Comparative studies on functions of two raw starch-digesting α-amylases and SBDs

SBDPG showed stronger disruptive ability.

Specifically, two non-binding residues of SBDPG were responsible for the disruption.

Two corresponding mutants of AmyPG enhanced the hydrolysis efficiency of raw starch.

Most raw starch-digesting enzymes possess at least one non-catalytic starch-binding domain (SBD), which enhances enzymatic hydrolysis of insoluble starch granules. Previous studies of SBD-starch interaction mainly focus on binding affinity for substrates, while the mechanism involved disruption of starch granules remains partially understood. Raw starch-digesting α-amylases AmyPG and AmyP were from Photobacterium gaetbulicola and an uncultured marine bacterium, respectively. Here, comparative studies on the two α-amylases and their SBDs (SBDPG and SBDAmyP) with high sequence identity were carried out. The degradation capacity of AmyPG towards raw starch was approximately 2-fold higher than that of AmyP, which was due to the stronger disruptive ability of SBDPG rather than the binding ability. Two non-binding amino acids (K626, T618) of SBDPG that specifically support the disruptive ability were first identified using affinity gel electrophoresis, amylose‑iodine absorbance spectra, and differential scanning calorimetry. The mutants SBDPG-K626A and SBDPG-T618A exhibited stronger disruptive ability, while the corresponding mutants of AmyPG enhanced the final hydrolysis degree of raw starch. The results confirmed that the disruptive ability of SBD can independently affect raw starch hydrolysis. This advancement in the functional characterization of SBDs contributes to a better understanding of enzyme-starch granule interactions, pushing forward designs of raw starch-digesting enzymes.

Starch-binding domain

Structure disruption

α-Amylase

Raw starch

© 2023 The Authors. Published by Elsevier B.V.