Study On Purification And Enzymatic Properties Of Alliinase

Garlic(Allium Sativum L) is a number of species of the liliaceae.It was used all over the world not only as a kind of food,but also as a popular remedy with a long history.Allicin and other sulfer-containing compounds were suggested as pharmacological active compounds in garlic,which were liberated from more stable alliin by alliinase when fresh garlic was crunched.Obviously,Alliinase plays an important role as a physical active material.Alliinase and alliin which are separately extracted from fresh garlic processed into garlic drugs and health products is an important development trend for the current garlic industry.Therefore,it has good commerical prospects.In this paper fresh garlic bulb was extracted by phosphate buffer and homogenated;then the deposition was filtered and removed by centrifugation;Solid ammounium sulphate was added to the supernatant gradely,and the deposition was collected by centrifugation.Further purification was through passing the Sephadex G-200 column with the samples of enzyme after ultrafiltered.The multiples and effect of purification of alliinase was determined by enzyme assay and gel electrophoresis of alliinase.Purified alliinase was used as testing solution,The enzymatic properties of alliinase from garlic were studied by synthetical S-allyl-L-cysteine sulphoxide as basic substrate.Considered by emzymatic properties of alliinase,the effect of enzymatic regulating technology on allicin yield was also researched by orthogonal design,the results show:1,The optimum purification conditions of alliinase:45%saturation of solid ammonium sulphate was added to the supernatant,the deposition was removed by centrifugation and dissolved by pH6.5 phosphate buffer,then the ultrafiltered enzyme was passed the Sephadex G-200 column.The optimum eluting time of alliinase by gel chromatography was 4.75h,which was mainly eluted between 3.25～5.75h.By enzyme assay and gel electrophoresis of alliinase,the results showed that a 16.2-fold purification of alliinase was obtained,and recovery was64.7;SDS-PAGE showed that no unpurposed propein was found,and one most intensive band at Mr=52.6kD was detected,which is consistent with literature reports.2,The emzymatic properties of alliinase:the carbohydrate content of alliinase was 2.25%by Phenol-sulfuric acid,and the isoelectric focusing point of alliinase is located in pH 4.9.The stability results of temperature and PH showed:the activity of alliinase was not steady in different temperature,which becomed lower with the increase of temperature and standing time;As usual,the activity of alliinase was steady relatively at 20℃.The optimum reactive temperature was 30℃,little acid conditions is effective to keep the activity of alliinase,the optimum pH was 6.3;The values of Km and Vmax were 5.91 mmol.L^-1 and 1.55umol.min^-1 by synthetical S-allyl-L-cysteine sulphoxide as basic substrate.Most metal ions had effect on alliinase,among of which activation effect of Mg²⁺,Zn²⁺,Fe²⁺,Fe³⁺,EDTA-Na were obivous,Cu²⁺ was badly inhibitive to alliinase activity.3,The effect of enzymatic regulating technology on allicin yield was examined: the results of orthogonal design showed,the effect of the factors of enzymatic regulating technology on allicin yield was:enzymatic temperature,enzymatic pH,enzymatic time,[Fe³⁺],the optimum effect is A₁B₂C₂D₃,which were as follows: temperature 30℃,pH was 6.3,enzymatic time was 20 min and[Fe³⁺]was 10 mM. The proven repeated experiment showed the allicin yield achieved 0.231%.