|Clark, Suzanne - UNIV. WISCONSIN|
|Hayes, Patrick - OREGON STATE UNIV.|
Submitted to: Crop Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 7, 2004
Publication Date: August 1, 2005
Citation: Clark, S., Hayes, P., Henson, C.A. 2005. Characterization of barley tissue-ubiquitous beta-amylase and the effects of single nucleotide polymorphisms on enzyme thermostability [abstract]. Crop Science. 45:1868-1876. Interpretive Summary: During seed germination, the degradation of starch, which is stored in cereal seeds, provides the sugars and energy required for seedling growth. This starch degradation process is also the basis of the malting and brewing industries, but instead of supporting seedling growth the sugars are used by brewer's yeast during fermentation. The industrial starch degradation process takes place at high temperatures which limit the function of some of the barley enzymes that degrade starch. The work conducted here was designed to identify more heat-tolerant enzymes in barley cultivars already adapted for growth and production in North America. Additional objectives were to characterize these enzymes and to provide information to plant breeders that they can use to select plants with more heat-tolerant enzymes. We identified one enzyme in a commonly grown barley variety that is significantly more heat-tolerant than the same enzyme from other barley varieties. We determined which parts of the primary structure of the enzyme are responsible for the enhanced heat-tolerance observed. These three parts of the enzyme's primary structure can be used as markers for the heat-tolerant enzyme by plant breeders who incorporate marker-assisted selection techniques in their breeding programs. Such techniques are used in essentially all of the public and private sector barley breeding programs in North America. It is expected that this tool will lead to the development of barley varieties better suited for use in industrial processing than those varieties currently available.
Technical Abstract: Barley seeds have two different genes that encode beta-amylases, which are one of the most important enzymes that degrade starch during germination. The endosperm-specific beta amylase (beta-amylase1) has been thoroughly characterized because it is the more abundant of the two isozymes in germinating cereal seeds. The second and lesser abundant isozyme is beta-amylase2 and this enzyme has not been biochemically characterized from any cereal seeds. Characterization of beta-amylase2 from seeds of two commonly grown barley cultivars was the primary objective of this study. The beta-amylase2 (Bmy2) cDNAs from a malt quality barley (Hordeum vulgare L.) cultivar, 'Morex', and a feed quality cultivar, 'Steptoe', were sequenced, expressed in Escherichia coli, and biochemical properties of the two recombinant enzymes were determined. The relative rates of hydrolysis of various homo-oligomeric and polymeric alpha-D-glucans by the 'Morex' and 'Steptoe' rBmy2 enzymes were not significantly different from each other nor did they significantly differ from the relative rates of hydrolysis reported for barley rBmy1 enzymes. The 'Morex' and 'Steptoe' rBmy2 enzymes both had broad pH activity profiles with maximal activity occurring from pH 5.0 - 6.5, which is very similar to the pH activity profiles reported for beta-amylase1. The rBmy2 from 'Morex' was 7 C more thermostable than the rBmy2 from 'Steptoe" as determined by differences in their T50 and T0 values, and is more thermostable than any beta-amylase1 reported to date. Only three amino acid differences were identified between the 'Morex' and 'Steptoe' Bmy2 sequences and the contributions of these three differences to enzyme thermostability were evaluated by site-directed mutagenesis. Each of the three amino acids contributed approximately 3 C to enzyme thermostability mutant enzymes with one amino acid substitution. Mutant enzymes with two amino acid substitutions contributed 5.5-5.7 C to the thermostability of the wild type rBmy2 from 'Morex'. The Bmy2 encoded by the 'Morex' allele, which has D in position 238, M in position 337, and Q in position 362, provides a discrete signature of a thermostable beta-amylase that can be used as a tool in breeding programs using marker assisted selection methods.