1a.Objectives (from AD-416):
Objective 1: Evaluation of the functional expression of disproportionating enzymes in germinating and malted barley.
Objective 2: Identify differences in metabolites in malting quality and non-malting quality barley.
Objective 3: Evaluate C-terminal processing of ß-amylase on enzyme thermostability in malting barley grain.
1b.Approach (from AD-416):
Functional expression of disproportionating enzyme will be by biochemical measures of activity during germination, malting and mashing and through the use of kinetic characterization of the enzymatic reaction via HPLC analyses. Immunological monitoring of the fate of the protein will be employed to assist in determining the impact of industrial processing upon the enzyme. Differences in metabolites in malting and non-malting quality barleys segregating for winter hardiness, an agronomic trait of increasing value to the barley producers in North America, will be through the use of established methods of derivatization, gas chromatography and mass spectral detection/identification of metabolites followed by statistical analyses to include QTL detection and multivariate analyses. Evaluation of C-terminal processing of beta-amylase on the enzyme’s thermostability will be through the use of a series of immunological probes specific for known sites throughout the protein’s primary structure combined with evaluation of enzyme activity throughout a variety of temperature regimes selected for relevancy to industrial processing.
Disproportionating enzyme extraction has been optimized for buffer, detergent and reductant requirement and assay has been optimized for pH activity, substrate concentration and for HPLC detection of the maltotriose dependent reaction product. Remaining optimization is for assay temperature. A sufficient amount of antigenic sequence was obtained and antibodies are being made. The first two productions of antibody have been obtained and analyzed relative to the pre-immune sera.