precipitation: The protein was precipitated (0-40%) using solid Ammonium Sulfate. The precipitated protein was dissolved in 0.02M Tris Hcl Buffer pH 8.5 and dialyzed over night with three changes using the same buffer.

Step 2:

Sephadex column chromatography: The partially purified protease from the above step was loaded on to a sephadex G-200 column (1.3x74cm) which is previously washed and equilibrated with 0.02 M Tris-Hcl (pH 8.5) and eluted with the same buffer and 3.0 ml fractions were collected. The enzyme activity was found in the fractions from 20 to 25 as shown in the Figure-1. These fractions were pooled and concentrated by lyophilization and used for further studies.



Electrophoresis: Protease activity was detected by Zymography (Activity staining) under non-denaturing conditions was carried at 4[degree]C as described by al., [20]. Activity staining was performed using 7% Acryl amide gel according to the method of Davis. et al (Native PAGE). In this method casein was incorporated in the gel, after performing the electrophoresis. The gel was incubated in the buffer at 37[degrees]C for 30 min and Ezee blue was used for staining the gel Ezee blue binds where substrate casein was present in the gel and leaves the clear portion of the gel which was hydrolyzed by the protease. Cathode and anode were at the top and bottom of the gel respectively. SDS PAGE was run according to the method of Weber K, laemmli [21] under denaturing conditions using Bovine serum albumin, 66 kDa ; Ovalbumin, 43 kDa ; Carbonic anhydrase, 29 kDa ; Trypsin inhibitor, 20.1 kDa and Lysozyme, 14.6 kDa as standard molecular weights.

Characterization of protease activity and stability

Effect of pH on protease activity and stability: The effect of pH on protease activity was measured over a pH range from 5.0-12.0 under standard assay conditions using 0.1M Phosphate buffer (pH 5.0 to 7.0), 0.2M Tris HCl buffer (pH 8.0), and 0.2M Glycine-NaOH buffer (pH 9 to 12). The pH stability of the purified protease was determined by the pre incubation of protease with buffers of different pH for 10h and residual activity was measured.

Effect of temperature on protease activity and stability: The effect of temperature on protease activity was determined by performing the standard assay procedure at a temperature range from 30[degrees]C to 100[degrees]C. Thermal stability was determined by preincubating the protease at temperature of 55[degrees]C, 60[degrees]C and 70[degrees]C for 3 h in the presence of 10 mM CaCl2 and residual activity was measured.

Effect of protease inhibitors and metal ions on protease activity: Protease was preincubated at 37[degrees]C for 30 min with each inhibitor and metal ions namely 5.0 mM, Phenyl Methane Sulphonyl fluoride (PMSF), 5.0 mM Ethylene Diamine Tetra Acetic acid (EDTA), 5.0 mM [beta] [beta]-mercaptoethanol ([beta] -ME) 5.0 mM and [Mg.sup.2+], [Mn.sup.2+], [Ca.sup.2+], [Zn.sup.2+],[Na.sup.2+], [Alc.sup.3+] to the reaction mixture at a final concentration of 5.0 mM and [Hg.sup.2+] and at final concentration of 0.2mM respectively and residual activity was measured at 660 nm.

Compatibility of alkaline protease activity with commercial detergents

Alkaline protease from Bacillus subtilis showed excellent compatibility in the presence of locally available detergents like Henko, Ariel, Rin, Nirma, Surf, Surf Excel in the presence of 10 mM CaCl2 and 0.2 M glycine NaoH buffer pH 10.5 at 55[degrees]C for 90 min.

Dehairing of goat skin

Goatskin was cut in to 5 x3 cm pieces and incubated with the purified protease (5 U/ml) in 0.2 M glycine NaoH buffer pH 10.5 37[degrees]C. The skin was checked for removal of hair at different incubation times.

Destaining of blood

A clean piece of white cloth (5x5) was stained with blood and allowed to dry the cloth. Then was soaked in 2%formaldehyde for 30 min and washed with water to remove excess formaldehyde. The cloth was incubated with the purified protease (5U/ml) and 0.5 gm of Ariel detergent at 37[degrees]C incubation. After incubation time, cloth was rinsed with water for 2 min and then dried. The same procedure was done for the control except incubation with the enzyme solution.

Decompose of gelatinous coating of X-ray films

A piece of x-ray film (2x1 cm) was incubated with the purified protease (5U/ml) and incubated at 37[degrees]C. The film was checked for decomposition of gelatinous coating for different incubation times.


The protease was purified to homogeneity by two step procedure involving ammonium sulphate precipitation and sephadex G-200 gel permeation chromatography. The purification profile of alkaline protease was presented in Table 1. The protease was purified by about 21 fold with 8.6% yield. The molecular weight of the purified protease was found to be 20.5 kDa by SDS PAGE and the activity staining using native PAGE also show a clear white band corresponding to 20.5 kDa. (Figure -2).

The highest protease activity was observed at pH 10.5 using Glycine-NaOH buffer (Figure: 3) and the protease was stable between pH 8.0 and pH 11.0 (Figure -4). The optimum activity observed by the protease at a temperature of 55[degrees]C (Figure -5). The protease was almost 100% stable at 55[degrees]C even after 180 minutes of incubation (Peek. K, et al., 1992) [22] (Figure -6). PMSF completely inhibited the protease activity (Gold, AM, et al.,21+964) [23]. Whereas, [Zn.sup.2+], [Hg.sup.2+], and [Na.sup.2+] slightly inhibited the protease activity. [Ca.sup.2+], P-mercaptoethanol and [Mn.sup.2+] activated the protease activity by 42%, 28% and 26% respectively. These results are similar to protease produced by Bacillus sp. SMIA-2 [24] .Whereas, [Mg.sup.2+], and EDTA slightly activated the protease activity [25] (Table 2). The Protease from Bacillus subtilis showed stability and compatibility with a wide range of commercial detergents at 55[degrees]C in the presence of 10 mM CaCl2. Protease retained more then 50% of the activity with most of the detergents tested even after 90 min of incubation at 55[degrees]C (Table-3).

Incubation of the protease with skin for dehairing showed that after 2-3h incubation of the protease (5U/ml) with goat skin, hair was removed very easily (Figure-7). In case of removing blood stain from cloth, it was seen that the protease enable to remove blood stain very easily with addition of 0.5 gm of detergent (Figure 8). Protease had completely decomposed the gelatinous coating on the x-ray film with an incubation of 24h (Figure-9).

EDTA (Ethylene Diamine Tetra Acetic acid), PMSF (Phenyl Methane Sulphonyl fluoride), Acryl amide, [beta] Merc ([beta] Mercapto ethanol).










The purified protease from Bacillus substilis has shown the optimum pH at 10.5 and the protease was stable between pH 8.0 and pH 11.0. These findings are in accordance with several earlier reports showing that pH optima of alkaline proteases of Bacillus species around 10.0 [26-29]. The important detergent enzymes, subtilisin Novo or BPN [30] also showed maximum activity at pH 10.5 and the protease was stable between pH 8.0 and pH 11.0 like other alkaline proteases [31] and used in liquid detergent preparation. The optimum temperature exhibited by the purified protease was 55[degrees]C. Most of the alkaline protease from different Bacillus species shows the highest activity at 50[degrees]C [32-34]. PMSF completely inhibited the protease activity. PMSF sulphonates the essential serine residues in the active site of the protease and has been reported to result in the complete loss of protease activity [35,36]. Beside pH, stability, a good detergent protease is expected to be stable in the presence of commercial detergents. Protease from Bacillus subtilis showed excellent stability and compatibility in the presence of commercial detergents like Henko, Rin, Nirma, Surf, Surf Excel and Ariel [37].

Protease from Bacillus subtilis has the ability of dehairing of goat skin. Protease showed high capability for removing proteins and blood stains from clothes. Anwar and Saleemuddin, [38] reported usefulness of protease from Spilosoma oblique for removal of blood stains from cotton cloth in the presence and absence of detergents but we believe that this protease is more effective. Protease from Bacillus subtilis also has the ability of decomposing of gelatinous coating of X-ray films. Fujiwara N, et al., [39] reported the use of an alkaline protease to decompose the gelatinous coating of X-ray films, from which silver was recovered.


The molecular weight of the purified protease was 20.5 kDa .The optimum protease activity was observed at pH 10.5 and temperature of 55[degrees]C. Protease activity was completely inhibited by PMSF, it shows that purified protease was a serine protease. Purified Protease has the commercial application of dehairing of goat skin, destaining of blood stains and decomposing of gelatinous coating on the X-ray films which is mainly useful in recovery of silver. So, purified protease can be used in Leather & textile industries and for recovering of silver.


The authors are grateful to GITAM University management for providing facilities required to carry out this work.


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* Ramakrishna D.P.N., Gopi Reddy N., Rajagopal S.V.

Department of Biotechnology, GITAM Institute of Science, GITAM University, Visakhapatnam, 530 045, India

* Corresponding author

E-mail:, E-mail:
Table 1: Purification profile of alkaline protease.

Purification Total Total specific
Step activity protein (mg) activity

Crude 55400 4620 11.9
Ammonium 38425 2050 18.7
Sephadex gel 4800 19. 5 246.1

Purification fold yield %
Step purification

Crude 1.0 100
Ammonium 1.5 69
Sephadex gel 20.6 8.6

Table 2: Effect of metal salts, Inhibitors and metal chelators.

Metal ion / chelator Con (mM) Relative Enzyme Activity

 Control ... 100
 [Mg.sup.2+] 5.0 112
 [Mn.sup.2+] 5.0 126
 [Ca.sup.2+] 5.0 142
 [Hg.sup.2+] 0.2 76
 [Zn.sup.2+] 5.0 82
 [Na.sup.2+] 5.0 96
 [Al.sup.3+] 5.0 89
 PMSF 5.0 00
 EDTA 5.0 108
 [beta] Merc 5.0 128

Table 3: Compatibility of alkaline protease activity with
commercial detergents in the presence of 10mM CaCl2 and 0.2 M
Glycine-NaoH buffer, pH 10.5 at 55[degrees]C.

 Relative Residual alkaline Protease Activity

Time Control Nirma Wheel Henko Surf Surf Ariel Rin
(min) Excel

0 100 100 100 100 100 100 100 100
15 96 90 94 89 94 91 92 84
30 95 86 89 84 82 88 90 80
45 92 80 86 79 78 81 87 76
60 88 75 82 75 74 77 80 72
75 79 62 60 59 64 65 72 60
90 74 53 50 51 52 58 69 53
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Author:Ramakrishna, D.P.N.; Gopi, Reddy N.; Rajagopal, S.V.
Publication:International Journal of Biotechnology & Biochemistry
Date:Oct 1, 2010
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