α-amylase

Safety

Results

Methods

α-amylase randomly breaks the bonds between glucose residues in starch. The product is a mixture of small saccharides including maltose. The appearance of reducing sugars can be measured, but the easiest method of following the reaction is to measure the disappearance of the blue/black starch-iodine complex.

α-amylase (EC 3.2.1.1) is present in saliva. Salivary amylase, (ptyalin), is readily available and is considered safe to use when simple precautions are followed. There are a number of different bacterial and fungal α-amylases available in liquid or powdered form.

The reaction between soluble starch and α-amylase can be followed by monitoring the disappearance of the blue/black starch-iodine complex or the appearance of reducing sugars using DNSA reagent or Benedict’s reagent .

Salivary amylase is good for showing the effects of temperature and pH on enzyme activity as it is relatively sensitive to both conditions, though the optimum temperature is considerably higher than is usually given in school textbooks.

Contrary to widespread belief there has never been a national recommendation in UK against the use of saliva in schools and it is considered to present low risk. The following precautions should be followed: Each student should collect and use only THEIR OWN saliva. After use test-tubes and other laboratory equipment that has been in contact with saliva should be soaked in a sterilising solution such as dilute bleach before being washed up.
	If powdered enzymes from bacteria or fungi are used then precautions must be taken when preparing solutions as the enzyme powders may provoke allergic reactions. Solutions should be prepared in a fume cupboard, or with care while wearing a dust mask. Skin contact with powdered enzymes and prepared solutions should be avoided.

Absorbance vs Time - 30°C, pH7

Effect of temperature

Effect of pH

To collect salivary amylase

• Rinse the mouth out first with water.
• Take a little water into the mouth, swirl it around and spit out into a container labelled to identify the person whose saliva it contains.
• After use the container should be soaked in a sterilising solution before being washed up.

Reaction mixture

A suitable reaction protocol for α-amylase is as follows;

• 0.5cm³ of a 5% solution of soluble starch
• 4.4 cm³ of pH7 buffer, (the reaction will work with water instead of buffer).
• 0.1 cm³ of amylase, (salivary amylase or a 0.1% solution of bacterial or fungal amylase).

Before adding it to the enzyme the substrate/buffer mixture should be equilibrated to the reaction temperature; 35°C works well, but lower temperatures, though slower, may be easier to maintain.

At 30 second intervals remove 0.1cm³ of the reaction mixture and add it to 3cm³ of iodine solution, (2% iodine stock solution in 0.1M HCl)

Read absorbance using red light (635nm).

Click to download a pdf file containing much of the information about this topic.