分光光度法检测酶促反应中底物的量

Spectrophotometry method to detect the substrate of enzymatic reaction

基于酶与底物结合出现游离氨基酸增多的现象建立了检测酶促反应底物存在的新方法。以蔗糖酶与α-淀粉酶为研究对象,采用茚三酮比色法考察了反应温度、pH、酶浓度等因素对因反应造成游离氨基酸量的影响。结果表明,在50℃、pH=8、0.1%的蔗糖酶条件下,因反应变化的游离氨基酸的量与蔗糖质量分数在0.02%～0.08%呈线性关系,方法检出限为37.07μg/mL,相对标准偏差为1.75%～5.11%,方法加标回收率为96.32%～101.31%。α-淀粉酶有相似的结果,方法检出限、相对标准偏差、加标回收率分别为1.317mg/mL、2.79%～5.88%、92.82%～100.86%。

A new method for determination of enzymatic reaction substrates based on the phenomenon that free amino acids increased in the process of enzyme and substrate mixture was proposed.The effects of reaction temperature,pH,enzyme concentration on the free amino acids were investigated using invertase andα-amylase as the research object by ninhydrin colorimetry.Results showed that a good linearity between the amount of free amino acids and sucrose concentration was observed within the range of the sucrose mass fraction of 0.02%-0.08% with the linear equation in the optimum conditions of 50 ℃,pH =8and 0.1% invertase.The detection limit was 37.07μg/mL and the relative standard deviation was 1.75%-5.11%,while the method of standard addition recoveries were96.32%-101.31%.The result ofα-amylase was similar with invertase,of which the method detection limit,relative standard deviation and standard addition recovery rate were 1.317 mg/mL,2.79%-5.88% and 92.82%-100.86%,respectively.