Enzyme specificity assay

Department of Physiology and Biochemistry, College of Plant Science and Technology, Heilongjiang Bayi Agricultural and Reclamation University, China

Operation method

Enzyme specificity assay

Principle

One of the main differences between enzymes and general catalysts is that enzymes are highly specific, i.e., an enzyme can only catalyze one or a class of compounds. For example, sucrase can only catalyze the hydrolysis of sucrose, but not the hydrolysis of starch, while amylase can catalyze the hydrolysis of starch, but not the hydrolysis of sucrose. This experiment illustrates the specificity of enzymes by using the effects of sucrase and amylase on sucrose and starch as examples.

Move

I. Instruments and reagents:

1. Instruments:

(1) mortar; (2) test tubes: 6; (3) test tube rack; (4) small beaker; (5) graduated pipettes: 1 ml × 5, 2 ml × 1; (6) funnel; (7) electrically heated thermostatic water bath.

2. Reagents:

(1) 1% starch solution (containing 0.3% NaCl): 1 g of soluble starch and 0.3 g of sodium chloride, suspended in 5 ml of distilled water, stirred and then slowly poured into the boiling 60 ml of distilled water, stirred and boiled for 1 minute. Allow to cool to room temperature and then add water to 100 ml and place in refrigerator for storage.

(2) 2% sucrose solution: Sucrose is a typical non-reducing sugar. If the reducing sugar content of commercial sucrose exceeds a certain standard, it will be reducing, and this kind of sucrose cannot be used. Therefore, it must be checked before the experiment. The sucrose used in this experiment should be at least analytically pure reagent, to be used now.

(3) Amylase solution: fresh saliva diluted 200 times.

(4) Sucrase solution: take 1 gram of fresh yeast into a mortar and pestle, add a small amount of quartz sand and distilled water, grind for about 10 minutes, dilute to 50 ml with distilled water, let it rest for a few moments before filtration, and the filtrate is the sucrase extract.

(5) Benedict's (Benedict) reagent: 17.3 grams of copper sulfate dissolved in 100 ml of distilled water. After cooling, dilute to 150 ml. Take 173 grams of sodium citrate and sodium carbonate (Na2CO3-H2O) 100 grams of water 600 milliliters, heating to dissolve, cooling, diluted to 850 milliliters. Finally, pour the copper sulfate solution slowly into the sodium citrate-sodium carbonate solution, stirring while adding, and filter if there is precipitation. This reagent can be stored for a long time.

Second, the operation steps:

Take 6 test tubes numbered and operate according to the following table:

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