The rate of sucrose hydrolysis was determined by measuring the amount of reducing sugars (glucose and fructose) produced, and in this experiment, the unit of sucrase activity refers to the amount of enzyme required for each milligram of glucose produced for 5 min of reaction under certain conditions. The aim of this experiment was to determine the protein content and sucrase activity of each grade.
Operation method
kosmochromate blue method (chemistry)
Principle
The rate of sucrose hydrolysis was determined by measuring the amount of reducing sugars (glucose and fructose) produced. In this experiment, the viability unit of sucrase refers to the amount of enzyme required to produce 1 mg of glucose in 5 min of reaction under certain conditions. Protein content was determined by the Caulophylline blue method, and specific activity was defined as the number of units of activity per milligram of protein.
Materials and Instruments
Protein Move 1. Determination of sucrase activity of each grade Ⅰ, Ⅱ and Ⅲ For more product details, please visit Aladdin Scientific website.
Acetic acid buffer Glucose Sucrose Dinitrosalicylic acid
Spectrophotometer Water bath Test tubes Cling film
Dilute the enzyme solution of each grade with 0.02 mol/ L, pH 4.9 acetic acid buffer (deionized water of pH 5-6 can also be used as a substitute), and determine the appropriate dilution times of the enzyme activities.
Ⅰ: 1,000 to 10,000 times
Ⅱ : 1,000~10,000 times
Ⅲ : 100 to 1,000 times
The above dilutions are for reference only.
The reagents were added into the test tubes in the order of measurement. To simplify the operation, the sealing of the plastic wrap could be canceled, and the heating of boiling water bath was changed to 90-95 ℃ water bath for 8-10 min. The relationship between the reaction rate and enzyme concentration was drawn with the number of milligrams of reducing sugar produced in 5 min as the vertical coordinate, and the enzyme concentration of 1 ml of reaction mixture in the test tubes (mg of protein/ml) as the horizontal coordinate. 2.
2. Calculate the specific activity, purification times and recoveries of each grade.
Determination of protein content is the same as experiment 3 of protein series. In order to measure and calculate the data in the following purification table, each fraction must be sampled, and every time a sample is taken, a portion of the volume will be lost for the next fraction, so the volume of the next fraction should be corrected, so that the calculation of the recovery rate will not be adversely affected.