Partial Purification and Characterization of Intracellular Alkaline Phosphatase from Newly Isolated Strain of Bacillus subtilis KIBGE-HAS
S Ali ul Qader, S Iqbal, Z Niazi
alkaline phosphatase, bacillus sp., characterization, purification
S Ali ul Qader, S Iqbal, Z Niazi. Partial Purification and Characterization of Intracellular Alkaline Phosphatase from Newly Isolated Strain of Bacillus subtilis KIBGE-HAS. The Internet Journal of Microbiology. 2008 Volume 7 Number 1.
The soluble alkaline phosphatase isolated from
Alkaline phosphatase (EC 126.96.36.199) is a broad term associated with non-specific phosphomonoesterase activity with activity optima at alkaline pH. It is a homodimeric metalloenzyme which hydrolyzes the phosphomonoester into inorganic phosphate and corresponding alcohol (1,2). Organic and inorganic phosphates are essential component of living organism; therefore it is a valuable reagent for the removal of terminal monoesterified phosphate from ribo-oligonucleotide, deoxyribo-oligonucleotides, alkaloids and proteins etc. (3,4).
Three types of alkaline phosphatase have been found in human, liver/kidney/ bones alkaline phosphatase, intestinal alkaline phosphatase and placental alkaline phosphatase. Serum alkaline phosphatase is markedly elevated in different types of liver and bones diseases while moderate elevation is noted in congestive heart failure, hyperparathyroidism and intestinal diseases.
Alkaline phosphatase have also been found in variety of micro-organism including
Alkaline phosphatase has become an important tool in molecular cloning and DNA sequencing. It also used as an important part of diagnostic kits component of different ELISA base kits (3).Bacterial alkaline phosphatase is used more commonly in research because it is comparatively resistant to inactivation, denaturation, degradation and higher rate of activity (3). Due to its so many industrial uses, it is necessary to purify it on large scale for commercial and research purpose.
In present study the partial purification and characterization of intracellular alkaline phosphatase (AP) enzyme from newly isolated strain of Bacillus
Materials and Method
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).
Medium for enzyme Production
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.
Extraction of Intracellular alkaline phosphatase
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
Purification of Intracellular Alkaline Phosphatase
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.
Molecular Mass Determination by SDS-PAGE
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.
Result and Discussion
Extraction of Intracellular enzyme
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.
Purification of Alkaline Phosphatase
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.
Molecular Mass Determination
Electrophoretic analysis of intracellular alkaline phosphatase from
Effect of Temperature on Enzyme Activity
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).
Effect of pH on Enzyme 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.
Effect of Buffer on Enzyme 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).
Effect of Reaction Time on Enzyme 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).
Effect of substrate concentration
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