Enhanced resistance of pea starch and cake to retrogradation and digestion by mutant 1,4-α-glucan branching enzyme from Bifidobacterium longum.

Abstract

The 1,4-α-glucan branching enzyme found in Bifidobacterium longum can modify the physicochemical properties of starch by forming denser and shorter branches. To enhance its activity and modification function with regard to pea starch, ten mutants were engineered using site-directed mutagenesis based on multiple sequence alignment, molecular docking, and dynamic simulation. The mutants E395D, S321D and M509L exhibited specific activities 1.32-, 1.23- and 0.81-fold higher than the wild type (WT). The catalytic efficiencies of S321D and E395D were 18.36 and 14.00 mg-1·min-1·mL-1, respectively, which were significantly higher than the WT's efficiency of 11.19 mg-1·min-1·mL-1. Docking analysis revealed that the mutations reorganised the hydrogen-bonding and electrostatic microenvironment at the active site, producing mutation-specific effects on substrate binding and transition-state stabilisation. These locally altered polar and nucleophilic properties ultimately dictated the catalytic efficiency of each mutant. Compared with WT, E395D and S321D increased DP 2 and 3 short branches, reduced FT-IR R₁₀₄₇/₁₀₂₂ and XRD crystallinity at 200-800 U/g starch, which expanded the amorphous region of starch. After seven days' storage at 4 °C, the retrogradation enthalpy of S321D-modified starch was 0.63 J/g lower than that of WT-modified starch of 0.71 J/g. Glucose release from S321D-modified starch was significantly lower than from WT-modified starch at the level of small intestinal α-glucosidase. The slowly digestible fraction of S321D-modified starch was 38.51%, significantly higher than the 29.19% observed in WT-modified starch. Fresh and stored pea cake modified by S321D had significantly lower hardness and higher slowly digestible properties than the WT-modified equivalent.