The Research On The Extraction, Characterization And Immobilization Of Myrosinase

Myrosinse(EC3.2.3.1), which exists in Cruciferae family, canhydrolyze glucosinolates into glucose, sulfate, isothiocyanates, nitrile andthiocyanate. The enzymatic products, especially isothiocynate, haveseveral biological activities. Sulforaphane, which is hydrolyzed fromglucoraphanin, has the best anticancer effects, and broccoli has the mostcontent of glucoraphanin. The daily process (like cooking) would affectglucosinolates transform into isothiocyanates. Insure the enoughisothiocyantes, the purified products should be studied. Therefore, usingmyrosinase to pruduce more anticancer products is necessary, which cangain the better anticancer effect. The extraction, purification,characterization and immobilization of myrosinase in broccoli have beenstudied.In order to gain more and purer myrosinase, the extraction andpurification of myrosinase in broccoli have been studied. The extraction of myrosinase can be well gained using the Tris-HCl extraction buffer(pH7.5). The cell can be well ground into fine powder using liquidnitrogen. The specific activity can be well gained using ethanol with 2fold. Myrosinase would be more active by adding DTT, glycerin, PMSFand EDTA. The purified myrosinase can be gained by three steps: Ethanolwith 2 fold, DEAE-Sepharose Ion exchange chromatography andSuperdex^TM-200 gel filtration chromatography. The purified myrosinasewere 1.75 times, 31.1 times and 45.65 times than the crude myrosinase,and the yield were 64％, 29％and 9％respectively. The specific activityof myrosinase in seed is higher than which in other organs.Only in certain conditions, myrosianse would have the best activity.The characterization of myrosinase has been studied. The research hassuggested that the molecular weight of myrosinase was about 50KDa.The optimum pH and temperature were 6.0 and 45℃, myrosinase werestable below 55℃and pH ranging from 5-7. The K_m value of myrosiansewas 1.95mmol/L with sinigrin as substrate. When adding 1mmol/L Vc,the total activity of myrosinase would be about twice as the one whichwithout adding 1mmol/L Vc.Enzyme can not be used time after time or automatization, theimmobilization of myrosinase has been studied. The myrosinase wasimmobilized on calcium alginate carrier by cross-link with glutaraldehyde.The effects of sodium alginate concentration and glutaraldehyde concentration on immobilization were analyzed. The optimum pH andtemperature, the pH stability and temperature stability of immobilizedmyrosinase were determined. The results show that the high recovery rateof immobilized myrosinase could be obtained under the conditions of 4％sodium alginate and 0.2％glutaraldehyde. The range of optimum pH ofimmobilized myrosinase was wider than before, and the pH stabilitywould stroger. The optimum temperature of immobilized myrosinase wasthe same as before, but the temperature stability would stronger. Below65℃, the immobilized myrosinase was stable. The K_m value was5.10mmol/L, the affinity to sinigrin was weaker. The total activity wouldbe 70％than before.