Characterization of Barley Tissue‐Ubiquitous β‐Amylase2 and Effects of the Single Nucleotide Polymorphisms on the Enzyme's Thermostability

There are two barley ( Hordeum vulgare L.) β‐amylase genes encoding important starch‐degrading enzymes. The endosperm‐specific β‐amylase (Bmy1), the more abundant isozyme in cereal seeds, has been thoroughly characterized. The lesser abundant β‐amylase2 (Bmy2) has not been biochemically characterized from any cereal seeds. Characterization of Bmy2 from two commonly grown barley cultivars, Morex and Steptoe, was a major objective of this study. The bmy2 cDNAs were sequenced, expressed in Escherichia coli , and the recombinant enzymes (rBmy2) characterized. The relative hydrolysis rates of various α‐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 T 50 values, and is more thermostable than any reported wild‐type β‐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 β‐amylase2 that could be targeted for marker assisted selection.