Characterization and immobilization of partially purified alkaline protease extracted from rhizospheric soil bacterium , Bacillus megaterium strain EN-2 and Bacillus subtilis strain EN-3

In this study, extracellular alkaline protease producing bacterial isolates EN-2 and EN-3 from the agricultural soil of C.R.C. Pantnagar were identified as Bacillus megaterium strains EN-2 and Bacillus subtilis strain EN-3 on the basis of 16S rDNA gene sequencing. During kinetic characterization, optimum pH for EN-2 and EN-3 protease activity was 10 and 9, respectively. While optimum temperature, for maximum protease activity in both isolates, was 50°C. The crude extracellular alkaline protease from isolates EN-2 and EN-3 were partially purified using ammonium sulphate fractionation and dialysis to 1.50 and 1.42 fold with 53.77% and 42% recovery respectively. The observed values of Vmax and Km for protease from isolate EN-2 were found to be 11.57 U/ml and 17.442 mg/ml, while for EN3 protease these were 42 U/ml and 10.62 mg/ml, respectively. The partially purified enzyme from both bacterial strains was then immobilized in sodium alginate beads with maximum immobilization efficiency at 3% (w/w) and some change in their kinetic properties. The immobilized alkaline protease from EN-2 and EN-3 showed their maximum protease activity at pH 9 and 10, and temperature 60 and 50°C, respectively. Due to these properties, isolated extracellular alkaline proteases from the two strains are ideal choice for application in detergent formulation, leather and food industries.


INTRODUCTION
Proteases (EC 3.2.21.24) are the single class of enzyme which occupies a pivotal position with respect to their applications in both physiological and commercial fields.These are degradative enzymes which catalyze the total hydrolysis of proteins by the cleavage of peptide bonds.Microbial alkaline proteases dominate the worldwide enzyme market, about 70% share of the detergent (Johnvesly and Naik, 2001).Among the various proteases, bacterial proteases are the most significant when compared with those of animals and plants (Gupta et al., 2002).This enzyme accounts for nearly 60% of the total worldwide enzyme sales (Adinarayana et al., 2003;Beg et al., 2003).There are acidic, alkaline and neutral proteases, but of all these, alkaline proteases are employed primarily as cleansing additives (Ward et al., 1995;Nehra et al., 2004).Alkaline protease of microbial origin possess considerable industrial potential due to their biochemical diversity and wide applications in tannery and food industries, medicinal formulations, detergents and processes like waste treatment, silver recovery and resolution of amino acid mixtures (Agarwal et al., 2004;Devi et al., 2008).One of the major drawbacks affecting the stability at alkaline pH of enzymes recovered from thermophiles is that enzymes from alkalophile confer stability in a wide pH range but are usually thermolabile (Griffin et al., 1992).One of the strategies to increase the enzyme stability and reusability is the immobilization.Some of the more significant advantages of the immobilized enzymes over their soluble counterparts includes enhanced stability under extreme conditions of temperature, pH and organic solvents, recovery and reuse (with their subsequent applicability in continuous processes) and in the case of proteases, removal or reduction of autodigestion (Quirega et al., 2011).
Within the different biopolymers that may be employed in such processes, Alginate is one of the most frequently used owing to that the immobilization is carried out under very mild conditions (physiological pH and temperature) without causing any deleterious alteration of the enzyme.Alginate beads have the advantages of being nontoxic, high biocompatibility and their inability to reswell in acidic environment, so acid sensitive compounds (drugs, enzymes, etc.) incorporated into the beads would be protected from gastric juice.Therefore, it is used as an entrapment matrix for cells and enzymes in pharmaceutical and food industry (Smidsrød et al., 1990).
With respect to the factors affecting culture conditions, productivity and properties of alkaline protease, purifying and characterizing this enzyme through kinetic studies by studying the effect of varying pH and temperature as the factors affecting their activity was considered significant.
In the present study, we aimed to purify and immobilize extracellular alkaline protease in alginate beads from the two agriculture soil bacterial isolates screened positive for protease production and the factors affecting their activity to present potential and possible application for industrial purposes were study.

Material, bacterial strain and growth conditions
Bovine serum albumin (BSA), and CaCl2 were purchased from Sigma-Aldrich India.Sodium alginate, glycine and Tris-HCl were supplied by Qualigen.Ammonium sulphate, Luria Broth (LB) media, and Nutrient Agar media were procured from HiMedia Laboratories, India.Other chemicals of interests used were of analytical grade.Previously isolated bacterial strain in our laboratory was taken following study of Pathak (2010).The strain was cultivated in Luria Broth (LB) media.

Screening and production of proteases
Isolated bacteria from soil samples were screened for the production of protease enzyme.Skim milk agar media (Adinarayana et al., 2003) was used to screen bacteria for protease production.For the observation of protease production, bacteria were inoculated on skim milk agar plates containing 10% (w/v) skim milk, 2% agar and 0.5% NaCl.Plates were incubated at 32°C for 48 h.Clear zones of skim milk hydrolysis around the colonies gave an indication of proteolytic activity.

Partial purification of alkaline protease
Protease production was achieved by cultivation of strain in Luria Broth (LB) media.All the purification steps were performed at 4°C.Crude alkaline protease in the cell free supernatant was precipitated by adding ammonium sulphate upto 70%.The precipitates were separated by centrifugation at 10,000 rpm for 15 min and resuspended in 0.1 M phosphate buffer (pH 8) and dialyzed against the same buffer overnight with two buffer change to concentrate the enzyme.

Biochemical analysis
The proteolytic activity was determined by the modified Kunitz method using casein as a substrate (Kunitz, 1947).The supernatant of centrifuged (8000 rpm for 10 min) overnight culture broth served as the crude enzyme source.The crude enzyme (0.5 ml) was mixed with 1 ml of 2% casein dissolved in 0.1 M phosphate buffer (pH 8) and was incubated at 37°C for 30 min.The reaction was stopped by adding 3 ml of 1% trichloroacetic acid.The reaction mixture was centrifuged at 8000 g for 10 min.The absorbance of supernatant was measured at 280 nm.Amount of tyrosine produced is calculated from tyrosine standard curve.One unit of protease activity is defined as the amount of enzyme that released 1 µg tyrosine/ml/min at assay condition.All experiments were carried out in triplicate.The activity of immobilized protease was determined using procedure given above except 0.2 g immobilized enzyme taken for assay.Bradford method was used for measuring the total protein by using bovine serum albumin (BSA) as the standard (Bradford, 1976).The protein concentration was estimated by observing the absorbance at 595 nm.
The calculation of Km and Vmax was done on the basis of Line-Weaver Burk Plot constructed by plotting the reciprocals of substrate (Casein) concentration on X-axis, and reciprocals of enzyme activity on Y-axis.

Enzyme immobilization
Partially purified protease in suitable amount was immobilized by dropping a 3% sodium alginate solution into a 0.25 M CaCl2 solution with continuous stirring.Curing of the beads were done for 4 h in CaCl2, washed several times with a 0.03 M CaCl2 solution until no protease activity was observed in the final washing and stored at 4°C in the same solution prior to use.A similar method was followed for the preparation of control beads without enzyme (Betigeri and Neau, 2002).

Effect of pH and temperature on free and immobilized extracellular protease activity
The optimum pH for protease activity was studied over a pH range of 3 to 12. Citrate buffer (pH 3-5), phosphate buffer (pH 6-8) and glycine-NaOH buffer (pH 9-12) were used to determine protease activity.The protease activity at different pH was determined.The optimum temperature for protease activity was determined by incubating the reaction mixture over the temperature range of 30-80°C at optimum pH.

Effect of pH and temperature on free and immobilized extracellular protease stability
The stability of the enzyme was determined by pre-incubating the enzyme for 1 h at 37°C with buffers of pH ranging from 3 to 12.After incubation, the residual enzyme activity (%) was measured at optimum pH.The thermal stability of the enzyme was studied by pre-incubating the enzyme at different temperature ranging from 30-80°C for 1 h at optimum pH.

Identification of bacterial isolates
Identification based on 16S rRNA gene sequence was also done.Briefly, genomic DNA from selected isolates was extracted and PCR amplification of 16S rRNA gene was carried out by using the primers: rDNA-1A (5'-AGA GTT TGA TCC TGG CTC AG-3') and RDNA-1B (5'-AAG GAG GTG ATC CAG CCG CA-3').The PCR was done as follows: a hot-start of 94°C for 3 min followed by 35 cycles of 94°C for 1 min, 54°C for 1 min, 72°C for 1.5 min, and a final extension for 10 min at 72°C (Kumar et al., 2013b).Amplified PCR products were purified with QIAquick Gel Extraction kit (Qiagen, Germany) and sequenced in an automated DNA sequencer (Applied Biosystems 3730) at DNA Sequencing Facility, University of Delhi (South Campus), New Delhi, India.The sequences obtained were compared with sequences in the GenBank database from the National Centre for Biotechnology Information (NCBI) using blastn program (http://blast.ncbi.nlm.nih.gov/) and then deposited in GenBank under accession number of JN642548, and JN642549 for EN-2 and EN-3 strains, respectively.Evolutionary analyses were conducted in MEGA5 (http://www.megasoftware.net/).

Screening for protease production and determination of protease activity
Bacterial strains EN-2 and EN-3 showed positive results for the production of protease by forming the zone of lysis around their colonies in skim milk agar plate.It was also confirmed by protease assay in production media in which EN-2 and EN-3 showed the protease activity (90.60 and 80.66 U/ml, respectively).

Identification of bacterial isolates
Bacterial isolates from rhizospheric soil of CRC, Pantnagar, were found to be Gram positive and rod shaped.Analysis of 16S rDNA sequence of isolates EN-2 and EN-3 with available 16S rDNA sequences in the NCBI database with 99% sequence similarity revealed identification of isolates as Bacillus megaterium strain EN-2 and Bacillus subtilis strain EN-3.The 16S rDNA sequence were submitted to NCBI GenBank database with Accession no.JN642549 for Bacillus megaterium strain EN-2 and Accession no.JN642548 for and B. subtilis strain EN-3.The phylogenetic tree of strain EN-2 and EN-3 16S rDNA sequences with 16S rDNA sequences of some protease producing isolates is given in Figure 1.

Partial purification and kinetic characterization of alkaline protease
The crude extracellular alkaline protease extracted from isolates EN-2 and EN-3 were partially purified by ammonium sulphate saturation (70%) followed by dialysis which gave 1.50 fold purification with recovery of 53.77% for EN-2 and 1.42 fold purification with recovery of 42% for EN-3 (Table 1).
On the basis of double reciprocal plot of enzyme kinetics, the value of V max and K m values for the partially purified extracellular enzyme from strain EN-2 were 11.57U/ml and 17.442 mg/ml, respectively.In the same way, V max and K m values of EN-3 protease were 42.00 U/ml and 10.67 mg/ml, respectively (Table 2).According to the above result we can say that the isolated protease from EN-3 has more affinity for its substrate than EN-2 because of its lesser K m value.Comparable result was reported for B. subtilis strain EN-4 by Kumar et al. (2013a) where the V max value of extracellular protease was 22.2 U/ml.

Immobilization of protease
By varying the concentration of sodium alginate solution (1-4%), the immobilization process of alkaline protease within calcium alginate beads was carried out.High yields of immobilization were defined as the ratio of the activity of immobilized enzyme to the activity of the free enzyme used.The percentage (%) immobilization (~55% for EN-2 and 94% for EN-3) was highest at 3% sodium alginate concentration (Table 3).Leakage of enzyme occurs at lower concentration of sodium alginate due to longer pores of less tightly cross linked gel which resulted in less immobilization efficiency.An approximate 5-40% loss of enzyme activity was noticed in all cases due to denaturation of enzyme during gel formation.3% sodium alginate concentration was found to be optimum.Although a good consolidation of the beads was obtained at 4% (w/w) sodium alginate, but a lower activity was observed.This decrease in immobilization yield with increasing sodium alginate concentration may be due to a higher density of sodium alginate at higher percentage and it did not allow the trapped enzyme to come out easily.Sodium alginate in concentration of 2-3% was used by several workers for enzyme immobilization    (Farag and Hassan, 2004;Mittal et al., 2005).

Effect of pH and temperature on activity and stability of free enzyme
For pH optimum, substrate solution was made in different buffers (pH 3.0-12.0),that is, citrate buffer (pH 3-5), phos- phate buffer (pH 6-8) and glycine-NaOH buffer (pH 9-12).Extracellular crude protease of EN-2 and EN-3 showed its maximum activity at pH 10.0 (90.60 U/ml) and 9.0 (80.66 U/ml), respectively (Figure 2).Incubation of extracted enzyme from both strains for 60 min in different  buffers before enzyme assay indicated the stability of the enzymes from pH 8 to 11 (Figure 3).Similar results have been reported for alkaline protease from Bacillus circulans MTCC7906 (Jaswal and Kocher, 2007), Bacillus clausii (Kumar et al., 2004) and Bacillus sp.P-2 (Kaur et al., 2001).Thus, both EN-2 and EN-3 showed moderate protease activity at alkaline pH in cell free extract that may further be increased by varying culture conditions, gene expression and other genetic manipulations.
The effect of varying temperature on the enzyme activity was determined in the range of 30 to 80°C and optimum temperature for the maximum protease activity was found to be 50°C for both the strains (Figure 4).The isolated enzymes from both strains were stable at the broad range of temperature between 45 to 70°C (Figure 5).Thus, the isolated alkaline protease from both strains was mesophillic in nature.For Bacillus sp.SSR1 (Singh et al., 2001) and Bacillus clausii (Joo et al., 2003), the temperature for the maximum protease activity were 40 and 45°C, respectively.The results are in accordance with the study of Nascimento et al. (2004), where extracellular protease from thermophilic Bacillus sp.SMIA  2 was stable for 2 h at 30°C, while at 40 and 80°C, 14 and 84% of the original activities were lost, respectively.

Optimum pH and temperature for immobilized alkaline protease activity
Optimum pH and temperature for immobilized alkaline protease from isolate EN-2 were 9.0 and 60°C, respectively (Figures 6 and 7) and for the enzyme of EN-3 the pH and temperature for maximum protease activity were 10 and 50°C, respectively (Figures 6 and 7).The values of optimum temperature and pH of immobilized alkaline protease were quite different from free enzyme.This was due to some change in enzyme stability and substrate availability which was because of its immobilization.
In general, all detergent compatible enzymes should be alkaline and thermostable in nature because of high pH (8-12) and temperatures (50-70°C) in the laundry environments.Though the present protease is optimally active at 50°C, it retains more than 75% activity in the temperature range of 50-80°C, thus could have potential  use as detergent additives.The appreciably enzyme activity and stability at different pH and temperatures makes these isolates industrially promising and of special interests for basic and applied research.

Conclusion
In our study, proteases produced by two different Bacillus strains showed activity at high temperatures and pH.The alkaline proteases isolated from strains EN-2 and EN-3 are thermostable proteases.These have a relevant economical and environmental impact which alleviates the pollution problem created by leather industries as it has desirable properties such as stability at alkaline pH and high temperature.
These properties indicate the possibility of using the protease in detergent, leather and in other biotechnological applications that would require higher working temperatures.Additional work is needed for full characterization (amino acid analysis, column chromatography) of the examined proteases.For the evaluation of a biotechno logical application of the protease of Bacillus sp., a more detailed understanding of the factors that enable this enzyme to act on compact substrates better than comparable enzymes of the same type would be helpful.Therefore, more research on the specific molecular characteristics of this interesting enzyme will be done.

Figure 1 .
Figure 1.Phylogenetic tree of bacterial 16S rDNA sequences by neighbor-joining method.

Figure 2 .
Figure 2. Effect of pH on activity of proteases from isolates EN-2 and EN-3.

Figure 3 .
Figure 3.Effect of pH on the stability of free enzyme from isolates EN-2 and EN-3.

Figure 4 .
Figure 4. Effect of temperature on activities of proteases from isolates EN-2 and EN-3.

Figure 5 .
Figure 5.Effect on temperature on the stability of free protease from isolates EN-2 and EN-3.

Figure 6 .
Figure 6.Effect of pH on the activity of immobilized proteases from isolates EN-2 and EN-3.

Figure 7 .
Figure 7. Effect of temperature on the activity of immobilized proteases from isolates EN-2 and EN-3.

Table 1 .
The purification chart of extracellular alkaline protease of EN-2 and EN-3 bacterial isolates.

Table 2 .
Enzyme kinetics of the partially purified extracellular protease.

Table 3 .
Immobilization efficiency (% immobilization) of alkaline protease at different concentration of sodium alginate.