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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

View larger version (75K): [in a new window]   Fig. 1. Aligned deduced amino acid sequences of the endosperm-specific (Bmy1) and tissue-ubiquitous (Bmy2) ß-amylases from the barley cultivars Morex and Steptoe. Morex Bmy1 is identified as M-Bmy1. Steptoe Bmy1 is identified as S-Bmy1. Morex Bmy2 is identified as M-Bmy2. Steptoe Bmy2 is identified as S-Bmy2. The asterisks label identical amino acids found in all four sequences. The two ß-amylase signature sequences are highlighted. The three single amino acid differences between the two Bmy1 and between the two Bmy2 enzymes are boxed.

View larger version (23K): [in a new window]   Fig. 2. Thermostability profiles of the ‘Morex’ and ‘Steptoe’ wild-type recombinant ß-amylase2 enzymes and the mutant enzymes made to assess the impact of single nucleotide polymorphisms on the thermostability. Enzymes were incubated for 10 min at temperatures from 0 to 70°C, and remaining activity was determined at 40°C. Means and standard deviations of activities from four to eight separate colonies are shown for each temperature. (A) Morex values are shown as squares and Steptoe values are shown as triangles. (B) Activities of the D238G are shown as open circles. M337T activities are shown as open triangles and the Q362R activities are shown as open squares. The Morex and Steptoe wild-type rBmy2 enzymes' thermostability profiles are shown for comparison. (C) Activities of the D238G/M337T double mutant rBmy2 are shown as open circles. Activities of the M337T/Q362R double mutant are shown as open squares. Activities of the D238G/Q362R double mutant are shown as open triangles. Morex and Steptoe wild-type rBmy2 profiles are shown for comparison. (D) Activities of the D238G/M337T/Q362R triple mutant are shown as open circles. Morex and Steptoe wild-type rBmy2 enzyme thermostability profiles are shown for comparison.

View larger version (26K): [in a new window]   Fig. 3. Enzyme activity expressed as a function of assay pH for the ‘Morex’ and ‘Steptoe’ wild-type rBmy2s. The solid and cross-hatched bars represent activities of the Morex and Steptoe rBmy2, respectively. Data are expressed as means ± standard deviations of activity expressed by three colonies.