Assay of Salivary Amylase enzyme activity

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. This protocol is the basic laboratory procedure for the assay of salivary amylase activity.

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.

assay of salivary amylase activity

What is Amylase?

Amylase is an enzyme responsible for breaking down carbohydrates, it is found in saliva and pancreatic juice, and it helps the body digest food. Amylase is also used in brewing and baking, as it helps to convert starch into sugar.

What is the salivary amylase structure?

Salivary amylase is an enzyme that is found in saliva. It is responsible for breaking down carbohydrates into simpler sugars. The structure of salivary amylase is a long, spiral-shaped molecule. It comprises two chains of amino acids, which are held together by weak bonds.

These bonds can be easily broken by water, so saliva is necessary for the enzyme to work. Salivary amylase is produced by the salivary glands and released into the mouth when we eat. It then breaks down carbohydrates in the food we eat into simpler sugars.

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.
  • The 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.

Basic Classification of Amylase

There are three basic types of amylase: alpha, beta, and gamma. 

  • Alpha-amylase is found in saliva and is responsible for the initial breakdown of starch into maltose. 
  • Beta amylase is located in the pancreas and produces maltose from the hydrolysis of the maltose molecule. 
  • Gamma amylase is found in the small intestine and completes the hydrolysis of maltose into glucose.

What are the functions of salivary amylase?

  • Salivary amylase is an enzyme secreted in the saliva of humans and other mammals. 
  • This enzyme is responsible for breaking down complex carbohydrates such as starch into simpler sugars such as maltose. 
  • Salivary amylase is present in high concentrations in the saliva of young children and decreases as we age. It is one of the reasons babies tend to drool more than adults.
  • Salivary amylase is essential for the digestion of carbohydrates. 
  • When we eat foods that contain carbohydrates, the salivary amylase begins to break down the complex carbohydrates into simpler sugars. 
  • This process starts in the mouth and continues in the stomach and intestines.
  • Without salivary amylase, we would not be able to digest carbohydrates properly and would not get the total nutritional value from these foods.

This article providing information on “Salivary amylase, functions, and Assay of Salivary amylase activity”.

Assay of Salivary Amylase activity

Aim

To determine activity of Amylase enzyme in Saliva

Principle

Amylase is the hydrolytic enzyme that breaks down many polysaccharides like Starch, Amylose, dextrins, and yields a disaccharide i.e., Maltose.

(C6H10O5)n  + H2O  →  n(C12 H22 O11)

Reagents

  1. 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.
  2. 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.
  3. 1% Sodium chloride: It is necessary for enzyme activity
  4. 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.
  5. Standard solution of Maltose: It is prepared by dissolving 200mg Maltose in 100ml of water (2mg / 1ml).

Procedure

  • 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 a 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.

Result

The amount of Maltose in the given unknown sample is _________ grams of Maltose formed per 100ml of enzyme per one hour.

Assay of amylase activity table

Calculation

  • 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

Get this protocol in PDF format. Just download this “Color Reactions of Carbohydrates” file, make a print, and distribute it to the students. It helps you protect your students from spelling mistakes and volumetric errors. All the best

Importance of Assay of Salivary amylase enzyme

This is a very important test to detect diabetes

Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycemia (high blood sugar levels) due to insulin resistance or absolute lack of insulin production. It has long been known that salivary gland dysfunction leads to glucose intolerance and ultimately to type 2 diabetes mellitus.

The diagnosis of saliva amylase enzyme activity deficiency requires an oral test with a standard protocol. Saliva can be collected using a calibrated collection device, and the resulting sample should ideally be analyzed within 15 minutes after collection.

This study aimed at evaluating the efficacy of salivary amylase enzyme activity assay to detect diabetes mellitus. One hundred twenty five patients were included in the present study.

Their age ranged between 25 – 65 years. All subjects were subjected to fasting blood glucose estimation followed by measurement of plasma glucose level after 75g of glucose ingestion. Based upon the plasma glucose value, all subjects were divided into three groups.

Those with normal plasma glucose had group I, those with impaired glucose tolerance had group II, and those who were diabetic had group III. After obtaining informed consent, unstimulated whole saliva was collected from each subject into a sterile container for 30 minutes and subsequently centrifuged immediately.

From these samples, salivary amylose enzyme assays were carried out. According to the obtained results, the sensitivity of salivary amylase assay was found to be 95% and specificity was 100%.

Salivary amylase analysis is a simple procedure and the results obtained can be used as an additional criterion in the diagnostic process of diabetes. Therefore, this method should be considered as a valuable tool in clinical practice.

Final words

This protocol helps you to understand the applications of amylase enzyme. It is an important enzyme and has wide range of functions like decomposing starch molecules in the human body. This article will discuss the various applications of amylase. It is an important enzyme and has wide range of functions like decomposing starch molecules in the human body. This article will discuss the different applications of amylase.

Amylase digests starch by catalyzing hydrolysis, which is splitting by the addition of a water molecule.

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