Characterization of active barley alpha-amylase 1 expressed and secreted by Saccharomyces cerevisiae

J Protein Chem. 2001 Nov;20(8):619-23. doi: 10.1023/a:1013712101741.

Abstract

Recombinant barley alpha-amylase 1 isozyme was constitutively secreted by Saccharomyces cerevisiae. The enzyme was purified to homogeneity by ultrafiltration and affinity chromatography. The protein had a correct N-terminal sequence of His-Gln-Val-Leu-Phe-Gln-Gly-Phe-Asn-Trp, indicating that the signal peptide was efficiently processed. The purified alpha-amylase had an enzyme activity of 1.9 mmol maltose/mg protein/min, equivalent to that observed for the native seed enzyme. The kcat/Km was 2.7 x 10(2) mM(-1) x s(-1), consistent with those of alpha-amylases from plants and other sources.

MeSH terms

  • Hordeum / enzymology*
  • Isoelectric Focusing
  • Plant Proteins / chemistry
  • Plant Proteins / genetics
  • Plant Proteins / isolation & purification
  • Plant Proteins / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • alpha-Amylases / chemistry
  • alpha-Amylases / genetics
  • alpha-Amylases / isolation & purification
  • alpha-Amylases / metabolism*

Substances

  • Plant Proteins
  • Recombinant Proteins
  • alpha-Amylases