植物源β-葡萄糖苷酶活性检测方法学研究  

Methodological Research on the Activity of β-Glycosidase from Plant Origin

作　　者：金璐[1] 王彦[1] 薛芸[1] 杨婵[1] 阎超[1] 

机构地区：[1]上海交通大学药学院,上海200240

出　　处：《现代生物医学进展》2016年第26期5032-5037,共6页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(21175092);上海市科委科研计划项目(15142200200)

摘　　要：目的:来源于植物β-葡萄糖苷酶活性检测的方法学研究。方法:以对硝基苯基β-D-葡萄糖苷(pNPG)为底物,β-葡萄糖苷酶为催化剂催化底物生成对硝基苯酚,用紫外分光光度法测定对硝基苯酚的含量,根据生成的对硝基苯酚含量计算酶活性,并对此进行方法学研究。结果:对硝基苯酚标准曲线的相关系数为0.999;催化反应的线性相关系数为0.999;反应精密度的相对标准偏差为0.05%;方法重复性的相对标准偏差为2.8%;方法耐用性考察中,酶活性均大于1200 U/g,九个不同方法参数的酶活性相对标准偏差为4.6%;中间精密度实验中,十二份样品酶活性的相对标准偏差为6.6%;稳定性实验中,β-葡萄糖苷酶与底物反应后,放置0小时、16小时、22小时后,测定的酶活性均大于1200 U/g,相对标准偏差为1.1%。结论:此方法线性较好,有良好的方法重复性、精密度、中间精密度、耐用性和溶液稳定性,适用于来源于植物的β-葡萄糖苷酶活性检测。Objective： Methodology research for the activity test of β-glycosidase from plants was conducted. Methods： The substrate of 4＇-Nitrophenyl-β-D-glucopyranoside （pNPG） was catalyzed by β-glycosidase to generate 4-nitrophenol. The enzymatic activity of β- glycosidase was determined through assay the concentration of 4-nitrophenol by the mean of ultraviolet spectrophotometry, and the methodology research for this method was fully evaluated. Results： The correlation coefficient of standard curve for 4-nitrophenol was 0.999; The linear correlation coefficient for catalytic reaction was 0.999; The relative standard deviation for precision was 0.05%; The relative standard deviation for method reproducibility was 2.8%; The enzyme activity was more than 1200 U/g and the relative standard deviation was 4.6% for nine different method parameters in the observation of method for durability; The relative standard deviation of enzyme activity for 12 samples was 6.6%, respectively, in the experiment of intermediate precision; For the stability experiment, three time points （0 hour, 16 hour, and 22 hour after the reaction ） were measured. The result shows that the activity of β-glycosidase is above 1200 unit/g after the reaction with the substrate （0 hour）, 16 hours later, as well as 22 hours later, where the RSD is 1.1%. Conclusion： The developed analytical method showed good linearity, repeatability, precision, intermediate precision, stability and durability. It can be applied in the determination of the activity for β-glycosidase from plants origin.

关 键 词：Β-葡萄糖苷酶 酶活性 方法学研究 

分 类 号：Q556.2[生物学—生物化学]