P-8
Characterization of barley tissue—Ubiquitous beta-amylase
Presenter: Cynthia Henson, USDA-ARS-CCRC, Madison, WI
Co-Authors: S.E. Clark, University of Wisconsin, Madison, WI; and P.M. Hayes, Oregon State University, Corvallis, OR

Barley has two genes that encode beta-amylases, which are important starch-degrading enzymes. The endosperm-specific beta-amylase (beta-amylase1; Bmy1), the more abundant isozyme in cereal seeds, has been thoroughly characterized. The lesser abundant isozyme, beta-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 sequenced, expressed in Escherichia coli, and the recombinant enzymes (rBmy2) were characterized. The relative hydrolysis rates of various alpha-D-glucans by 'Morex' and 'Steptoe' rBmy2s were the same and were not significantly different from hydrolysis rates of barley rBmy1. The rBmy2s had maximal activity between pH 5.0 – 6.5, similar to rBmy1. The rBmy2 from 'Morex' as 7°C more thermostable than that from 'Steptoe' as determined by differences in their T(50) values, and is more thermostable than any wild-type beta-amylase1 reported to date. Three amino acid differences were identified between the two Bmy2 sequences. The contributions of these differences to enzyme thermostability were evaluated by site-directed mutagenesis. Examination of mutant enzymes with one amino acid substitution revealed that each of the three amino acids contributed ~3°C to the thermostability of the wild-type rBmy2 from 'Morex'. Mutant enzymes with two amino acid substitutions contributed ~5.6°C to the thermostability. The 'Morex' Bmy2 allele, containing D238, M337, and Q362, provides a discrete signature of a thermostable beta-amylase2 for use in breeding programs employing marker-assisted selection.

Cynthia Henson is a research plant physiologist with USDA-Agricultural Research Service and is a member of the Cereal Crops Research Unit in Madison, WI. She is also an associate professor of agronomy at the University of Wisconsin-Madison.