Improved Hydrolysis of Granular Starches by a Psychrophilic α-Amylase Starch Binding Domain-Fusion

Degradation of starch granules by a psychrophilic a-amylase, AHA, from the Antarctic bacterium Pseudoalteromonas haloplanktis TAB23 was facilitated by C-terminal fusion to a starch-binding domain (SBD) from either Aspergillus niger glucoamylase (SBDGA) or Arabidopsis thaliana glucan, water dikinase 3 (SBDGWD3) via a decapeptide linker. Depending on the waxy, normal or high-amylose starch type and the botanical source, the AHA-SBD fusion enzymes showed up to 3 times higher activity than AHA wild-type. The SBD-fusion thus increased the density of enzyme attack-sites and binding-sites on the starch granules by up to 5- and 7-fold, respectively, as measured using an interfacial catalysis approach that combined conventional Michaelis-Menten kinetics, with the substrate in excess, and inverse kinetics, having enzyme in excess, with enzyme-starch granule adsorption isotherms. Higher substrate affinity of the SBDGA compared to SBDGWD3 was accompanied by the superior activity of AHA-SBDGA in agreement with the Sabatier principle of adsorption limited heterogenous catalysis.

Automated summary

The degradation of starch granules by the psychrophilic a-amylase AHA from Antarctic bacterium Pseudoalteromonas haloplanktis TAB23 was enhanced by fusion with a starch-binding domain (SBD) from either Aspergillus niger glucoamylase (SBDGA) or Arabidopsis thaliana glucan, water dikinase 3 (SBDGWD3) through a decapeptide linker. The AHA-SBD fusion enzymes exhibited up to 3 times higher activity than AHA wild-type depending on the type of starch and its botanical source. The density of enzyme attack-sites and binding-sites on the starch granules was increased up to 5- and 7-fold, respectively, by the SBD-fusion.