Exploring the roles of amylopectin in starch modification with Limosilactobacillus reuteri 121 4,6-α-glucanotransferase via developed methods

Limosilactobacillus reuteri 121 4,6-α-glucanotransferase (GtfBΔN) modifies starch by cleaving (α1 → 4) linkages and introducing non-branched (α1 → 6) linkages to produce functional starch derivatives. Research has mainly focused on GtfBΔN converting amylose (linear substrate), whereas the conversion of amylopectin (branched substrate) has not been studied in detail. In this study, we used GtfBΔN to understand amylopectin modification and performed a set of experiments to analyze this modification pattern. The donor substrates were segments from the non-reducing ends to the nearest branch point in amylopectin as shown from the results of the chain length distribution of GtfBΔN-modified starches. Decreased and increased contents of β-limit dextrin and reducing sugars, respectively, during the incubation of β-limit dextrin with GtfBΔN indicated that the segments from the reducing end to the nearest branch point in amylopectin act as donor substrates. Dextranase was involved in the hydrolysis of the GtfBΔN conversion products of three different substrates groups, maltohexaose (G6), amylopectin, and G6 plus amylopectin. No reducing sugars were detected, therefore, amylopectin was not used as an acceptor substrate, and no non-branched (α1 → 6) linkages were introduced into it. Thus, these methods provide a reasonable and effective approach to studying GtfB-like 4,6-α-glucanotransferase in analyzing the roles and contribution of branched substrates.