The Effect Of Ultrasound On The Properties And Conformation Of Alliinase

In this paper, alliinase was purified from fresh garlic using ammonium sulfate fractionation and column chromatography with Sephacry1 S-200. The purified alliinase showed homogeneity on SDS-PAGE with molecular weight of subunit of 54,000. The effect of ultrasound on the enzymatic property, conformation, and chemical modification was studied, and the effect of exogenetic alliinase on the yield of allicin was also investigated.The optimum ultrasound conditions for mono-frequent ultrasound (frequency of 135 kHz, intensity of 0.5 W/cm²) and dual-frequency ultrasound (28KHz/40KHz with recombination action) were obtained. Under the optimum ultrasound conditions, the alliinase activity was enhanced by 46%-47%.The optimal temperature of the alliinase was 35℃and the optimal pH value was and 6.5. The ultrasounic treatment couldn't change the optimum temperature and pH of the enzme, while could enhance the thermostability of the enzyme. The effect of K⁺, Fe²⁺, Mg²⁺ and PLP and L-cysteine on the activity of alliinase was changed under the ultrasound conditions. The activation energy of the enzyme was reduced with ultrasonic treatment.The fluorescence spectrum of alliinase indicated that the fluorescence signal came from Trp, Tyr, Phe, and PLP, and ultrasound treatment may change the microenvironment of alliinase, leading to the alteration of the enzyme conformation. The CD spectrum of ultrasonicated alliinase showed a different composition with increasedα-helix andβ-turn and reducedβ-sheet fractions in secondary structure compared with alliinase without ultrasonic treatment. The X-ray diffraction showed that the three-dimensional conformation of alliinase was changed after ultrasound treatment.The modification of NBS, ch-T, DEPC and IAA resulted in a great loss of alliinase activity, suggesting that Trp, Met, His, and amino- amino acid involved in the enzyme active sites. The active site of alliinase more required Met and His under ultrasound conditions..The appropriate concentration of exogenous alliinase could increase the yield of allicin. And ultrasound could improve the yield of alliicin further.