Assay of Salivary Amylase enzyme activity
This article providing the information on “Salivary amylase, functions, and Assay of Salivary amylase enzyme activity”.
An enzyme is a protein molecule that is a biological catalyst with three characteristics.
- The basic function of an enzyme is to increase the rate of a reaction.
- Most enzymes act specifically with only one reactant, called a substrate, to produce products.
- Most remarkable characteristic is that enzymes are regulated from a state of low activity to high activity and vice versa.
The activity of enzymes is strongly affected by changes in pH and temperature. Each enzyme works best at a certain pH and temperature, its activity decreasing at values above and below that point due to denaturation. For enzymes, denaturation can be defined as the loss of enough structure rendering the enzyme inactive. This is not surprising considering the importance of tertiary structure in enzyme function and non-covalent forces in determining the shape of enzymes
Salivary amylase is the enzyme produced by the salivary glands. Formerly known as ptyalin, it breaks down starch into maltose and isomaltose. Amylase, like other enzymes, works as a catalyst. All catalysts are enzymes, but not all enzymes are catalysts. A catalyst is a substance that hastens a chemical reaction but does not become part of the end product.
Amylase digests starch by catalyzing hydrolysis, which is splitting by the addition of a water molecule.
The presence and absence of starch can be confirmed by several tests such as the
- Iodine test, Benedict’s and Fehling’s test. In general, a blue-black color indicates the presence of starch
The below given salivary amylase experiment is most common practical protocol. You can download the experiment protocol in PDF format.
Assay of Salivary Amylase enzyme activity
To determine activity of Amylase enzyme in Saliva
Amylase is the hydrolytic enzyme which breaks down many polysaccharides like Starch, Amylose, dextrins and yields a disaccharide i.e., Maltose.
(C6H10O5)n + H2O → n(C12 H22 O11)
- Substrate (Starch) : Mix 1 gm of soluble starch in 200ml of 0.1M Phosphate buffer (pH 6.8) boil for 3 minutes and cool to room temperature and filter it necessary.
- Enzyme: Saliva is the best and easily available source of amylase. Collect some saliva in a beaker and dilute it to 1:20 dilution with distilled water.
- 1% Sodium chloride: It is necessary for enzyme activity
- DNS (Dinitro Salicylic acid): Dissolve 1.6 gm of NaOH in 20ml of distilled water. Take 1gm of 3,5 DNS in NaOH solution. In other beaker take 30gm of Sodium potassium tartrate. Dissolve in 50ml of distilled water. Mix this DNS solution and finally make the volume up to 100ml with distilled water.
- Standard solution of Maltose: It is prepared by dissolving 200mg Maltose in 100ml of water (2mg / 1ml).
Take 0.5ml of substrate and 0.2ml of 1% NaCl in a test tube and pre-incubated at 370C for 10 minutes then add 0.3ml of diluted saliva and incubate for 15 minutes at 370C. Stop the reaction by addition of 1 ml of DNS reagent mix well and keep the test tubes in boiling water bath for 10 minutes. Cool and dilute with 10ml of distilled water. Read the color developed at 520 nm. Simultaneously setup the color developed at 520nm. Simultaneously setup the blank as per the test by adding DNS prior to the addition of enzyme simultaneously. Set up the standards of different test tubes and repeat the experiment as per the test and measure the color developed at 520nm absorbance.
Preparation of Phosphate buffer:
Dissolve 0.2M (2.7218 grams) of KH2PO4 in 100ml of distilled water to this solution add 0.5M (2.8053 grams) KOH drop by drop till the pH is set to 6.8. Then make it to 200ml with distilled water. So the final concentration is 0.1M of 200ml Phosphate buffer.
The amount of Maltose in the given unknown sample is _________ grams of Maltose formed per 100ml of enzyme per one hour.
- 1.5 mg of Maltose formed / 0.3. ml / 15 minutes
- 1.5 X 4 mg of Maltose formed / 0.3 ml of Enzyme / 1 hour
- 1.5 X 4 X 3.3 mg of Maltose formed / 1ml of Enzyme / 1 hour
- 1.5 X 4 X 3.3 X 100 mg of Maltose formed / 100ml of Enzyme / 1 hour