title: The activity of barley alpha-amylase on starch granules is enhanced by fusion of a starch binding domain from Aspergillus niger glucoamylase.
abstract: 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 alpha-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 x 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 x nmol(-1) and K(d) = 0.13 mg x mL(-1), respectively, were both improved with respect to the values 0.015 U x nmol(-1) and 0.67 mg x 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.
authors.affiliation: Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA, UK.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Juge
authors.forename: Jane
authors.initials: J
authors.lastname: Nøhr
authors.forename: Marie-Françoise
authors.initials: MF
authors.lastname: Le Gal-Coëffet
authors.forename: Birte
authors.initials: B
authors.lastname: Kramhøft
authors.forename: Caroline S M
authors.initials: CS
authors.lastname: Furniss
authors.forename: Véronique
authors.initials: V
authors.lastname: Planchot
authors.forename: David B
authors.initials: DB
authors.lastname: Archer
authors.forename: Gary
authors.initials: G
authors.lastname: Williamson
authors.forename: Birte
authors.initials: B
authors.lastname: Svensson
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology*; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001467:Hordeum / Q000201:enzymology*; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D013213:Starch / Q000737:chemistry*; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D005656:Fungal Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D013213:Starch; D000516:alpha-Amylases; D005087:Glucan 1,4-alpha-Glucosidase