A Novel Versatile Neopullulanase with Transglycosylation Activity from <i>Bacillus Inaquosorum</i> : Heterologous Expression, Characterization, and Synergistic Application with α-Transglucosidase from <i> <i>Aspergillus niger</i> </i> in Bread Making

To explore transglycosidase application potential in starch processing and baking, a <i>Bacillus inaquosorum</i> strain with high amylopectin debranching activity was isolated and identified from the soil. Its neopullulanase gene (<i>Binp</i>) was cloned and heterologously expressed in <i><i>Escherichia coli</i></i> BL21. The recombinant enzyme reBinp, which requires a metal ion, had optimal catalytic activity at 35 °C and pH 7.0, efficiently hydrolyzed α-(1,6)-glycosidic bonds. Synergistic hydrolysis and transglycosylation effects were displayed when reBinp acted with <i><i>Aspergillus niger</i></i> α-transglucosidase (Antg). Adding reBinp and Antg (each of 1000 U/kg wheat flour) significantly improved bread quality and increased specific volume, hardness, gumminess, chewiness, cohesiveness, and storage stability. Mechanistically, the mixed enzymes decreased the relative crystallinity and gelatinization enthalpy of dough to 14.5% and 3.95 mJ/mg, respectively, while increasing the FTIR <i>R</i>_1047/1022 ratio to 1.70. This study provides a solid basis for the synergistic application of neopullulanase and α-transglucosidase in starch biotransformation and the baking industry.