Purification and characterisation of alliinase produced by Cupriavidus necator and its application for generation of cytotoxic agent : Allicin

الملخص EN

Allicin, an extremely active constituent of freshly crushed garlic, is produced upon reaction of alliin with the enzyme alliinase (EC 4.4.1.4).

A bacterium Cupriavidus necator with the ability of alliinase production was isolated from a soil sample and was identified by morphological, biochemical and 16S rRNA sequence.

Alliinase production was optimised and it was further purified to apparent homogeneity with 103-fold purification and specific activity of 209 U/mg of protein by using DEAE Cellulose and Sephadex G-100 chromatography.

The enzyme is a homodimer of molecular weight 110 kDa with two subunits of molecular weight 55 kDa each.

The optimum activity of the purified enzyme was found at pH 7 and the optimum temperature was 35 C.

The enzyme exhibited maximum reaction rate (Vmax) at 74.65 U/mg and Michaelis–Menten constant (Km) was determined to be 0.83 mM when alliin was used as a substrate.

The cytotoxic activity of in-situ generated allicin using purified alliinase and alliin was assessed on MIA PaCa-2 cell line using MTT assay and Acridine orange–ethidium bromide staining.

This approach of in-situ allicin generation suggests a novel therapeutic strategy wherein alliin and alliinase work together synergistically to produce cytotoxic agent allicin.