ARS | Publication request: CHARACTERIZATION OF BARLEY TISSUE-UIBIQUITOUS B-AMYLASE2

Submitted to: North American Barley Research Workshop Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: February 15, 2005
Publication Date: July 17, 2005
Citation: Clark, S.E., Hayes, P.M., Henson, C.A. 2005. Characterization of barley tissue-uibiquitous b-amylase2 [abstract]. North American Barley Research Workshop Proceedings. Paper No. 5.

Technical Abstract: There are two barley b-amylases genes, encoding important starch degrading enzymes. The endosperm-specific b-amylase (Bmy1), the more abundant isozyme in cereal seeds, has been thoroughly characterized. The lesser abundant b-amylase2 (Bmy2), has not been biochemically characterized from any cereal seeds. Characterization of Bmy2 from two commonly grown barley (Hordeum vulgare L.) cultivars, ‘Morex’ and ‘Steptoe’, was a major objective of this study. The bmy2 cDNAs were sequence, expressed in Escherichia coli, and the recombinant enzymes (rBmy2) characterized. The relative hydrolysis rates of various a-D-glucans and the pH activity optima of ‘Morex’ and ‘Steptoe’ rBmy2s were the same and not significantly different from barley rBmy1. The ‘Morex’ rBmy2 was 7'C more thermostable than the ‘Steptoe’ rBmy2, determined by differences in their T50 values, and is more thermostable than any reported wild type b-amylase1. Three amino acid differences were identified between the two Bmy2 sequences and the contributions to enzyme thermostability evaluated by site-directed mutagenesis. Examination of mutant enzymes with one amino acid substitution revealed that each of the three residues contributed ~3'C to the thermostability of the ‘Morex’ wild type rBmy2. Mutant enzymes with two amino acid substitutions contributed ~5.6'C and the triple amino acid mutant enzyme contributed ~8.7'C to thermostability. To date, no quantitative trait loci (QTL) for malting quality traits have been associated with the bmy2 locus. Should an association be discovered, the ‘Morex’ bmy2 allele, containing D238, M337 and Q362, provides a discrete signature of a thermostable b-amylase2 that could be targeted for marker assisted selection.