Comparison of the agar‐plate diffusion method for α‐Amylase assay with the colorimetric stationary‐stage method

The general reaction conditions of the diffusion test on agar plate were studied for the determination of α‐amylase enzyme activity, and a standardised agar‐plate diffusion method for this assay was developed. Four wheats and five commercial α‐amylase preparations were used as test material for comparing the plate‐test method with the colorimetric stationary‐stage method. A straight‐line relationship between the log enzyme quantity and the plate‐test response, measured as total area of the reaction spot surface, was observed. An arbitrary enzyme activity unit (the reciprocal value of the absolute enzyme quantity, which corresponds to the plate‐test response) showed a fair correlation with the activity value obtained by the colorimetric stationary‐stage method. The use of the straight‐line relationship enabled the standardisation of the plate‐test method without an enzyme standard as reference, i.e. on a substrate basis, while still yielding, in a dilution series of the sample solution, a fair estimate for the enzyme activity in S.K.B. units (Sandstedt‐Kneen‐Blish) directly from the plate‐test results. The sensitivity of the plate test was much higher than that of the colorimetric stationary‐stage method. This was because the straight‐line portion of the plate‐test curve easily covered an enzyme concentration range diluted several logarithmic units lower than that of the colorimetric curve which seldom covered more than one logarithmic unit.