在该研究中使用包括ONPG和乳糖的两种底物测量β-半乳糖苷酶活性。通过遵循从ONPG释放的邻硝基苯酚的量来测量ONPG的β-半乳糖苷酶活性。反应混合物由100μL酶溶液和400μLONPG溶液(2.5g/L,在pH6.8的100mM Tris-HCl缓冲液中)组成。在78℃温育15分钟后,通过加入等体积的1M Na 2 CO 3终止反应。通过在A405测量来定量测定释放的邻硝基苯酚。一个活性单位定义为在测定条件下每分钟产生1μmol邻硝基苯酚所需的酶量。比活性表示为每毫克蛋白质的单位。在含有100μL酶溶液和5%乳糖的相同缓冲液中进行对乳糖活性的测定,并通过煮沸10分钟终止反应,并使用葡萄糖氧化酶 – 过氧化物酶测定试剂盒测定葡萄糖浓度。通过测量A492定量测定释放的葡萄糖。一个酶活性单位定义为每分钟释放1μmol葡萄糖所需的活性量[1]。
[1]. Zhang X, et al. Metagenomic approach for the isolation of a thermostable β-galactosidase with high tolerance of galactose and glucose from soil samples of Turpan Basin. BMC Microbiol. 2013 Oct 24;13:237. doi: 10.1186/1471-2180-13-237
α-Glucosidase inhibitory assay(5 mM PNPG,37℃, 30 min)
10 μL of α-glucosidase, 10 μL of 0.1 mg/mL Up, Up-3, Up-4, and Up-5, 80 μL of PBS buffer (0.02 mol/L, pH 6.8) and 10 μL of 5 mM PNPG were mixed in a 96-well plate, and then the mixture was incubated at 37℃ for 30 min. The presence of the product, p-nitrophenol (PNP) was monitored by measuring absorbance at 405 nm in a plate reader. Acarbose was used as a positive control. Reactions without inhibitor were also performed in parallel serving as controls. The PNP concentration was determined from a standard curve. The relative activity was expressed as a percentage of the PNP formation in the presence of acarbose or polysaccharides compared with that of the control reactions. Subsequently, the α-glucosidase inhibitory activity of acarbose, Up-3 and Up-4 at different concentrations (0.01, 0.03, 0.05, 0.08, 0.1, 0.3, 0.5, 1.0, and 2.0 mg/mL) were determined according to the method described above to calculate the IC50 value.
来源文献:Zhong QW, Zhou TS, Qiu WH, Wang YK, Xu QL, Ke SZ, Wang SJ, Jin WH, Chen JW, Zhang HW, Wei B, Wang H. Characterization and hypoglycemic effects of sulfated polysaccharides derived from brown seaweed Undaria pinnatifida. Food Chem. 2021 Mar 30;341(Pt 1):128148. doi: 10.1016/j.foodchem.2020.128148. Epub 2020 Sep 22. PMID: 33038776.