title: Overexpression, purification, and characterization of a barley alpha-glucosidase secreted by Pichia pastoris.
abstract: alpha-Glucosidases (EC 3.2.1.20) are recognized as important in starch degradation during cereal seed germination. A barley (Hordeum vulgare) alpha-glucosidase expressed in Pichia pastoris was cultured in flasks; however, the yield was low necessitating the use of multiple batches. Problems arose because of significant variation between batches. We solved these problems by switching to a fermentation system producing a sufficient quantity of a uniform sample. Here we present the expression and purification of a recombinant alpha-glucosidase grown under fermentation conditions. We also present the results of experiments to characterize the thermostability, pH optimum, and substrate specificity of the recombinant enzyme. The optimal pH for the hydrolysis of maltose by recombinant alpha-glucosidase is between 3.5 and 4.5. The thermostability of recombinant alpha-glucosidase was determined at pH 4, where activity is optimal, and at pH 5 and 6, which better mimic the conditions used to convert barley starch to fermentable sugars during industrial processing. The results indicate the enzyme is most thermolabile at pH 4. However, the enzyme is protected from heat inactivation at pH 4 by high concentrations of sucrose. The purified enzyme hydrolyzed maltose three times more rapidly than nigerose and 20 times more rapidly than trehalose and isomaltose. Concentrations of maltose greater than 20 mM inhibited maltose hydrolysis. This is the first report of substrate inhibition for any alpha-glucosidase. The results indicate that the only significant difference between the recombinant enzyme and the previously characterized barley isoforms was the V(max) for maltose hydrolysis.
authors.affiliation: Department of Agronomy, University of Wisconsin, 1575 Linden Drive, Madison, Wisconsin, 53706, USA.
authors.forename: E H
authors.initials: EH
authors.lastname: Muslin
authors.forename: A M
authors.initials: AM
authors.lastname: Kanikula
authors.forename: S E
authors.initials: SE
authors.lastname: Clark
authors.forename: C A
authors.initials: CA
authors.lastname: Henson
mesh_terms: D004187:Disaccharides / Q000378:metabolism; D004795:Enzyme Stability; D005285:Fermentation; D015870:Gene Expression; D017343:Genes, Plant; D001467:Hordeum / Q000201:enzymology* / Q000235:genetics*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007534:Isomaltose / Q000378:metabolism; D008320:Maltose / Q000378:metabolism; D010843:Pichia / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014199:Trehalose / Q000378:metabolism; D000520:alpha-Glucosidases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D004187:Disaccharides; D011994:Recombinant Proteins; C014637:laminaribiose; D007534:Isomaltose; D008320:Maltose; D014199:Trehalose; D000520:alpha-Glucosidases