Activity and thermal stability of genetically truncated forms of Aspergillus glucoamylase

Rebecca Evans, Clark Ford, Michael Sierks, Zivko Nikolov, Birte Svensson

Research output: Contribution to journalArticle

36 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)131-134
Number of pages4
JournalGene
Volume91
Issue number1
DOIs
StatePublished - 1990
Externally publishedYes

Fingerprint

Glucan 1,4-alpha-Glucosidase
Aspergillus
Starch
Hot Temperature
Enzymes
Terminator Codon
Hydrolases
Genes
Saccharomyces cerevisiae
Catalytic Domain
Mutation

Keywords

  • enzyme
  • gene
  • protein function
  • Recombinant DNA
  • restriction site
  • starch hydrolysis
  • stop codon
  • truncation

ASJC Scopus subject areas

  • Genetics

Cite this

Activity and thermal stability of genetically truncated forms of Aspergillus glucoamylase. / Evans, Rebecca; Ford, Clark; Sierks, Michael; Nikolov, Zivko; Svensson, Birte.

In: Gene, Vol. 91, No. 1, 1990, p. 131-134.

Research output: Contribution to journalArticle

Evans, Rebecca ; Ford, Clark ; Sierks, Michael ; Nikolov, Zivko ; Svensson, Birte. / Activity and thermal stability of genetically truncated forms of Aspergillus glucoamylase. In: Gene. 1990 ; Vol. 91, No. 1. pp. 131-134.
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