In the related biochemical testing of enzyme preparations, in addition to measuring the content, concentration, and stability of various indicators to be tested, there are also indicators for detecting enzyme activity, such as blood sugar, blood lipids, and creatinine. For indicators such as transaminase and sarcosine oxidase, enzyme activity needs to be measured.
Generally speaking, enzyme activity is the activity of enzyme catalysis, which involves a kinetic parameter of the enzyme-the Michaelis constant Km, which means the concentration of the substrate (S) when the enzymatic reaction reaches half of the maximum speed (Vm) . The speed of the enzyme reaction is not uniform, not linear like the concentration, but the number of meters in Changshu is constant, which is a characteristic physical quantity of the enzyme. The Michaelis constant of the enzyme changes with the measured substrate type, reaction temperature, pH and ionic strength. Under the same conditions, no matter what the concentration of the enzyme is, the concentration of the required substrate is the same when the maximum reaction rate is reached.
Measurement method of Michaelis constant of sarcosine oxidase:
Prepare a sarcosine solution with a concentration of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0mmo1/L with Tris-HC1 buffer of pH 8.0, and react at 37°C for 5 minutes Determine the initial oxidation activity of SOX at different sarcosine concentrations. The reaction rate V and 1/V are obtained. According to the Lineweaver-Burk double reciprocal mapping method, with 1/[s] as the abscissa and 1/V as the ordinate, the Km and Vmax of SOX to sarcosine were calculated.
Conclusion of Michaelis constant of sarcosine oxidase:
The Michaelis constant can be used to judge the specificity of the enzyme. The smaller the Km value, the greater the affinity between the enzyme and the substrate, and the more suitable the substrate is as the natural substrate of the enzyme. According to the test and calculation of sarcosine oxidase above, the Michaelis equation is y=1232.5X+8.7012 and the correlation coefficient is 0.9947: the calculated Km and Vmax values of SOX to sarcosine are 141.6mmol/L and 0.115mmol, respectively /(L.min).
It should be noted that sarcosine oxidase SOX from different sources has a certain difference in the Michaelis constant of sarcosine. For example, the Km value of SOX from Corynebaclerium to sarcosine is 21mmol/L. Since the Michaelis constant has nothing to do with the concentration of the enzyme and the type of enzyme, this point can also be used for enzyme identification or determination. Desheng Biochemical focuses on the field of biochemical detection, and provides a variety of enzyme preparations including SOX, cholesterol esterase, glucose oxidase, etc.
