Journal article
The activity of barley alpha-amylase on starch granules is enhanced by fusion of a starch binding domain from Aspergillus niger glucoamylase
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.
Language: | English |
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Year: | 2006 |
Pages: | 275-284 |
ISSN: | 18781454 , 15709639 , 18782434 and 00063002 |
Types: | Journal article |
DOI: | 10.1016/j.bbapap.2005.11.008 |
ORCIDs: | Svensson, Birte |
Aspergillus niger Carbohydrate binding module family 20 Fungal Proteins Glucan 1,4-alpha-Glucosidase Glycoside hydrolase family 13 Hordeum O-glycosylated linker region Plant Proteins Protein Conformation Protein Structure, Tertiary Recombinant Fusion Proteins Starch Starch binding domain fusion Starch granule hydrolysis alpha-Amylases