Food-grade expression and characterization of cyclomaltodextrinase from B. sphaericus E−244 in Bacillussubtilis

Abstract

As the utilization of maltodextrins in food, pharmaceuticals, and agriculture continues to expand, substantial research has been conducted to enhance their functionality. Among the methods presented, the one-pot approach for the synthesis of nonreducing maltoheptaose offers a novel solution to the challenge of maltodextrins with varying degrees of polymerization and reducing ends. Nevertheless, the key enzyme in this method was currently only expressed in Escherichia coli, which restricts the applicability of this method in the food and pharmaceutical industries. In this study, the food-grade expression of cyclomaltodextrinase (CDase, EC 3.2.1.54), one of the key enzymes, was achieved using Bacillus subtilis as a host. The enzymatic properties of the recombinant CDase were then investigated, and the extracellular secretion of the CDase was enhanced in order to make it more widely available for use in the food industry. The enzyme exhibited optimal activity at a pH of 8.0 and a temperature range of 35–45 °C. After incubation at 25–35 °C for 10 h, 90% of the enzyme activity was retained. Additionally, the enzyme retained 80% of its initial activity after 24 h at pH 5.5–9.5. Finally, Cu2+ completely inhibited the enzyme activity. The extracellular secretion efficiency of recombinant CDase was significantly increased by the addition of Mn2+ to the fermentation medium. The percentage of extracellular enzyme activity increased to 63.75% when the final concentration of Mn2+ in the fermentation medium was 5 mM, which was 5.3-fold higher than that of the unadded one.