Fig. 4: Amylosome CAZymes exhibit synergistic effects in RS degradation.

a An assay to test the RS-degradation capability of the amylosome CAZymes: the purified enzymes were incubated with resistant starch either individually or in combinations for 3 h at 37 °C. After the reaction time, product formation was assessed qualitatively by TLC and ion chromatography and quantitatively using a BCA assay. Created in BioRender. W, B. (2025). https://BioRender.com/c49f659. b The release of reducing sugars quantified by the BCA assay. Only Amy4Δdoc (p = 0.0005) and Amy16 (p < 0.0001) show significant reducing sugar production (four biological replicates, one-way ANOVA with Dunnett’s multiple comparison test). c If all enzymes are incubated together, more substrate is produced than expected by the sum of activities (four biological replicates, two-sided paired t test, p = 0.0098). d The synergistic interactions were quantified as the ratio of observed over expected product formation. In a leave-one-out series, no significant synergism was detected without Amy4Δdoc (four biological replicates, two-sided paired t tests). Exact p-values: p = 0.7447 (no Amy4Δdoc), p = 0.0173 (no Amy9), p = 0.0160 (no Amy16), p = 0.0046 (no Amy10), p = 0.0046 (no Amy12). e Pairwise synergism with Amy4Δdoc is only observed for Amy16, all other combinations not significant (two-sided unpaired t test, Amy10 and Amy12: three technical replicates, Amy16: five technical replicates from two biological replicates, Amy9: 2 technical replicates after outlier removal). Exact p-values: p = 0.3194 (Amy9) p = 0.0023 (Amy16), p = 0.3239 (Amy10), p = 0.5599 (Amy12). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant.