Isolation, characterization, and ELISA applications of alkaline phosphatase and acetylcholinesterase from Moniezia expansa

Abstract

Moniezia expansa worms are a significant source of alkaline phosphatase (ALP) and acetylcholinesterase (AChE) enzymes. The current study presents a simple and reproducible ALP and AChE purification method from Moniezia expansa helminthes by precipitating the proteins with ammonium sulfate and chromatography on the Sephacryl S-300 column. The M. expansa ALP purified at 1070.8 U/mg, displaying 6.0 purification folds and 53.6% yield, while M. expansa AChE is at 5250 U/mg, displaying 2.0 purification folds and 43% yield. The M. expansa ALP isoenzyme displayed its optimum activity at pH 9.6, while the M. expansa AChE isoenzyme displayed its optimum activity at pH 8.0. The affinity of M. expansa ALP for several substrates revealed that p-nitrophenyl phosphate preferentially cleaved with a Km value of 4.4 mM. M. expansa AChE preferentially cleaved acetylthiocholine iodide with a Km value of 0.9 mM. M. expansa ALP is strongly stimulated with Co 2+ , Mn 2+ , Ni 2+ , and Mg 2+ and reduced with Zn 2+ , Cu 2+ , Ca 2+ , EDTA and DTT. On the other hand, M. expansa AChE is significantly induced with Co 2+ , Zn 2+ , and Ni 2+ and inhibited with Mg 2+ , Ca 2+ , EDTA, 1,10-phenanthroline and eserine. The antisera of the purified M. expansa ALP and AChE found effective for determining the two enzymes in different unknown sera from different animal species, including humans, sheep and fish. These results may provide a possible future application of such enzymes in producing ALP and AChE-coated ELISA plates for research purposes.