关键词: 高效液相色谱, 活性, 葡萄糖苷转移酶, 三糖, 糖化酶

Abstract:

An analytical method for the determination of the activity of transglycosidase in diastatic enzyme by high performance liquid chromatography (HPLC) was established. Taken as the substrate, maltose was transformed into trisaccharide by transglycosidase in a 37 ℃ water bath and acetic acid buffer solution (pH=4.8) with acarbose as transglycosidase inhibitor. The transformation of the trisaccharide was detected on a SUGAR SH1011 column (300 mm×8.0 mm, 6 μm) with 0.01 mol/L sulfuric acid solution as mobile phase at a flow rate of 0.6 mL/min and a differential refractive index detector (RID), in order that the activity of transglycosidase can be measured indirectly. The conditions such as the chromatographic conditions, the concentration of substrate, the usage of inhibitor, and the incubation time were investigated. Under the optimized separation conditions, the calibration curve of the trisaccharide showed good linearity within the mass concentrations of 0.1-10 g/L (r=0.9998). The limit of detection and the limit of quantitation for transglycosidase activity were 0.013 U and 0.043 U, respectively. The relative standard deviation was 0.63% for six parallel tests. The activities of transglycosidase from different batches of diastatic enzyme were also determined with good result. The method can be applied to determine the activity of transglycosidase in the diastatic enzyme of the producers’ raw materials with the advantages of convenience, simplicity and stability.

Key words: activity, diastatic enzyme, high performance liquid chromatography (HPLC), transglycosidase, trisaccharide