The Activity And Property Of Myrosinase From Cruciferous Seeds

Myrosinase is aβ-thioglucosidase glucohydrolase that catalyses the hydrolysis of the glucosinolates. Some aliphatic glucosinolates, such as sinigrin, glucoraphenin (GRA), and glucoraphenin (GRE) have attracted much attention owing to their myrosinase-mediated breakdown products which have been considered as promising compounds for fighting cancer. Therefore, the study of myrosinase have attracted the interest of many medicinal scientists, food chemists and biologists. In the dissertation, the enzymatic properties of myrosinases from cruciferous vegetable seeds towards three glucosinolates were researched.In this study, myrosinases from eight kinds of cruciferous vegetable seeds were extracted and purified preliminarily by ammonium sulfate sedimentation and dialysis. Three kinds of glucosinolate were used as substrates to compare the activities of myrosinase samples separated from different cruciferous vegetable seeds. Then, broccoli seeds myrosinase was purified to homogeneity by Sephadex G-100 gel filtration chromatography. The specific activity of purified myrosinase was 102U·g-1, which was 5.1 times than before and the recovery rate of 30%. The property of myrosinase and influence of ascorbic acid, NaCl, EDTA and glucosinolates breakdown products on broccoli myrosinase were investigated.Experimental results showed that the hydrolysis abilities of myrosinase from eight cruciferous seeds were significantly different towards different substrates mentioned above. With sinigrin as the substrate, myrosinase from broccoli seeds displayed the highest specific activity which value was over 8 times higher than myrosinase from yellow mustard seeds. Myrosinase from broccoli seeds exhibited high activity in the range of (pH 5.0-8.0) and temperature (45 and 60℃). The stability of myrosinase was all right at pH 6.0-8.0, with temperature rising, the stability of myrosinase was decreased. Sinigrin as the optimal substrate, the Km and Vmax values for the purified broccoli enzyme were estimated to be 0.067mM and 0.729μmol·min-1, respectively. The activity of myrosinase was also affected by some chemistry reagent, at optimum ascorbic acid concentration (1.6mM), the specific activity of broccoli myrosinase enhanced 4.3 times compared with no ascorbic acid. At concentration broad 1-2mM, the activation effect of ascorbic acid to myrosinase was better, but no activation when concentration was over 4.5mM. NaCl existed salt stress effect on myrosinase, and high salt concentration made against activity of myrosinase. There was activation effect of EDTA to myrosinase at concentration broad 0-1.4mM, but the activation effect went stabilization over 1.4mM. Among the three breakdown products of glucosinolate, only two kinds of isothiocyanates had suppression effect on activity of myrosinase. With glucose and SO42-, there was no suppression effect.The activity and enzymatic property study between myrosinase and substrates provided an ideal myrosinase source and enzymatic reaction conditions. Therefore, it is of great importance for the further study of three glucosinolate degradation products (Sulforaphane, Sulforaphene, AITC).