4.7 Article

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

Journal

JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
Volume 71, Issue 23, Pages 9040-9050

Publisher

AMER CHEMICAL SOC
DOI: 10.1021/acs.jafc.3c01898

Keywords

carbohydrate-binding module; waxy starch; normalstarch; high-amylose starch; Pseudoalteromonashaloplanktis alpha-amylase; heterogenous catalysis; Sabatier principle

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Degradation of starch granules can be increased by fusing the psychrophilic a-amylase AHA from Antarctic bacterium Pseudoalteromonas haloplanktis TAB23 with starch-binding domains (SBD) from either Aspergillus niger glucoamylase (SBDGA) or Arabidopsis thaliana glucan, water dikinase 3 (SBDGWD3). The fusion enzymes showed higher activity than the wild-type AHA, depending on the starch type and source. The increased density of enzyme attack-sites and binding-sites on the starch granules was measured using an interfacial catalysis approach.
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.

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