The activity of barley α-amylase on starch granules is enhanced by fusion of a starch binding domain from Aspergillus niger glucoamylase

Nathalie Juge, Jane Nøhr, Marie Françoise Le Gal-Coëffet, Birte Kramhøft, Caroline S.M. Furniss, Véronique Planchot, David B. Archer, Gary Williamson, Birte Svensson

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High affinity for starch granules of certain amylolytic enzymes is mediated by a separate starch binding domain (SBD). In Aspergillus niger glucoamylase (GA-I), a 70 amino acid O-glycosylated peptide linker connects SBD with the catalytic domain. A gene was constructed to encode barley α-amylase 1 (AMY1) fused C-terminally to this SBD via a 37 residue GA-I linker segment. AMY1-SBD was expressed in A. niger, secreted using the AMY1 signal sequence at 25 mg × L-1 and purified in 50% yield. AMY1-SBD contained 23% carbohydrate and consisted of correctly N-terminally processed multiple forms of isoelectric points in the range 4.1-5.2. Activity and apparent affinity of AMY1-SBD (50 nM) for barley starch granules of 0.034 U × nmol-1 and Kd = 0.13 mg × mL-1, respectively, were both improved with respect to the values 0.015 U × nmol-1 and 0.67 mg × mL-1 for rAMY1 (recombinant AMY1 produced in A. niger). AMY1-SBD showed a 2-fold increased activity for soluble starch at low (0.5%) but not at high (1%) concentration. AMY1-SBD hydrolysed amylose DP440 with an increased degree of multiple attack of 3 compared to 1.9 for rAMY1. Remarkably, at low concentration (2 nM), AMY1-SBD hydrolysed barley starch granules 15-fold faster than rAMY1, while higher amounts of AMY-SBD caused molecular overcrowding of the starch granule surface.

Original languageEnglish
Pages (from-to)275-284
Number of pages10
JournalBBA Proteins and Proteomics
Issue number2
Publication statusPublished - 1 Feb 2006
Externally publishedYes


  • Aspergillus niger
  • Carbohydrate binding module family 20
  • Glycoside hydrolase family 13
  • O-glycosylated linker region
  • Starch binding domain fusion
  • Starch granule hydrolysis

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