title: Activity and thermal stability of genetically truncated forms of Aspergillus glucoamylase.
abstract: Glucoamylase (GA) from Aspergillus awamori (EC 3.2.1.3) is a secreted starch hydrolase with a large catalytic domain (aa 1-440), a starch-binding domain (aa 513-616), and a highly O-glycosylated region of 72 aa of unknown function that links the catalytic and starch-binding domains. We have genetically engineered a series of truncated forms of GA to determine how much of the highly O-glycosylated region is necessary for the activity or stability of GAII, a fully active form of the enzyme that lacks the starch-binding domain. Mutations were made by inserting stop-codon linkers into restriction sites within the coding region of the GA gene, and mutated genes were expressed in Saccharomyces cerevisiae for analysis of the truncated enzymes. Our results show that up to 30 aa from the C-terminal end of GAII can be deleted with little effect on the activity, thermal stability, or secretion of the enzyme. Further deletions resulted in diminution or loss of enzyme activity on starch plates, and loss of detectable enzyme in culture supernatants, indicating that these residues are essential for GAII function.
authors.affiliation: Department of Genetics, Iowa State University, Ames 50011.
authors.forename: R
authors.initials: R
authors.lastname: Evans
authors.forename: C
authors.initials: C
authors.lastname: Ford
authors.forename: M
authors.initials: M
authors.lastname: Sierks
authors.forename: Z
authors.initials: Z
authors.lastname: Nikolov
authors.forename: B
authors.initials: B
authors.lastname: Svensson
mesh_terms: D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D004795:Enzyme Stability; D005822:Genetic Vectors; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D013816:Thermodynamics
chemical_list: D005087:Glucan 1,4-alpha-Glucosidase