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In this study, we explored the hydrolytic mechanism of glucan 1,4-α-maltotiohydrolase (AMTS). Kinetic parameters analysis of AMTS were performed using the amylopectin with different external chain lengths (ECL). Then, the waxy maize starch was gelatinized and hydrolyzed by AMTS to various degrees; retrogradation of starch hydrolysates were determined. Michaelis constant ( K m ) of the β -dextrin-3 (ECL 2.3) was eight folds than the amylopectin (ECL 17.8), but its catalytic efficiency ( k cat / K m ) was only 6.5% of amylopectin. AMTS preferred the external chains to the internal chains for hydrolysis, and amylopectin was hydrolyzed by AMTS through combined exo- and endo-action. In the initial hydrolysis stage, the exo-action of AMTS was predominant with slow reduction in M w and little changes in the internal chains of amylopection. The percentage of maltotriose in the soluble oligosaccharides (PM) was 90.5% at 2.1% degree of hydrolysis (DH), and gradually decreased from 90.5% to 71.2% at 35.4% DH. The PM decreased could be partially attributed to the increased transglycosylation reactions of AMTS. Complete inhibition of amylopectin retrogradation was observed with >15.9% hydrolysis for the increased shorter outer chains and low molecular weight dextrins. These results indicate that AMTS may be effective in retarding starch retrogradation in baked products. [ABSTRACT FROM AUTHOR]