Interfacial enzyme kinetics reveals degradation mechanisms behind resistant starch

The digestive resistance of so-called resistant starch (RS) provides an important nutritional principle. However, as yet the mechanisms underlying digestive resistance of the granular starches are not well described. We address this problem by employing a novel enzyme kinetics approach, which in contrast to conventional enzyme kinetics takes the interfacial nature of the enzyme reaction into account. Structurally different starch granules with different amylose contents were modified by the hydrolytic enzyme glucoamylase (GA). Kinetic-, adsorbtion-, imaging-, particle size-, and spectroscopic data combinedly revealed important origins of hydrolytic resistance of high-amylose starch. Our data demonstrate that amylose restricted the enzymatic catalysis efficiency on starch granules chiefly by providing less hydrolytic effective attack sites for the enzyme at granular surface. Specifically, the total binding sites (^adsΓ_max) (20.9 nmol/g) on starch with 0% amylose content (AC) were almost all effective attack sites (^kinΓ_max) (20.2 nmol/g). At 26% AC, the binding sites on the granules decreased to 13.9 nmol/g, and attack sites decreased to 7.4%. At 72% AC, the binding sites were only slightly reduced to 11.2 nmol/g, however, the attack sites were remarkably decreased to 2.6 nmol/g. Beyond the initial catalytic events, i.e., for further degraded granules, the binding and catalysis efficiency differed notably for the three starch types. At 0% AC, both binding and attack increased demonstrating increased hydrolytic susceptibility of the granules. At 26% AC only binding increased, while attack was unchanged. Interestingly, at 72% AC, binding increased, while attack deceased notably with hydrolysis time, demonstrating decreased efficiency of interfacial catalysis during the hydrolysis process.