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Measurement of α-amylase in pre-harvest sprouted wheat
V. A. MCKIE (1), B. V. McCleary (2), C. Cornaggia (1), D. Mangan (1), R. Ivory (1). (1) Megazyme International Ireland, Bray, County Wicklow, Ireland; (2) Megazyme International Ireland Ltd., Bray, County Wicklow, Ireland

The aim of this research was to develop sensitive and rapid manual and automated procedures for the measurement of α-amylase in sprout damaged (weather damaged) wheat.

Method: 4,6-<i>O</i>-benzylidene (Bz) methylumbelliferyl-G7 (BzMUG7) and Bz-2-chloro-4-nitrophenyl-G7 (BzCNPG7) were synthesised and used along with benzylidene-(BzNPG7) and ethylidene-4-NPG7. Each of these was prepared in the presence of saturating levels of α-glucosidase and ß-glucosidase (where appropriate) and used for the measurement of the trace levels of α-amylase present in sound and sprout damaged wheat flour. The major focus was on reducing the incubation time required to get reaction absorbance (fluorescence) values substantially higher than blank absorbance values; reducing absorbance due to interfering compounds in the extract, and reducing reaction incubation times to allow the method to be suitable for use in breeding programmes and at grain receivable points.

Results: With substrates based on Bz-4-methylumbelliferyl-G7 (BzMUG7), sensitivity was increased approx. 8-fold using a fluorimetric detector, meaning that incubation times could be reduced to 3-5 min. A similar reduction in incubation time could be achieved with  Ethylidene-4-NPG7 by altering the volumes of the reagents and sample extract used in the assay. This assay was further simplified by adapting it to a ChemWell-T autoanalyser, which had the added advantage of reducing the amount of reagent required per test. There was good correlation of results obtained with each substrate mixture across 30 samples of sound and sprout damaged wheat.

Conclusion: Simple procedures for manual and automated measurement of α-amylase in sprout damaged wheat have been developed. These methods should prove useful for evaluation of wheat lines in wheat breeding programmes and for rapid evaluation of wheat at grain collection points.

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