Contribution to the study of alliinase, the active principle of garlic

Abstract

Alliinase is a crucial enzyme in the Allium genus. The conversion of its substrates, cysteine sulfoxides, into volatile thiosulfinates is an important mechanism in the defence of the plant. It also provides the typical pungent flavour of garlic. As a matter of fact, the thiosulfinates decompose in a range of organosulfur compounds (OSC) known for their biological activities (antioxidant, anticancer, anti-diabetes properties, etc.). The environment of the decomposition of the thiosulfinates determines the type and amount of products released. A deeper knowledge of the conditions that form each OSC could help us in the preparation of garlic-based nutraceutics in the perspective of healthier food consumption. This study aims to improve the extraction and purification the enzyme alliinase in order to perform the reaction between the enzyme and its substrates and assess the potentialities of garlic preparations. Three methods have been applied to evaluate the efficiency of the extractions and purifications of the enzyme. A first idea of the purity of the enzyme is given by an electrophoresis separation of each sample on a polyacrylamide gel (SDS-PAGE). The protein content is then measured by UV-vis spectrometry with Lowry-Folin reagent for the coloration and BSA as an external standard. Finally, the specific activity is assessed by an indirect measure of the pyruvate (released as a co-product): the addition of NADH and lactate dehydrogenase turns the pyruvate in lactate, and the disappearance of NADH is measured by UV-vis spectrometry at 340 nm. The extraction of the enzyme from garlic was performed either by PEG 8000 precipitation or by ammonium sulphate precipitation. Two purifications were tested: affinity chromatography (on ConA) and size-exclusion chromatography. The combination of the ammonium sulphate process with the ConA chromatography provided the purest enzyme, with the best activity but a lower yield than the size-exclusion process. Finally the stability of the enzyme has been assessed at 4, -20 and -80 °C, showing that the enzyme could be kept at -80 °C for over 4 months without deterioration, while activity loss was observed at higher temperature