CONCLUSION

Alkaline phosphatase is a typical enzyme which readily hydrolyzes the phosphate group from the
Substrates containing the phosphate group. It plays a prominent role in the diagnosis of liver and
bone disorders. It is also used efficiently in dairy industries as an indicator of pasteurization as it
is a heat stable thermophilic enzyme. Various sources have revealed the existence and
availability of the alkaline phosphatase for commercial uses and for diagnostic aspects. Based
the study carried out, the enzyme was isolated from soil as means of bacterial source. The
Bacillus sps was found to produce the enzyme crudely which hydrolyzed the
Phosphate group of the substrate p-nitrophenolphosphate to p-nitrophenol by indicating the
growth of yellow colored colonies. The crude enzyme was isolated and further purified
sequentially using purification stages such as salting in and salting out, dialysis, ion exchange
chromatography and size exclusion chromatography. The purified enzyme was checked for
kinetic properties including effect of pH, temperature, incubation time and substrate
concentration. The optimum conditions of the purified enzyme was determined. The enzyme
showed optimum pH at 8, temperature at 35C along with incubation time
and substrate

concentration. The purified enzyme was successfully immobilized into

calcium alginate beads

by entrapment method and stored at 4C for further commercial purposes.

Page 1 of 57

REFERENCES

1 .Kim EE,

Wyckoff HW (March 1991). "Reaction mechanism of alkaline

phosphatase based on

crystal structures. Two-metal ion catalysis". J. Mol. Biol. 218

RE
2. Horiuchi T, Horiuchi S, Mizuno D (May 1959). "A possible negative
feedback phenomenon

controlling formation of alkaline phosphomonoesterase in Escherichia

coli". Nature183

3. "A fine-structure genetic and chemical study of the enzyme alkaline

phosphatase of E. coli.

Purification and characterization of alkaline phosphatase". Biochem. Biophys.

Acta 38: 47083.

4. Kay, H. (1935). "Some Results of the Application of a Simple Test for

Efficiency of

Pasteurisation". The Lancet 225.

5. Hoy, W. A.; Neave, F. K. (1937). "The Phosphatase Test for Efficient

Pasteurisation". The

Lancet 230.

6. Iqbal, J (2011) An enzyme immobilized microassay in capillary

electrophoresis for

characterization and inhibition studies of alkaline phosphatases J Anal.

Biochem. 414, 226-231.

7. . Mahjoub S, Masrour RoudsariJ. Quantification of liver alkaline phosphatase isoenzyme

activity using heat inactivation and phenylalanine inhibition techniques: Comparison of two
methods. World Appl Sci J. 2012;17:9416.
8. Mahjoub S, Ghasempour M, Mohammadi A. Salivary alkaline phosphatase activity and
inorganic phosphorus concentration in children with different dental caries. J Babol Univ Med
Sci. 2007;9:238.
9. Kubo K, Yuki K, Ikebukuro T. Changes in bone alkaline phosphatase and procollagen type-1
C-peptide after static and dynamic exercises. Res Q Exerc Sport. 2012;83:4954.
10. Baoudene-Assali, F., J. Baratti, and G. P. F. Michel. 1993. Purification and
properties of a phosphateirrepressible membrane-bound alkaline phosphatase from Zymomonas mobilis. J.
Gen. Microbiol.
139:229235.
11. Bradford, M. 1976. A rapid and sensitive method for the quantitation of
microgram quantities of
protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248254.

12. Cole, G. T. 1986. Preparation of microfungi for scanning electron microscopy, p.

1415. In H. C.
Aldrich and W. J. Todd (ed.), Ultrastructure techniques for microorganisms. Plenum
Press, New York.
13. Collins, C. H., P. M. Lyne, and J. M. Grange (ed.). 1991. Collins and Lynes
microbiological methods,
6th ed. Butterworth-Heinemann, Oxford. [Reprint of 1989 edition.]
14. Fiedler, F., K. Schleifer, and O. Kandler. 1973. Amino acid sequence of the
threonine-containing
mureins of coryneform bacteria. J. Bacteriol. 113:817.
15. Fitt, P. S., and P. I. Peterkin. 1976. Isolation and properties of a small manganeseion-stimulated
bacterial alkaline phosphatase. Biochem. J. 157: 161167.
16. Glew, R. H., and E. C. Heath. 1971. Studies on the extracellular alkaline
phosphatase of Micrococcus
sodonensis. II. Factors affecting secretion. J. Biol. Chem. 246:15661574.
17. Glew, R. H., and E. C. Heath.1971. Studies on the extracellular alkaline
phosphatase of Micrococcus
sodonensis. I. Isolation and characterization. J. Biol. Chem. 246:15561565.
18. Goldman, S., K. Hecht, H. Eisenberg, and M. Mevarech. 1990. Extracellular Ca21dependent inducible
alkaline phosphatase from the extremely halophilic archeabacterium Haloarcula
marismortui. J.
Bacteriol. 172:70657070.
19. Hulett, F. M., C. Bookstein, and K. Jensen. 1990. Evidence of two structural genes
for alkaline
phosphatase in Bacillus subtilis. J. Bacteriol. 172:735740.
20. Hulett, F. M., E. E. Kim, C. Bookstein, N. V. Kapp, C. W. Edwards, and VOL. 62,
1996 PHOSPHATASES
FROM A PSYCHROPHILIC ARTHROBACTER STRAIN 3737 on May 31, 2016 by guest

21. Hulett, F. M., P.-Z. Wang, M. Sussman, and J. K. Lee. 1985. Two alkaline
phosphatase genes
positioned in tandem in Bacillus licheniformis MC14 require different RNA
polymerase holoenzymes for
transcription. Proc. Natl. Acad. Sci. USA 82:10351039.
22 . Jones, D., and R. M. Keddie. 1992. The genus arthrobacter, p. 12831299. In A.
Balows, H. Truper,
M. Dworkin, W. Harder, and K.-H. Schleifer (ed.), The prokaryotes, vol. 2. SpringerVerlag, New York.
23. Keddie, R. M., M. D. Collins, and D. Jones. 1986. Genus Arthrobacter Conn and
Dimmick 1947, 300AL,
p. 12881301. In P. H. A. Sneath, N. S. Mair, E. M. Sharpe, and J. G. Holt (ed.),
Bergeys manual of
systematic bacteriology, vol. 2. The Williams & Wilkins Co., Baltimore.
24. Kobori, H., C. W. Sullivan, and H. Shizuya. 1984. Heat-labile alkaline phosphatase
from Antarctic
bacteria: rapid 59 end-labeling of nucleic acids. Proc. Natl. Acad. Sci. USA 81:6691
6695.

25. Kobori, H., and N. Taga. 1980. Extracellular alkaline phosphatase from marine
bacteria: purification
and properties of extracellular phosphatase from a marine Pseudomonas sp. Can. J.
Microbiol. 26:833838
26. Kormendy, A. C. 1975. Microorganisms, p. 82108. In M. A. Hayat (ed.),
Principles and techniques of
scanning electron microscopy. Van Nostrand Reinhold Company, New York.
27. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the
head of bacteriophag
e T4. Nature (London) 227:680685.

28. Loveland, J., K. Gutshall, J. Kasmir, P. Prema, and J. E. Brenchley. 1994.

Characterization of
psychrotrophic microorganisms producing b-galactosidase activities. Appl. Environ.
Microbiol. 60:1218.
29. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.
30. Neidhardt, F. C., J. L. Ingraham, and M. Schaechter. 1990. Physiology of the
bacterial cell, 1st ed., p.
506. Sinauer Assoc. Inc., Sunderland, Mass.
31. Nomoto, M., M. Ohsawa, H.-L. Wang, C.-C. Chen, and K.-W. Yeh. 1988.
Purification and
characterization of extracellular alkaline phosphatase from an alkalophilic
bacterium. Agric. Biol. Chem.
52:16431647.

7. RESULTS AND DISCUSSION

7.1 Isolation of Bacteria:
Strains of Bacillus species were isolated and identified from the soil sample which
0
0.2
0.4
0.6
0.8
1
1.2
Fig3: Changes in
4
6
8
10
12
14
16

18
Fig4: Changes in Reducing Sugar Content

Page 39 of 57
There was a marked increase in the fold purification of the samples and with the sequential
7.4 Characterization of purified enzyme:
1.2
1.4
1.6
1.8