Streamlined alpha-amylase assays for wheat preharvest sprouting and late maturity alpha-amylase detection

Authors: HAUVERMALE, AMBER - Washington State University; PARVEEN, REHANA - Washington State University; HARRIS, TRACY - Washington State University; TUTTLE, KEIKO - Washington State University; Mikhaylenko, Galina; NAIR, SINDHU - Washington State University; MCCUBBIN, ANDREW - Washington State University; PUMPHREY, MICHAEL - Washington State University; Steber, Camille

Submitted to: Agrosystems, Geosciences & Environment
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/2/2022
Publication Date: 1/1/2023
Citation: Hauvermale, A.L., Parveen, R.S., Harris, T., Tuttle, K.M., Mikhaylenko, G.G., Nair, S., McCubbin, A.G., Pumphrey, M.O., Steber, C.M. 2023. Streamlined alpha-amylase assays for wheat preharvest sprouting and late maturity alpha-amylase detection. Agrosystems, Geosciences & Environment. 6(1). Article e20327. https://doi.org/10.1002/agg2.20327.
DOI: https://doi.org/10.1002/agg2.20327

Interpretive Summary: Farmers received serious discounts when Late maturity alpha-amylase (LMA) and preharvest sprouting (PHS) lead to elevated alpha-amylase in wheat grain. Risk of poor end-product quality due to elevated alpha-amylase is detected in the wheat industry using the Hagberg-Perten falling number (FN) method. The FN method requires larger samples of 20 to 300 grams. Selection for PHS and LMA tolerance requires faster methods requiring a smaller sample size than the FN method. This study developed and evaluated methods that are faster and use smaller amounts of grain (one gram) than the FN method. We developed a 96-well method for the Phadebas™ alpha-amylase enzyme assay for wheat grain and compared this method to FN and to the Megazyme™ SD assay performed on the automated ChemWell-T robot. The Phadebas enzyme activity was highly reproducible and gave similar results to the SD enzyme assay and FN method regardless of milling method. Practical considerations for choosing an enzyme assay were described, and two methods for normalizing batch to batch variation in Phadebas substrate were described and validated. Both the 96-well Phadebas and Megazyme SD assays are suitable for alpha-amylase detection from small samples. The use of a coffee grinder to process small one gram samples gave similar results to mills requiring at least a 20 grams sample.

Technical Abstract: Background and Objectives: Late maturity alpha-amylase (LMA) and preharvest sprouting (PHS) lead to elevated alpha-amylase in wheat grain. Risk of poor end-product quality due to elevated alpha-amylase is detected in the wheat industry using the Hagberg-Perten falling number (FN) method. Selection for PHS and LMA tolerance requires higher throughput methods requiring a smaller sample size than the FN method. This study developed and evaluated a 96-well method for the Phadebas™ alpha-amylase enzyme assay for wheat grain and compared this method to FN and the Megazyme™ SD assay performed on the automated ChemWell-T Analyzer. Findings: The Phadebas enzyme activity was highly reproducible and showed a strong correlation to the SD enzyme assay and FN method regardless of milling method. Practical considerations for choosing an enzyme assay were described, and two methods for normalizing batch to batch variation in Phadebas substrate were described and validated. Conclusions: Both the 96-well Phadebas and Megazyme SD assays are suitable for alpha-amylase detection from small samples, and the use of coffee grinders to process small samples does not significantly impact assay performance. Significance and Novelty: Enzymes assays useful for wheat breeding are described, including a novel 96-well Phadebas assay.