A New Method Of Using The Characteristic Of Enzyme To Detect Substrate

Enzyme, catalyst with high specificity and strong specificity, often be referred as an analytical reagent or analysis tools and can be used to detect the content of certain substances in the samples. People often used the characteristics of the enzyme test material existence. But the most are by measuring the quantity of the enzymatic reaction production of complex to indirectly detect the presence of trace material. It is hard to ensure the accuracy and precision of this method.This paper used alpha amylase and sucrose as the research object, trying to establish a new method for detecting substrate. Work content is as follows:After enzyme promoting reaction, the structure changes, fluorescence spectrum analysis method was used to study the fluorescence spectra of various enzyme and the effects of substrate on the quenching rate of the fluorescence of the enzyme. We found that different enzyme fluorescence quenching rate is different with the substrate influence on rate of enzyme fluorescence quenching irregularity, so this method cannot be used to detect the substrate.We found the phenomenon that the number of free amino acids increases in the enzymatic reaction liquid and the amount of substrate proportional to the amount of free amino acid in the reaction liquid, so we characterize the amount of the substrate by measuring the amount of free amino acids, and use ninhydrin colorimetric method to investigate the influence factors such as reaction temperature, pH, concentration of sucrose enzyme on the quantity of free amino acids.Results show that under the optimized conditions, a good linearity between the amount of free amino acid y and sucrose concentration x was observed within the range of the mass fraction of 0.02%~0.08% with the linear equation of y=351.67 + 0.0217x(R2 = 0.9997)and the detection limit of 37.07 μg/m L and the relative standard deviation of 1.75%~5.11%. The method of standard addition recoveries were in the range of 96.32% ~101.31%.Amylase also has this phenomenon, the method detection limit and relative standard deviation, standard addition recovery rate is 1317 μg/mL, respectively, 2.79%~5.88% and 92.82% ~ 100.86%. This method is simple, rapid, accurate and reliable, economical and practical.