Abstract: In the present study, ammonium sulfate fractional precipitation, PEG 6000 precipitation and Sephadex G-200 column chromatography were sequentially carried out to separate and purify alliinase from crude garlic extract. SDS-PAGE was used to identify the enzyme and analyze its purity. The optimal extraction and separation conditions were determined as follows: alliinase was extracted with pH 6.5 phosphate buffer at 4 ℃, precipitated with 20% PEG 6000, and centrifugated at 2000 × g for 30 min. After purification by Sephadex G-200 column chromatography, the purification factor of the purified alliinase relative to the crude alliinase extract was 16.85 fold and activity recovery 41.26%. The subunit molecular weight of the alliinase was approximately 54.7 kD. An orthogonal array design was use to optimize the effects of three metal ions, glucose and sucrose on its activity. Moreover, the effect of metal ions on its reaction kinetics and UV-visible spectrum was also explored. The results showed that combinations of metal ions at various concentrations revealed a significant impact on alliinase activity. Mn2+ was the most important factor. In the presence of 10 mmol/L Mn2+, 10 mmol/L Fe3+, 5 mmol/L Ca2+, and 5 mmol/L sucrose, the highest relative activity of 155.59% was obtained. However, 10 mmol/L Cu2+ significantly inhibited alliinase activity. The Km was measured to be 0.25 mmol/L and Vmax was determined to be 1.29 μmol/min using alliin as the substrate. UV-visible spectroscopy was used to confirm the existence of the cofactor pyridoxal phosphate (PLP). The activation or inhibition of alliinase by metal ions may be due to changes in the structure of 5,-PLP and its binding to the main chain of alliinase.

Key words: metal ion, alliinase, purification, enzymatic properties