title: The effect of proline insertions on the thermostability of a barley alpha-glucosidase.
abstract: The thermal stability of alpha-glucosidase is important because the conversion of starch to fermentable sugars during industrial production of ethanol (e.g. brewing, fuel ethanol production) typically takes place at temperatures of 65-73 degrees C. In this study we investigate the thermostability of alpha-glucosidases from four plant species, compare their deduced amino acid sequences, and test the effect of substituting a proline for the residue present in the wild-type enzyme on the thermostability of alpha-glucosidase. The alpha-glucosidase from barley (Hordeum vulgare) was significantly less thermostable than the other three alpha-glucosidases. A comparison of the published deduced amino acid sequences of these four alpha-glucosidases revealed conserved proline residues in the three most thermostable alpha-glucosidases that were not found in the barley enzyme. Site-directed mutagenesis was done on recombinant barley alpha-glucosidase to create proteins with prolines at these conserved positions. The thermostability (T(50)) of one of these mutant enzymes, T340P, was 10 degrees C higher than the non-mutated enzyme.
authors.affiliation: Department of Agronomy, University of Wisconsin, 1575 Linden Drive, Madison, WI 53706, USA.
authors.forename: E H
authors.initials: EH
authors.lastname: Muslin
authors.forename: S E
authors.initials: SE
authors.lastname: Clark
authors.forename: C A
authors.initials: CA
authors.lastname: Henson
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017124:Conserved Sequence; D004795:Enzyme Stability; D001467:Hordeum / Q000201:enzymology*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010944:Plants / Q000201:enzymology; D011392:Proline / Q000235:genetics*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D000520:alpha-Glucosidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D011392:Proline; D000520:alpha-Glucosidases