Preliminary Studies On Activity,Purification And Characterization Of β-glucosidase In Chimonanthus Praecox L.

β - glucosidase is one of the key enzymes, which plays an important role in alcoholic aroma formation of fragrant flowers. Under the optimized conditions for the enzyme extracting and activity analysis, we compared the changes of β- glucosidase activity during different stages of florescence among different cultivars of Chimonanthus praecox (L.). Furthermore, β - glucosidase was purified and its characterization was studied for the first time. This will establish theoretical foundation for further research on the mechanism of aroma formation in the flowers of Chimonanthus praecox (L.) in the future. The main results are as follows:1 Optimization of conditions for extracting and activity analysis of P - glucosidaseUsing p-nitrophanyl β -D-gluopyranoside (pNPG) as substrate, the conditions for extracting and activity analysis of β - glucosidase in the fresh flowers of Chimonanthus praecox (L.) were studied. The results indicated that the amount of polyvinypolypyrrolidone (PVP) had great impact on β- glucosidase activity when the enzyme was extracted. With the amount of PVP increasing, the enzyme activity had the trend to ascend. It attained the apex when the amount of PVP was equal to that of fresh flowers. The results also showed that β -glucosidase activity was effected by different buffers and pH values. As pH value rose to 5.0, β - glucosidase activity increased to max., and then decreased. At the same pH value, enzyme activity was the highest with citric acid- citric acid Na buffer. Enzyme activity analysis revealed that the highest asorbance value could be observed when testing wavelength was 410nm. It was found that the enzymatic reaction was stable at 37℃ in 120min. In conclusion, the extracting conditions of β - glucosidase were that the amount of PVP should be equal to that of fresh flowers using citric acid- citric acid Na (pH 5.0) as extracting buffer. The enzymatic reaction should be performed at 37℃ in 120min and the testing wavelength ought to be 410nm.2 Changes of β - glucosidase activity during different stages of florescence among different cultivarsThe changes of β - glucosidase activity among four cultivars of Chimonanthus praecox (L.), namely Chimonanthus praecox var. concolor I , Chimonanthus praecox var. concolor II, Chimonanthus praecox var. intermedius and Chimonanthus praecox var. grandiflora, were investigated in this paper. As a result, from yellow bud to full-blown,the tendency of the 3 - glucosidase activity was Chimonanthus praecox var. concolor II > Chimonanthus praecox var. grandiflora > Chimonanthus praecox var. concolor I > Chimonanthus praecox var. intermedius. During different stages of florescence, the changes of 6- glucosidase activity were similar among the four cultivars. The trend was yellow bud > initial stages of florescence > full-blown > green bud > withering. From yellow bud to full-blown the enzyme activity preserved rather high though it decreased slowly, then rapidly from full-blown to withering. It hinted that the change of aroma in Chimonanthus praecox (L.) flowers was related to the endogenous B- glucosidase activity.3 Isolation and purification of 3 - glucosidaseBy the methods of ammonium sulfate precipetation and column chromatography on CM-52 Cellulose, 3 - glucosidase in the flowers of Chimonanthus praecox var. concolor II was purified. Three factions, namely Glucosidase- I ,Glucosidase- II and Glucosidase-HI, were obtained. Through Sephadex G-100 gel filtration chromatography, Glucosidase-III, the main faction of 3 - glucosidase, was father purified 12.6 fold with a specific activity of 0.244U/mg and a yield of 2.4%.4 Research on characterization of 3 -glucosidaseResearch on the characterization of the purified Glucosidase-III proved that the enzyme showed optimal activity at pH 4.0 and 50X2. It was stable in the pH range from 4.0 to 6.0 and temperature range from 30'C to 40*C. The enzyme might be consisted of two monomers, which molecular weights were 64.5 and 42.0 kDa, respectively. It was also indicated that the apparent Michaelis constant for pNPG was 0.23mmol/L and Vmax was 12.24U/L. The enzyme activity was inhibited by Hg2+, Ag+and SDS strongly, but slightly by Cu2+and Fe2+. The survival rate of enzyme activity remained over 80% when stored at 4°C for a month. So it could be concluded that Glucosidase-III had a strong affinity for pNPG, exhibited poor thermostability and was evidently inhibited by some metal ions and inhibitors.