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Updated: Mar 23, 2026
Colorimetric Analysis of Alkaline Phosphatase Activity in S. aureus Biofilm
Published on: April 12, 2019
PARTIAL PURIFICATION AND CHARACTERIZATION OF A CALCIUM-DEPENDENT ALKALINE PHOSPHATASE FROM THE CYANOBACTERIUM
Antonia D Asencio1, Asuncióín Morte1, Francisco García-Carmona1
1División Botánica, Departamento de Biología Aplicada, Facultad de Ciencias Experimentales, Universidad Miguel Hernández, Campus de Elche, Avenida de la Universidad s/n, 03202 Elche, SpainDepartamento de Biología Vegetal, Facultad de Biología, Universidad de Murcia, Campus de Espinardo, 30100 Murcia, SpainDepartamento de Bioquímica y Biología Molecular-A, Facultad de Biología, Universidad de Murcia, Campus de Espinardo, 30100 Murcia, Spain.
Abstract:
In the present study, Triton X-114 (TX-114) is used to extract and partially purify alkaline phosphatase (ALP) from a membranous fraction of Arthrospira platensis Gomont containing cell wall, plasma membrane, thylakoids, and sheath. TX-114 has a double effect: solubilizing cell components to liberate the enzyme and, after phase partitioning, removing chl and other pigments present in the crude extract. The recovery of the enzyme in the aqueous phase suggests the overall hydrophilic character of this enzyme. ALP was kinetically characterized at pH 11.0 using p-nitrophenyl phosphate as substrate, giving a Km value of 1.7 mM. Orthovanadate was seen to be a competitive inhibitor of ALP, with a Ki of 0.8 mM. The enzyme was almost completely inactivated in the presence of 70 μM EDTA, although the addition of Ca(2+) reverted this inactivation; these results indicate that ALP from A. platensis is a calcium-dependent metalloenzyme. When the effect of Ca(2+) was investigated in detail, a value of 0.067 μM(-1) for the affinity constant was obtained. The enzyme was histochemically localized in the cytoplasm, cell wall, and sheath using the enzyme-labeled fluorescent substrate (ELF) method. It is assumed that the same enzyme is either soluble in the cytoplasm and in some way "trapped" in the cell wall or in the sheath. ALP localization within the sheath and the subsequent release of phosphorus (P) may benefit the neighboring cells surrounding this layer.
