Purification And Properties Of Alliinase From Fresh Garlic

Garlic (Allium Sativum L.) is a number of species of the liliaceae. It was a widely distributed plant and used in all parts of the world not only as a spice and a food, but also as a popular remedy. In addition, it effected on blood coagulation, lipid- and cholesterol-lowering, blood pressure, antithrombic, and anticancer activities were under discussion. Allicin and other sulfur containing compounds were suggested as active compounds in garlic. The extremely unstable allicin itself is liberated from the more stable alliin by the enzyme alliinase if fresh garlic is crunched. Therefore , products of allicin is due to alliinase. This paper included two parts, one part research was to purify the alliinase from fresh garlic, another one was to research optimum reactive conditions and reaction kinetics of alliinase.Garlic bulb was blended, the precipitate was removed by centrifugation. Solid ammonium sulphate was added to the supernatant to give 25~55% saturation, and the precipitate was collected by centrifugation, then suspended in the phosphate-glycerol solution. Further purification was achieved by passing samples of the enzyme through sephadex G-200 column. By enzyme assay and gel electrophoresis of alliinase, the results showed that a 14-fold purification of alliinase was obtained, and one most intensive band at Mr =53kD was detected which is in accordance with literature values.Purified alliinase optimum reactive conditions and reaction kinetics by alliin as basic substance were studied. The Km and Vm values for enzyme activity on alliin at 35℃ were 0.693mmol/L, 0.353?mol/min, which accords with Michaclis-Menten equation. The optimum temperature for activity was 30℃, range of thermal stability was below 50℃. Isoelectric focusing of alliinase, the major bands were visualized at pH 4.82. Identification of an essential residue in alliinase by chemical modification, the results indicated that Trp residue played an important role in enzyme catalysis.We had purified alliinase from fresh garlic, and studied on its properties. This study provided theoretical foundation for development and application of Garlic in xinjiang.