离子色谱-脉冲安培法同时测定蔗糖酶和果聚糖酶活力的方法建立

本文旨在建立一种采用离子色谱仪同时测定蔗糖酶、果聚糖酶活力的方法。以蔗糖为底物，先加入蔗糖酶酶解并以硼氢化钠还原酶解产物葡萄糖和果糖得到甘露醇和山梨醇，再将蔗糖酶高温失活后加入果聚糖酶，得到含甘露醇、山梨醇、葡萄糖和果糖四种化合物的混合溶液。以水-氢氧化钠溶液-乙酸钠溶液为流动相进行梯度洗脱，在流速为1.0 mL/min、柱温为30 ℃条件下经CarboPaCTM PA1 250 mm×2 mm分离以测定四种化合物的含量。最后以甘露醇和山梨醇总量计算蔗糖酶活力，以葡萄糖和果糖的总量计算果聚糖酶的活力。结果表明:甘露醇、山梨醇、葡萄糖和果糖四种化合物在20 min内完成测定，且线性相关系数均达到0.9995以上，分离度均在1.0以上；果聚糖酶活力的测定可以采用蔗糖为底物；加入1.2 mL 10 mg/mL的硼氢化钠溶液并60 ℃水浴30 min后，葡萄糖和果糖能够分别完全转化为甘露醇和山梨醇；本方法测定的蔗糖酶活力、果聚糖酶活力与3,5-二硝基水杨酸比色法（DNS法）测定的蔗糖酶活力、果聚糖酶活力结果无显著性差异（P>0.05）；且测定的蔗糖酶活力、果聚糖酶活力含量结果的RSD分别为2.0%和1.7%，精密度好，且本方法不涉及传统方法所用的有毒有害试剂。因此，所建方法可以同时用于测定蔗糖酶和果聚糖酶的活力，同时也为其它酶活力测定提供了一种新的思路。

Establishment of Ion Chromatography-Pulse Amperometric Method for Simultaneous Determination of Sucrase and Inulinase Activity

This study aimed to establish a new method for simultaneous determination of sucrase and inulinase activity using ion chromatography. Sucrose was used as the substrate and enzymatic hydrolysised by sucrase. Sodium borohydride solution was applied for converting glucose and fructose to mannitol and sorbitol. Afterwards, sucrase was inactivated at high temperature, and inulinase was added to get the mixed solution containing mannitol, sorbitol, glucose and fructose. To determine the concentration of four compounds in the mixed solution, chromatographic separation was carried out using CarboPaCTM PA1 250 mm×2 mm column with water, sodium hydroxide and sodium acetate as gradient eluents (the flow rate of 1.0 mL/min and the column temperature of 30 ℃). Finally, sucrase activity was calculated based on the total amount of mannitol and sorbitol, and inulinase activity was calculated according to the total amount of glucose and fructose. The results showed that mannitol, sorbitol, glucose and fructose could be determined within 20 min. All the linear correlation coefficients were all above 0.9995, and resolutions were above 1.0. Thus, sucrose could be used as substrate for the determination of inulinase activity. Glucose and fructose could be completely converted into mannitol and sorbitol after adding 1.2 mL of 10 mg/mL sodium borohydride solution in a water bath at 60 °C for 30 min. The activities of sucrase and inulinase determined by this method did not show significantly differences compared to 3,5-dinitrosalicylic acid colorimetry determination method (DNS method) (P>0.05). The relative standard deviation of sucrase activity and inulinase activity determined were 2.0% and 1.7% respectively. The method did not involve toxic and harmful reagents used in traditional methods. Therefore, this method could be used to determine the activities of sucrase and inulinase simultaneously, and would provide a new idea for the determination of other enzyme activities.