Partial Purification and Characterization of a Haloalkaline Protease from Pseudomonas aeruginosa

A study was conducted to partially purify and characterize a haloalkaline protease isolated from Pseudomonas aeruginosa.


INTRODUCTION
Halophilic microorganisms require very high salt (2 to 5 M NaCl) concentrations for growth and are found in salterns and hypersaline lakes. Many extreme and moderate halophiles have been isolated and investigated for possible biotechnological applications. Proteases encompass a group of industrial enzymes, which alone form about 60% of the total world-wide enzyme yield [1]. Among the variety of enzymes, haloalkaline proteases constitute an important group of enzymes that have high catalytic potential at high salinity. Microbial proteases are an important group of enzymes that can have application in various industries such as leather processing, food processing, pharmaceutical and bioremediation [2]. The applications of proteases include their protein hydrolysate use in cheesemaking, cosmetic industries, beer clarification and pharmaceutical products [3]. These potential applications of the proteolytic enzymes in biotechnology are widely common. Hence the search for new haloalkaline proteolytic organisms and enzymes is still a point of interest. In contrast to other types of proteases, the activity of the aspartic proteases is dependent on pH conditions [4]. Proteases have also been extensively studied for their role in inflammatory diseases and blood clotting [5]. Looking into the depth of microbial diversity in India, the chance of haloalkaline protease producing bacterial strains and its exploitation due to their wide variety of industrial application, an attempt was undertaken to isolate microorganisms capable of producing haloalkaline proteases.

MATERIALS AND METHODS
The elephant dung was collected from the Shenbagathoppu hills, Srivilliputur, Tamilnadu, India by using sterile container and used. From this sample microbes were isolated and plated on nutrient agar initially and on skim milk agar selective medium which has the capacity to degrade protease.

Isolation and Screening of Protease Producing Bacteria
The isolated colonies were streaked on the skim milk agar plates and incubated at 37°C for 48 hours. The zone around the bacterial growth indicated a positive reaction for protease activity [6].

Protease Enzyme Assay
The haloalkaline protease enzyme activity was studied by the modified method of Hagihara [7] using casein as the substrate. One unit of protease enzyme activity is defined as the enzyme that releases 1 µg/ml of tyrosine per min under standard assay conditions [8].

Partial Purification of Crude Protease and Characterization
The crude protease extract was partially purified by acetone (75% m/v) precipitation followed by dialysis. The precipitate was collected by centrifugation at 10,000 rpm for 10 min at 4°C and dissolved in 0.01 M phosphate buffer (pH 7.0). The solution was dialyzed against the same buffer at 4°C for 8 h with 3 changes of the dialysis buffer, and protein concentration was measured as per the method of Lowry et al. [9] using Bovine Serum Albumin as standard protein.

Enzyme Purification and Molecular Mass Determination of Protease
Purification of enzyme was carried out at 4°C. Enzymes (supernatant of fermented broth obtained after centrifugation at 10,000 g for 10 min) were precipitated by adding two volumes of acetone and kept for 1 h at 0-4°C to allow complete precipitation. The resulting precipitate was collected by centrifugation (10,000 rpm, for 30 min) and the pellet was air dried and resuspended in a minimal volume of 20 mM Tris HCl buffer, pH 7.0. After the removal of insoluble materials by centrifugation (10,000 rpm for 30 min), the supernatant was subjected to ion exchange chromatography. Ion exchange chromatography was performed on a column of DEAE-Sephadox (2.5 to 30 cm), which was equilibrated with 20 mM Tris HCl pH 7.2. After loading the sample, the column was washed with the same buffer until the optical density of the elution at 280 nm was zero. The bound proteins were then eluted with a linear gradient of sodium chloride in the range of 0.1-1 M as the equilibrating buffer. Fractions (4.0 ml each) were collected and the enzyme activity and protein content of each fraction were determined. The approximate molecular weight of the partially purified enzyme was determined by (Sodium Dodecyl Sulphate -Polyacrylamide Gel Electrophoresis) SDS-PAGE [10].

Effect of SDS on Enzyme Activity
The enzyme was pre-incubated with various concentrations of 10 mM, 25 mM, 50 mM, 100 mM and 150 mM Sodium Dodecyl Sulphate (SDS). The enzyme activity was then determined at 37°C by using 0.5 ml of casein as the substrate. The protease activity was checked using carbonate buffer. The residual activities were quantified under standard assay conditions.

Effect of EDTA on Enzyme Activity
The enzyme was pre-incubated with various concentrations of 10 mM, 25 mM, 50 mM, 100 mM and 150 mM EDTA. The enzyme activity was then determined at 37°C by using 0.5 ml of casein as the substrate. The protease activity was checked using carbonate buffer. The residual activities were quantified under standard assay conditions.

Effect of pH and Temperature on Partially Purified Enzyme Activity
Stability of the purified protease was studied and the pH of the reaction mixture was adjusted using one of the following buffers: Sodium acetate, sodium phosphate and Tris HCl. To ensure the pH stability and the enzyme reaction mixtures were incubated at 40°C for 1 h and the relative activity was measured. The enzyme stability of the purified enzyme was determined by incubating the reaction mixture at different temperatures ranging from 25, 30, 35, 40, 45 and 50°C for 1 h and then relative activities were assayed at standard assay conditions.

Compatibility of Protease Enzyme with Ariel and Power Commercial Detergents Activity
Ariel and Power detergent powder was used for protease compatibility studies. The enzymes were pre-incubated with various concentrations of 10 µl, 25 µl, 50 µl, 100 µl and 150 µl of Ariel and Power detergent. The enzyme activity was then determined at 37°C by using 0.5 ml of casein as the substrate. The protease activity was checked using carbonate buffer. The residual activities were quantified under standard assay conditions.

Isolation and Screening of Protease Producing Bacterial Strains
A total of ten bacterial strains were isolated from the elephant dung sample of Shenbagathoppu hills, Srivilliputur, Tamilnadu, India and screened. Only one bacterial strain was selected on the basis of maximum protease production by hydrolysis of casein and used for successive studies.

Identification of Protease Positive Colony
Depending upon the morphological, physiological and biochemical tests the selected colony was identified as Pseudomonas aeruginosa following the standard keys of Bergey's Manual of Determinative Bacteriology (Table  1). Consequently, the strain was identified as a protease producer and was utilised for further experimental studies. Phylogenetic studies revealed that the 16S rRNA gene sequence of Pseudomonas aeruginosa has 97% similarity with the nearest match in the Gene bank.

Enzyme Purification
The protease activity of the crude enzyme extract was concentrated by acetone precipitation. Eighty nine percentage (89%) of the protease recovery was achieved with 1.6-fold purification ( Table 2). Following acetone precipitation the resuspended solution was applied to a column of DEAE-Sephadex. Fig. 1 shows that the maximum protease activity was observed fraction in 30 kDa. This step resulted protease recovery (2.2 -Purification of fold) with a specific activity of 4.4 U/mg -1 of protein. The purified protease migrated as a single band of 30 kDa in the SDS-PAGE (Fig. 2) under reducing conditions, suggesting that the purified protein was homogenous. This report is similar for Bacillus cerus resulted in molecular weight of 34 to 45 kDa. Table 3 shows the effect of pH on the protease activity and stability under various experimental pH. Results inferred that protease activity of Pseudomonas sp. was more active at pH 9.0 after 30 min of incubation. The minimum protease activity was recorded in pH 6.0.

Effect of Temperature on Protease Activity
Effect of different temperatures on protease activity was studied after 30 min of incubation. Among different temperatures tested, the maximum activity of Pseudomonas sp. was registered at 35°C, whereas the variation in temperature above and below 40°C showed reduction in the protease activity (Table 4).

Effect of Surfactants (SDS) and EDTA on Enzyme Activity
Enzyme was tested at various concentrations of SDS at 1 h incubation. The maximum enzyme activity was recorded at 50 mM, while the least activity was noticed at 150 mM concentration (Table 5). Similarly, the effect of EDTA was tested against the purified enzyme. Table 6 shows maximum enzyme activity at 50 mM, while the least enzyme activity was noticed at 150 mM concentration.

Effect of Commercial Detergents on the Enzyme Activity
The enzyme was tested at various concentrations of detergents like Ariel and Power detergent after 10 min of incubation. Both detergents showed maximum enzyme activity at 50 µl and least activity at 150 µl (Table 7).

DISCUSSION
In the partial purification of the highly haloalkaliphilic protease produced by Pseudomonas aeruginosa, precipitation by acetone was important in the exclusion of contaminating exopolysaccharides and proteins [11]. The above mentioned method was previously employed for the purification of the halophilic serine protease from Natronococcus occultus. An advantage of the enzyme purification is an increase in its enzyme activity, making the after purification more specific for industrial applications. In order to elucidate the specific functions of the enzyme, there is a need to characterize an enzyme after purification.

Fig. 2. SDS -PAGE for molecular weight detrermination of Pseudomonas aeruginosa protease
In the present investigation, the enzyme was purified in a 2-step procedure involving acetone precipitation and DEAE -Sephadex G-100 chromatography. The enzyme was shown to be homogenous and had a relatively low molecular weight of 30 kDa. From this study, it was observed that the partially purified protease of Pseudomonas aeruginosa was homogenous and recorded a single band with molecular weight of 30 kDa. This is similar to the protease produced by the Bacillus pumilis that was found to be 31 kDa [12].
In this study, the effect of temperature on the partially purified protease showed that the optimum protease activity was found to be at 35°C and the optimum pH for the enzyme activity recorded was at 9.0 with casein as the substrate. The present study is in accordance with the previous report of Jadwiga [13], who has reported a similar optimum temperature range from 40 to 50°C for the purified protease, from B. cereus [14]. Some marine bacterial strains such as Vibrio, Pseudomonas and Bacillus sp. have produced noval thermal protease [15]. He also reported that the optimum pH of alkaline protease from B. subtilis CN2 and B. subtilis PE-11 was found to be at pH 10.
In the present study, the maximum protease activity was recorded at 50 mM EDTA added mixture. This result is identical to the earlier report of Hoshino et al. [16] who have also reported the maximum protease activity obtained in reaction mixture containing EDTA after 48 hours of incubation.
The partially purified protease showed admirable stability and compatibility in the presence of locally available detergents such as Power and Ariel. The present report is analogous to the previous report of Adinarayana et al. [17], who reported that the alkaline protease from B. subtilis PE-11 showed good stability and compatibility with a broad range of locally available detergents in the presence of CaCl 2 and glycine as stabilizers at 60°C. Bhosale et al. [18] also reported that the activity of alkaline protease from Conidiobolus coronatus (NCL 86.8.20) showed good compatibility and stability with locally available commercial detergents.

CONCLUSION
The halophilic protease purified from Pseudomonas aeruginosa in this study exhibited stability towards ionic, non ionic surfactant and commercial detergents. Considering the promising properties of Pseudomonas aeruginosa enzymatic preparation, we suggest that the purified enzyme could be a better choice for biodetergent potentiality.