Laboratory of Food

Chemistry and
Biochemistry

Standard operating
protocol
Determination of
Amylase activity

SOP no. LMCB-2

Determination of Amylase activity with Amylazyme tablets

Principle
The amylase activity is measured spectrophotometrically by the
Amylazyme method (Megazyme, Bray, Ireland). The coloured
substrate used is an Amylazyme tablet. This tablet contains
azurine-crosslinked amylose, which readily hydrates in water but is
water insoluble. Hydrolysis by -amylase produces water soluble
dyed fragments, and the rate of release (observed as an increase in
absorbance at 590 nm) can be related directly to enzyme activity.
This substrate specifically measures -amylase activity in the
presence of large excesses of -amylase and amyloglucosidase.

Materials
Water bath
Test tubes 20 mL
Centrifuge tubes
Centrifuge
Shaker
Automatic pipets and tips
Vortex
Timer
Filter paper
Small funnels
Small beakers
Spectrophotometer at 590 nm
Disposable cuvets (4 mL)

Products
Amylazyme tablets
Amylazyme tablets are available directly from Megazyme in
pack sizes of 200 or 1000 tablets. The tablets are unbuffered,
allowing their use over a wide pH range. The substrate is very
stable and can be used at temperatures up to 60C, however
the substrate is not stable in alkaline solutions at high
temperatures.

Buffers and solutions

- Sodium maleate buffer (100 mM, pH 6.0) containing 5mM
CaCl2
Dissolve maleic acid (11.6 g, S104) in 700 mL of distilled
water add calcium chloride dihydrate (0.74 g, S23) and
adjust the pH to 6.0 with sodium hydroxide solution (2 M),
which is prepared by dissolving 80 g sodium hydroxide
pellets (S121) per L deionised water, (approximately 80 mL
is required). Adjust the volume to 1 litre. Store at 4C
temperature.
!Wear protective gloves!
-

Tris-hydroxymethylaminomethaan (TRIS
2% solution;
S174):
Put 20.0 g TRIS in a volumetric flask and add deionised
water to 1000 mL
!Wear protective gloves!

Samples
- Grains or course materials should be ground before
extraction with IKA mill or cyclotec mill into powders
- Powder (flour, bran and wholemeal) extracts:
- Put 1 g powder + 10 mL sodium maleate buffer (100
mM, pH 6.0, 5mM CaCl2) in a big falcone tube and
manually shake the sample shortly to ensure all
powder is hydrated. Work in duplicate or triplicate, and
make different extracts of the same sample.
- Shake the tubes for 30 min on the shaker at speed 150
at 7C in the fridge. In mean time, cool the centrifuge
to 7C by bringing empty buckets in balance and
centrifuge them for 25 minutes on 5 or 6C.
- Centrifuge the samples for 10 min at 4000 rpm, 7C
(always do the same for your samples!)
- Filtrate the samples
- Samples with high sprout damage: dilute the extract
20 times (1 ml extract + 19 ml sodium maleate buffer).
- Use extracts immediately for further analysis and store
extracts in fridge (7C) only if necessary, amylase
activity decreases upon storage in fridge.

Procedure
Powder (flour, bran and wholemeal) extracts:
- Pipet 1.00 mL of powder extract in a big test tube. If you
work in duplicate, you need three tubes (2 samples + 1
specific blank) per extract and also 3 tubes with buffer
instead of extract as control (2 buffer + 1 buffer blank).
- Pre-incubate the tubes for 5 min at 40C
- Add an Amylazyme tablet as substrate in 2 of the 3 tubes, 1
tube should be incubated without tablet (= specific blank).
- Incubate the samples for exactly 30 min (work correct!!)
(leave 30 sec between the different tubes, and for the blank
leave 30 sec and do nothing)
- Stop the reaction by adding 10 ml TRIS (2% solution), vortex
- Filtrate
- Measure the extinction values at 590 nm
Important:
- There is no need to add a tablet to the blank samples after
the reaction has been stopped with TRIS. This is redundant
as it adds the same value to the E590 of the sample and the
control, which are subtracted from each other.
- If the signal is too high, you should incubate the sample for
shorter times rather than diluting the sample. If you divide
the time by 5, it is ok to multiply the obtained activity by 5. If
you dilute the extract 5 times, multiplying the activity with 5
will result in an overestimation.
- If its necessary to dilute the samples, you have to use
sodium maleate buffer (100 mM, pH 6.0, 5mM CaCl2)
Calculations
Amylase activity of extracts is expressed as E 590 per gram
sample and per hour for extracts
[E590 sample E590 blank sample (E590 control E590 blank
Result =control)] x 10 x V
weight sample x hours of incubation
With:
E590 sample =

extinction at 590 nm of sample with tablet

(average of 3 values)
E590 blank sample =
extinction at 590 nm of sample
without tablet (1 value)
E590 control =
extinction at 590 nm of control with tablet
(average of 3)
E590 blank control =
extinction at 590 nm of control without
tablet (1 value)
10 =
dilution factor made by making extract

weight sample =

extact weight of the milled sample

used in the test

V = further dilution of the initial extraction solution

Waste
- Enzyme dilutions and extracts: waste depends on the buffer used
- Tris 2%: non-halogenated organic solutions (waste category 3, red
waste bin)