高效液相色谱法测定糖化酶中葡萄糖苷转移酶的活性

摘要

An analytical method for the determination of the activity of transglycosidase in dia-static 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 trans-formation of the trisaccharide was detected on a SUGAR SH1011 column( 300 mm × 8. 0 mm,6μm)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. 999 8). The limit of detection and the limit of quantita-tion 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 bat-ches 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.%建立了糖化酶中葡萄糖苷转移酶活性测定的高效液相色谱方法。以麦芽糖为底物，阿卡波糖为抑制剂，在37℃、pH=4．8醋酸缓冲液介质中葡萄糖苷转移酶作用下将麦芽糖转化为三糖。采用 SUGAR SH1011色谱柱（300 mm×8.0 mm，6μm）分离，以0.01 mol/L硫酸水溶液为流动相，流速0.6 mL/min，用示差折光检测器检测测定三糖的转化量，间接测定葡萄糖苷转移酶的活性。对色谱条件、底物浓度、抑制剂用量及培养时间等条件进行了系统考察。在优化的条件下，三糖在0.1~10 g/L范围内线性关系良好（ r=0.9998），葡萄糖苷转移酶活性检出限和定量限分别为0.013 U和0.043 U，6次平行测定的相对标准偏差（ RSD）为0.63％。在优化条件下测定了不同批次糖化酶中葡萄糖苷转移酶的活性，结果良好。该方法操作简便，稳定性高，可用于葡萄糖生产原料糖化酶中葡萄糖苷转移酶活性的测定。