Enhanced suppression of retrogradation and digestibility in waxy rice starch by engineered mutants of GtfC type 4,6-α-glucanotransferase from Geobacillus sp.

Abstract

GtfC type 4,6-α-glucanotransferase could induce short branches in starch by catalyzing inter- or inside-chain transglycosylation. To improve activity of wild-type (WT) GsGtfC from Geobacillus sp., 15 mutants were engineered by site-directed mutagenesis based on amino acid alignments, enzyme-substrate docking and dynamic simulation. At 2 U/g starch, five mutants with increased specific activity by 1.02–1.63-fold reduced molecular weight of starch from 8.85 × 10⁷ to 4.18 × 10⁷–9.58 × 10⁶ and increased DP ≤ 12 short branches from 33.18 to 35.77–43.65%, compared with WT. Retrogradation enthalpy of starch modified by K342V and WT reduced to 0.27 J/g and 0.69 J/g on the 14th day. RDS (rapidly digestible starch) of starch modified by K520A and WT decreased to 75.91% and 83.20%, and SDS (slowly digestible starch) increased to 22.88% and 11.85%. Glucose release from starch modified by K342V, K520A and D350N in a decreased order was significantly lower than that of WT from 120 to 360 min at small intestinal α-glucosidase level. Amino acid substitution strengthened enzyme-substrate affinity and stabilized critical hydrophobic interactions to enhance activity.