核苷磷酸化酶酶活力检测方法的建立-《中国调味品》

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摘要:: 建立了一种核苷磷酸化酶酶活的检测方法，解决了工业化磷酸化酶质量无法判定的难题。建立核苷磷酸化酶的反应体系，以10 g/L肌苷、10 g/L三聚磷酸钠为底物，最适反应温度40 ℃，pH4.5，反应10 min，使肌苷在磷酸化酶的作用下生成肌苷酸二钠（IMP）。通过高效液相色谱法（HPLC）测定IMP的生成量，液相色谱条件：采用C18色谱柱，以0.02 mol/L磷酸二氢钾:乙腈=99.5:0.5（v/v）为流动相，1 mL/min等度洗脱，40℃，紫外检测器（λ=254 nm），进样量2 μL。此方法测定结果准确，回收率＞99.5%，重现性好，RSD＜0.5%。核苷磷酸化酶酶活检测方法的建立，能精准的判定酶液产品指标，准确的计算酶加量，解决了酶催化反应终点无法判定的问题。

Abstract:: A method for the detection of nucleoside phosphorylase activity was established, which solved the problem that the quality of phosphorylase could not be determined. The reaction system of nucleoside phosphorylase was established to make inosine under the action of phosphorylase to generate inosinic acid disodium (IMP). 10 g/L inosine and 10 g/L sodium tripolyphosphate were used as substrates, the optimal reaction temperature was 40℃, pH4.5, and the reaction was 10 min. The IMP production was determined by high performance liquid chromatography (HPLC), C18 column was used with 0.02 mol/L potassium dihydrogen phosphate : acetonitrile = 99.5 : 0.5 (V/V) as the mobile phase, 1 mL/min isocratic elution, 40℃, UV detector (λ=254 nm), and the injection volume was 2μL. This method is accurate, the recovery is more than 99.5%, and the RSD is less than 0.5% . The establishment of nucleoside phosphorylase activity detection method can accurately determine the product index of enzyme solution, accurately calculate the enzyme dosage, and solve the problem that the end point of enzyme catalytic reaction cannot be determined