Bacillus specie used in this study was isolated from the air and its growth was optimized at 37°C at pH 7. The stock culture was maintained on nutrient agar slant containing 2 % agar and pH was adjusted to 7.0.

Buffers that used through out in this study was as fallows; 50 Mm Tris/HCl buffer (p H 8.0), 50 m M phosphate buffer (p H 7.5), DEA buffer ( p H 8.0), Glycine/NaOH buffer (p H 8.0).

The growth medium used for the production of intracellular alkaline phosphatase was composed of: Peptone 5.0 g/l, yeast extract 1.0 g/l, starch 15.0 g/l, MgSO4 0.5 g/l, NaCl 0.5 g/l, CaCl₂ 0.01 g/l. The medium was adjusted to pH 7.0 and then autoclaved.

For the production of alkaline phosphatase, 10ml of 24hrs culture inoculum was transferred to 90ml medium and incubated for 24hrs at 37°C. After 24hrs of incubation the cells were removed from broth and washed 2-3 times with phosphate buffer. Washed cells were resuspended in 20ml of the same buffer and homogenized for 10 minutes using Ultra Turrax T-25 Basic 9IKA. Homogenized cells were centrifuged at 10,000 x g for 15min. at 2◦C and alkaline phosphatase activity was performed in supernatant and cells debris was discarded.

Alkaline phosphatase activity was measured spectrophotometrically by incubating 10µl of enzyme solution with 1000µl of 20 mM p-Nitrophenyl phosphate (prepared in 50mM Tris-HCl buffer) at 37°C for 2 minutes by detecting the concentration of p-nitrophenol, liberated from p-nitrophenyl Phosphate during reaction at 405nm (1, 11, 12).

One unit of alkaline phosphatase activity is defined as, “the amount of enzyme that liberates 1µmole p-nitrophenol from p-nitrophenyl phosphate in one minute at 37 ºC”. The specific activity is given as U/mg protein.

Total protein level was measured by the Lowry et al. method (13) by using bovine serum albumin (250µg/ml) as standard.

Crude soluble enzyme (20ml) was brought to 40% saturation by the addition of solid ammonium sulphate (NH₄)₂ SO₄ with continuous stirring for 5 minutes and then kept at 2- 8°C for 1hr (15). The obtained protein precipitates were separated out by centrifugation (16,000xg for 15 minutes) and dissolved in 10ml phosphate buffer (pH 7.5). A dialysis tube was used for the concentration of enzymatic protein by removing the salts for 24 hours at 2C using same buffer.

Partial purified protein was resolved on 10% polyacrylamide gel electrophoresis according to the method of Laemmli (14) and then gel was stained with Coomassie Blue R-250.

After initial step of cell homogenization 883 U/L of alkaline phosphatase activity was detected (table1) which supported the idea that tissue homogenizer can be used for the extraction of intracellular enzyme. In the previous studies lysozyme & DNase have been used for the extraction of intracellular enzyme but these lytic enzymes cannot be used again and again. Enzymatic treatment is also time consuming because it requires 3-10 hrs incubation for the cells lysis (9) while tissue homogenizer required 10-15 minutes for the extraction of enzyme from cells lysis.

For the purification of alkaline phosphatase, ammonium sulphate was used for the precipitation of enzyme and after precipitation enzyme activity was increased upto1737 U/L that reflects that the specific protein-protein aggregation became predominant over protein-water and protein-salt interaction.

After salt precipitation the enzyme was dialyzed and the activity of enzyme increased upto 1689 U/L with specific activity of 11.29 U/mg (table1) and purification reaches to 5.1 fold.

Figure 1

Table 1: Partial Purification of Alkaline Phosphatase

Electrophoretic analysis of intracellular alkaline phosphatase from Bacillus subtilis KIBGE-HAS been carried out after partial purification. Molecular mass was determined by comparing the relative mobility of the protein with the molecular weight markers (Figure 1, Lane B). Three protein bands were detected in the partially purified sample (Figure 1, Lane A) and the estimated molecular weight of protein bands is approximately 59,500 Da , 23,700 Da and 22,300 Da. Alkaline phosphatase exist in both dimeric and monomeric forms, the homodimeric forms of alkaline phosphatase, with a monomer molecular mass of 54,000 Da have been previously reported in E.pyrococcus abyssi (1,15,16). The SDS-PAGE of B. stearothermophilus alkaline phosphatase showed a single protein band of 32,000 Da (17).

Figure 2

Figure: SDS-PAGE Profile of Alkaline Phosphatase: Lane M, Molecular weight standards (kDa): Lane A, partially purified Alkaline Phosphatase (Coomassie Blue staining)

The effect of temperature on the partially purified enzyme was observed by incubating the enzyme on different temperatures ranging from 25°C-50°C for 2 min. The results showed that the enzyme activity was increased with the increase in temperature and optimum activity was observed at 37°C (9). Further increase in temperature, resulted in the decrease of enzyme activity and 20 % activity was loss at 50°C (Fig.2). The high temperature increases the kinetic energy of the molecules that break the bond that holding the active amino acid resulting in a loss of enzyme activity (18).

Figure 3

Figure 2: Effect of temperature on intracellular alkaline phosphatase activity

The activity profile of the enzyme on different pH ranging from 7.5 to 10 was performed. The Fig-3 showed that the maximum enzyme activity (3629 U/L) was found at pH 8.0 and as the pH increases the sharp decrease in activity was observed and only 30 % enzyme activity was detected at pH 10 (9,19). It was reported previously that maximum activity of calf intestinal alkaline phosphatase was found in the range of 10 to 10.1 (20) .The increase in pH causes internal electrostatic repulsion or loss of internal electrostatic attraction of the charges on the side chains of amino acid and due to which proteins opens up and resulting in decrease of enzyme activity, therefore when the pH increased from the optimum pH the activity of enzyme decreased.

Figure 4

Figure 3: Effect of pH on intracellular alkaline phosphatase activity

Enzyme assay was carried out with three different buffers of 50 mM (Tris-HCl, DEA & Glycine-NaOH buffer) of same pH (8.0) for the selection of appropriate buffer to get the maximum enzyme activity. It was observed that the Tris-HCl environment (1,17,19) is optimum for the maximum enzyme activity(1049U/l) while Glycine-NaOH and DEA buffer 30% and 83% activity was detected respectively with reference to the activity in Tris-HCl buffer (Fig.4).

Figure 5

Figure 4: Effect of buffers on intracellular alkaline phosphatase activity

Enzyme substrate mixture was incubated at 37°C for one minute to five minutes interval and maximum activity was observed when enzyme substrate mixture was kept for 2.0 minute at 37°C (Fig 5). With the lengthening of incubation time enzyme activity was markedly decreased and only 37% activity was detected after 5.0 minutes. It might be due to the thermal labile nature of the enzyme with reference of time and as the time increases the temperature start breaking the bonds between the two amino acids (9).

Figure 6

Figure 5: Effect of reaction time on intracellular alkaline phosphatase activity

Alkaline phosphatase has high substrate specificity to p-nitro phenyl phosphate (1,12). It was observed that the alkaline phosphatase activity was increased with the increase in substrate concentration and maxima were attained at 20mM-substrate concentration (Fig 6) but no further increased in alkaline phosphatase activity was observed with increase in substrate concentration beyond 20mM. The Michealis constant for p-Nitro phenyl phosphate at pH 8.0 was calculated by Lineweaver-Burk plot and Km for PNP was 2.74 mM and Vmax 3299 U/L (Fig 6a & 6b). The Km value for the purified alkaline phosphatase of B. licheniformis was calculated 6.05×10-4M (9). Some workers also reported that Km value of alkaline phosphatase from B. stearothermophilus was 1.11 mM (17).

Figure 7

Figure 6A: Substrate Saturation Kinetics of Intracellular Alkaline Phosphatase

Figure 8

Figure 6B: Lineweaver Burk plot of Intracellular Alkaline Phosphatase