BIOCHEMICAL PROPERTIES OF RECOMBINANT ALKALINE PHOSPHATASE FROM BACILLUS LICHENIFORMIS T5
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Authors
S.O. Kirillov
National Center for Biotechnology, 13/5, Korgalzhyn road, Astana, 010000, Kazakhstan
B.B. Khassenov
National Center for Biotechnology, 13/5, Korgalzhyn road, Astana, 010000, Kazakhstan
D.V. Silayev
National Center for Biotechnology, 13/5, Korgalzhyn road, Astana, 010000, Kazakhstan
Abstract
In this work, recombinant alkaline phosphatase (phoB) from Bacillus licheniformis was successfully expressed in Escherichia coli, purified and biochemically characterized. Gene coding alkaline phosphatase was amplified from genomic DNA of B. licheniformis and cloned into expression vector pET-28c (+). Using the recombinant vector, a BL21 (DE3)/pAlPh strain-producer was obtained with over expression of the gene. Optimal cultivation parameters for producing recombinant alkaline phosphatase have been determined; 10 mg of protein was purified from 1 liter of culture. The activity of the recombinant alkaline phosphatase is 100 U/mg at standard conditions. Biochemical characteristics of recombinant alkaline phosphatase showed that enzyme has maximum activity at pH=10.0 and temperature +60°C. The kinetics of p-nitrophenyl phosphate hydrolysis have been studied, the Michaelis constant Km was 0.91±0.13 mM and the limiting value of the maximal rate of the enzymatic reaction Vmax was 21.4±1.19 mM. Experiments were carried out to determine the dependence of the enzyme activity on various divalent metals.
Keywords
alkaline phosphatase, strain-producer, biochemical characteristics, enzymatic activity, purification, expression, kinetic parameters
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References
Zernov Yu.P., Dedkov V.S., Antonova Yu.A. et al. Thermolabile alkaline phosphatase from the psychrophilic microorganism Alteromonas undina P2. Biotechnology, 2005, vol. 2, pp. 38-43.
Angelina S., Moreno R., Gouffi K. et al. Export of Thermus thermophilus alkaline phosphatase via the twin-arginin translocation pathway in Escherichia coli. FEBS Lett., 2001, vol. 506, pp. 103-107.
Lequin R.M. Enzyme Immunoassay (EIA) /Enzyme-Linked Immunosorbent Assay (ELISA). Clinical Chemistry, 2005, vol. 51, pp. 2415-2418.
Schmidt S.D., Mazzella M.J., Nixon R.A., Mathews P.M. Aβ measurement by enzyme-linked immunosorbent assay. Methods in Molecular Biology, 2012, vol. 849, pp. 507-527.
Holtz K.M., Kantrowitz E.R. The mechanism of the alkaline phosphatase reaction: insights from NMR, crystallography and site-specific mutagenesis. FEBS Letters, 1999, vol. 462, pp. 7-11.
Pandey S.K., Banik R.M. Extractive fermentation for enhanced production of alkaline phosphatase from Bacillus licheniformis MTCC 1483 using aqueous two-phase systems. Bioresour Technol., 2011, vol. 102, pp. 4226-4231.
Maniatis T., Fritsch E.E., Sambrook J. Molecular cloning. A laboratory manual. New York, Cold Spring Harbor Laboratory, 1982, 545 p.
Bessey O.A., Lowry O.H., Brock M.J. A method for the rapid determination of alkaline phosphates with five cubic millimeters of serum. J. Biol. Chem., 1946, vol. 164, pp. 321-329.
Divya A., Santhiagu A. Prakash S.J. Cloning, expression and characterization of a highly active thermostable alkaline phosphatase from Bacillus licheniformis MTCC 1483 in Escherichia coli BL21 (DE3). Applied Biochemistry and Microbiology, 2016, vol. 52, pp. 358-365.