ARS | Publication request: THE EFFECT OF PROLINE INSERTIONS ON THE THERMOSTABILITY OF BARLEY ALPHA-GLUCOSIDASE

Submitted to: Plant Physiology Supplement
Publication Type: Abstract Only
Publication Acceptance Date: July 21, 2001
Publication Date: N/A

Technical Abstract: The thermostability of malt carbohydrases is important because the conversion of starch to fermentable sugars during mashing typically takes place at temperatures of 65-73 degrees Celsius. Of the four enzymes known to be involved in the degradation of malt starch, only alpha-amylase is sufficiently thermostable to function throughout the starch conversion phase of mashing. In this study, we developed a recombinant alpha- glucosidase with enhanced thermostability by site directed mutagenesis of the alpha-glucosidase gene previously cloned from barley. Our strategy for selection of mutagenesis targets involved comparing the deduced amino acid sequences of alpha-glucosidases from four plant species and the thermostability of the enzymes encoded, followed by identification of key amino acids that were present in the thermostable enzymes but absent in the barley alpha-glucosidase. A total of five mutant enzymes were created to assess the role of prolines in stablizing barley alpha-glucosidase. The thermostability (T(50)) of one of these mutant enzymes was 10 degrees Celsius higher than the non-mutated enzyme.