We think the Ceralpha Reagent would probably be best for this application.

The Ceralpha method is excellent for all alpha-amylases and will work fine for porcine pancreas alpha-amylase at pH 6.9.

This is the extinction coefficient, i.e. absorbance of a 1 mM solution of p-nitrophenol in a 1 cm light path at 400 nm.

The level of alpha-amylase in wheat flour is quite low, so in bread baked from this flour it is likely to be much lower.  The Ceralpha test is very sensitive, but you can increase sensitivity by increasing incubation time up to several hours (6 hours).  This should allow measurement of the enzyme if there is any there.

Cereal alpha-amylase assay reagent cannot be used at pH 3-4.  However, you can perform a similar assay using blocked p-nitophenol maltoheptaoside (BPNPG7). Basically dissolve the BPNPG7 vial contents in 10 mL with water.  Incubate 0.2 mL of your enzyme (buffered) with 0.2 mL of BPNPG7 for 10 min at a set temperature. Terminate the reaction by heating in a boiling water bath for 2 min.  Then add 0.2 mL of alpha-glucosidase (E-TSAGL; at 10 U/mL in 0.2 M tris buffer, pH 7.0) and incubate for 10 min.  Add 3 mL stopping reagent (Trizma base pH 8.5) and read colour at 400 nm.

The Ceralpha Kit does not distinguish between the different forms of alpha amylase, however some idea of relative levels of fungal and bacterial alpha-amylase can be obtained by performing the assay at different pH values.  Please refer to the Ceralpha kit booklet on this web site.

Cereal grains do need to be milled to allow effective extraction of alpha-amylase (and other constituents).  We recommend milling to pass a 0.5 mm screen.  You may have some success with a cheap coffee grinder.  However, the reproducibility will not be as good (it may be adequate for your requirements).

The Ceralpha method is routinely run at 40˚C, but it can be used at temperatures up to 60˚C.  If it is run at higher temperatures (up to 60˚C) then higher activity values will be obtained.  This assumes that the enzymes being analysed (alpha-amylase) are stable at these higher temperatures.  If you need to measure activity at temperatures above 60˚C you can use the Megazyme Amylazyme tablets or Red Starch.

The standard method should be fine.  You can best check this by performing a time course hydrolysis of the alpha-amylase.  This should be linear if the alpha-glucosidase in the kit reagent is not attacked by the protease.

Yes, only alpha-amylase can cleave the internal glycosidic bonds.

When alpha-amylase cleaves the glycosidic linkage in the blocked substrate, any other enzymes in the extract do not accelerate the hydrolysis to give free p-nitrophenol.  The reason for this is that the level of thermostable alpha-glucosidase in the substrate mixture is saturating.

Rice alpha-amylase can be assayed with Ceralpha kit.  Alpha-glucosidase in the rice extract will not interfere.

We have found that we get effective extraction from a range of cereal flours at room temperature (approx. 22˚C) and that the enzyme was very stable at this temperature for several hours.  For wheat flour, the optimal temperature for extraction is 40˚C (refer to the Ceralpha Booklet).

The Ceralpha method can be used for oats.  The only likely problem is the high viscosity of the extract.  If the extract looks very viscous, double the ratio of extraction buffer to flour.  Allow for this in the calculations.  If the absorbances are low then increase the incubation time to say 30 minutes.  Then allow for this increased time of incubation in the calculations.

We suggest that the fermentation broth is simply adjusted to 5.0 with acid or base and this can be used directly in the assay. The sensitivity can then be increased by increasing incubation time up to 4 hours.  Appropriate adjustments then need to be made to the calculations.

You can zero the spectrophotometer directly with the blank.  However, it is wise to know how high the blank is to ensure that the substrate is OK, i.e. the blank is usually 0.05-0.06.  If it is above 0.1, it has probably been contaminated with some alpha-amylase enzyme.

If the absorbance value is above 1.2 then the assay will be limited by the amount of available substrate.  Thus, you cannot simply dilute the colour.

To measure alpha-amylase on glass beads I would recommend the use of the Ceralpha method.  The kit is complete and extremely easy to use.  Perhaps to assay, you may freeze dry a small sample of the beads and then weigh an appropriate amount (say 5 mg) into a test tube.  Add 0.2 mL of buffer pH 5-6 and 0.2 mL Ceralpha reagent.  Incubate at 40˚C for 10 min.

The Ceralpha method should be fine for acid stable alpha-amylase.  However, assays should be performed in the pH range of 5.2 to 7.5.

For measurement of residual enzymes in bakery products, you should use the Amylazyme method rather than the Ceralpha method.  With the standard assay formats available, the sensitivity of the Amylazyme method is 10-20 times greater than can be achieved with the Ceralpha method.  You will need this greater sensitivity to measure the extremely low levels of activity present.

The level of alpha-amylase in malted barley is about 150 Ceralpha units per gram, which would be the expected level in germinated barley grain.

The pH of the assay solution after the sample is added should be the same as that of the assay buffer that is supplied with the kit.
Low sample volumes (e.g. 0.1 mL) are not likely to affect the pH of the assay solution and therefore may not require pH adjustment.