| Stevia rebaudiana Bertoni is a perennial root herb species in the genus Stevia of the Asteraceae.Stevia is well known for its sweet leaves,and it gets more concern because it is the Contains glycosides and product with the character of high sweetness(250?400 times of sucrose)and low calorie(1/300 of sucrose),and the medicinal value of prevention and adjuvant treatment of diabetes,hypertension and so on.Steviol glycosides is a general name of a variety of tetracyclic two terpenoid glycosides with steviol as aglycones.It’s worth mentioning that there are differences in characteristics and functions of steviol glycosides caused by different glycosyl groups linked to the C19 and C13 sites of steviol.Stevioside(St)and Rebaudioside A(RA),content of total glycosides from 60%to 80%,are the two major components in stevioside.RA is more glucoside than St at C-13,C3’ in structure.And there are reciprocal transformation by glycosylation and deglycosylation between them.β-glucosidase is an important glucoside hydrolase,and must participate in the mutual transformation of the two glycosides.In this paper,the β-glucosidase was purified from stevia plant by ion exchange chromatography and gel filtration chromatography with the cultivar ’Zhongshan No.5’as material,and the physicochemical properties and the enzyme activity difference among cultivar,organs,and growth periods of stevia were studied.The main research contents are as follows:1.The separation and purification of β-glucosidase was made from the leaves of’Zhongshan No.5’.a high St and low RA variety,which was Budding period.Firstly,the enzymewas coarsely extracted by fraction precipitation by ammonium sulfate,and the extraction conditions were optimized.And stevia p-glucosidase was further purificated by dialytic desalination,ion exchange chromatography and gel filtration chromatography.The results show that the activity of the enzyme is higher when the ammonium sulfate is gradually added to the system until the saturation is 40%.Meanwhile,the enzyme activity was higher when the extract buffer was PBS with pH 7.0,and theenzyme activity was relatively higher at 37℃ for lh.The crude enzyme was desalted fully with dialysis,and the enzyme was further purified by the anion exchange agent DEAE-52 and Sephadex G-200 dextran gel,which was prepared for the follow test.After purification,the molecular weight of stevia β-glucosidaseis about 55KD,and the purification fold is 10.22 times。2.The properties of p-glucosidase from stevia were analyzed by the influence of temperature,pH,metal ions and inhibitors to enzyme activity,as well as pH stability,thermal stability and preservation stability of the enzyme.The results show that at the range of 20℃ to 40℃ enzyme activity was relatively high,enzyme activity decreased gradually after.And the reaction system were put into ice bath and water bath of 30℃,40℃,50℃,60℃,determined the enzyme activity after 30,60,90,150 and 180min incubated,respectively.The result shown that the enzyme activity decreased gradually,but incubated at 60℃ for 180min,the enzyme activity was 71.52%.Then we have used different pH to extract the β-glucosidase from stevia.When the extract pH was 7.0,the enzyme activity was relatively high.And the enzyme pH stability was studied by solution enzyme with buffer of different pH,and kept at 4℃ for 24h,then measured its enzyme activity.The results showed that enzyme activity was higher with pH 6.0～8.0.When we add Mn2+,Fe2+and SDS into the reaction system,the enzyme activities were all larger than those of the control,which promoted the enzyme activity,and the enzyme activity was inhibited in the reaction system containing Hg2+,Cu2+ and Co2+,respectively.The enzyme activity was measured every two days in 28 days under the condition of 4oC,and the results showed that the enzyme activity gradually decreased with the time.3.The β-glucosidase activity from different organs,different growth periods and different varieties of stevia were analyzed.The resuts indicated the enzyme activity in the leaves was relatively higher than root,stem,and flower of stevia cultivar ’Zhongshan No.5’.The enzyme activities of leaves of stevia plants at different growth stages were analyzed.The results showed that the activities of enymes in flowering stage was highest,and their activities:The Stevia rebaudianum of β-glucosidase activity in the flowering stage was the largest,followed by the budding period,and finally the rapid growth period.We also analyzed the enzyme activities of the leaves from ’Zhongshan No.5’,’Zhongshan No.6’,’Zhongshan No.8’,’Huajing’,’Jiangtian No.2’ and ’Shoutian No.3’ and results reflected’Huajing’and ’Zhongshan No.5’ had the highest enzyme activity,while ’Huajing’ and’Zhongshan No.5’,were high St varieties.Followed by ’Zhongshan No.8’ and ’Zhongshan No.6’,the enzyme activities o’f Shoutian No.3’ and ’Jiangtian No.2’ were relatively low. |
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