β-amylase

Safety

Results

Methods

β-amylase breaks every second bond in starch producing maltose, but cannot pass the branches (1,6 linkages) in amylopectin so the blue/black iodine complex with soluble starch is only partially reduced. The appearance of maltose is usually used to measure the rate of reaction, but the disappearance of the blue iodine complex can be used if amylose is the substrate.

Sweet potato is an excellent source of β-amylase, (EC 3.2.1.2).

Enzyme activity can be shown using soluble starch as the substrate and DNSA or Benedicts reagent to measure the maltose produced. There is some reduction in the colour with iodine solution during the reaction which can be measured with the colorimeter.

An alternative method is to use amylose prepared from potato starch as the substrate as this is completely broken down to maltose, (since there are no branches in the chains to stop the progress of the enzyme), and the blue colour obtained with iodine solution will disappear completely.

If you have a centrifuge the β-amylase from sweet potato can easily be partially isolated by precipitation with ammonium sulphate, (or completely isolated and crystallised if you are prepared to go through a rather lengthy series of precipitation and resuspension stages).

Sweet potato β-amylase is a stable enzyme and will keep well in a refrigerator.

Reaction of amylose with sweet potato β-amylase

Enzyme extraction

Sweet potato (Ipomoea batatas) is crushed in a pestle and mortar, or blended, with water using 1cm³ of water for each gram of sweet potato. 30g of sweet potato gives a useful amount of extract and we have found that large sweet potatoes give a better yield of enzyme than small, young potatoes, though small ones still give good yields.

The extract is filtered through several layers of butter muslin.

If a centrifuge is available it can be used to remove cell debris but the extract will give good results without this stage.

The extract will keep for several weeks refrigerated without much loss of activity.

Reaction mixture

Suitable conditions for this reaction are a temperature of 35°C in pH6 buffer.

The buffer/substrate mixture should be allowed to equilibrate to the reaction temperature before being added to the enzyme.

Using soluble starch or amylose as the substrate and measuring the production of maltose.

• Mix 1.5cm³ of a 5% solution of soluble starch (or 1.5cm³ of amylose from potato starch) with 1.5cm³ of buffer.
• Add this to 0.1cm³ of the enzyme extract to start the reaction.
At intervals of 1 minute remove 0.3cm³ and add to 0.3cm³ DNSA reagent.

Using amylose as the substrate and measuring the colour of the blue complex formed between amylose and iodine.

• Mix 2cm³ of amylose from potato starch with 2cm³ of buffer.
• Add this to 0.1cm3 of the enzyme extract to start the reaction.
• At intervals of 30 seconds remove 0.1cm³ into 3cm³ of iodine solution, (2% iodine stock solution in 0.1MHCl).
• Read absorbance using red light (635nm).

Preparation of amylose from potato starch

0.16M NaOH and 0.6M HCl are IRRITANT

Katakuriko flour used in Japanese cooking is potato starch and works well with this method.

• Mix 0.5 grams of potato starch with 5cm³ of water.
• Add 55cm³ of 0.16M NaOH and swirl the mixture gently. The suspension will separate into a clear layer and a cloudy precipitate.
• Allow this to stand with occasional swirling for 5 minutes. During this time amylose will separate from amylopectin.
• Neutralise by stirring in 15cm³ of 0.6M HCl containing 0.75g NaCl.
• Centrifuge the mixture – amylopectin will sediment as a glutinous pellet and the supernanant will contain a solution of amylose which can be used as the substrate for β-amylase.

Amylose gives a bright blue colour with iodine solution.

Purification of β-amylase by precipitation with ammonium sulphate

Ammonium sulphate is widely used for salting out proteins. Ammonium sulphate takes up the water molecules around the protein exposing hydrophobic sites. These hydrophobic groups cause the protein molecules to stick together and come out of solution.
The method described below will concentrate and partially purify β-amylase from sweet potato, but much greater degrees of purification are possible using narrower ranges of ammonium sulphate concentration, (and precipitation with propanone).


Powdered ammonium sulphate should be used.
All procedures should be carried out ice cold.


• Add ice cold sweet potato extract (pre-centrifuged to remove cell debris) to powdered ammonium sulphate (1.76g per 10cm³ of extract) with stirring to dissolve. This solution is 30% saturated.
• Allow this to stand for 15 minutes then centrifuge, discard the pellet and add the supernatant to ammonium sulphate, (1.62g per 10cm³ of supernatant), stirring to dissolve. This solution is 55% saturated.
• Allow to stand, ice cold, for 15 minutes then centrifuge, discard the supernatant, and resuspend the pellet in pH6 buffer.
• This solution, the 30-55% saturation fraction, should contain virtually all of the enzyme activity.

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