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GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY

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

^a Dep. of Agronomy, Univ. of Wisconsin-Madison, 1575 Linden Drive, Madison, WI 53706
^b USDA-ARS Cereal Crops Research Unit and Dep. of Agronomy, Univ. of Wisconsin-Madison, 1575 Linden Drive, Madison, WI 53706
^c Dep. of Crop and Soil Science, Oregon State Univ., Corvallis, OR 97331

^* Corresponding author (cahenson{at}wisc.edu)

There are two barley (Hordeum vulgare L.) ß-amylase genes encoding important starch-degrading enzymes. The endosperm-specific ß-amylase (Bmy1), the more abundant isozyme in cereal seeds, has been thoroughly characterized. The lesser abundant ß-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 -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 ß-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 ß-amylase2 that could be targeted for marker assisted selection.

Abbreviations: ESD, early seed development specific • MW, molecular weight • pI, isoelectric point • QTL, quantitative trait loci • SNP, single nucleotide polymorphism