Application of Enzyme Engineering and Synthetic Biology for Modulated Transformation of Fructooligosaccharides (FOSs) to Elucidate the Catalytic Mechanism of Fructofuranosidases.

Fructooligosaccharides (FOSs) are plant-based prebiotics widely utilized in the food and pharmaceutical industries. As a major sugar-producing region, Guangxi holds significant potential for enzymatic production of FOS from sucrose. This study engineered a mutant enzyme, 142P-242K, to address the low catalytic activity characteristic of wild-type enzymes. The mutation upregulated the FOS conversion efficiency from 29 to 52%, respectively. Optimal enzymatic activity was observed at 45 °C, pH 6.0, and in the presence of 1 mM Na+. Mechanistic investigations revealed that modifications to the catalytic domain pocket and shifts in substrate affinity were the primary factors driving enhanced FOS production. The accumulation of 1-Kestose (GF2) was attributed to the enhanced flexibility of the 142P-242K loop, which facilitates substrate access to the active site. However, the synthesis of nystose (GF3) from GF2 is hindered by the hydrophobic nature of the active site and strong hydrogen bonds binding GF2. Comparing the enzyme's ability to produce FOS using sugarcane juice, sugarcane molasses, and adsorption-heating sugarcane molasses, it was determined that heat-adsorbed molasses yielded the highest FOS concentration (30.77%). This study offers a practical and cost-effective strategy for enzyme modification and efficient valorization of molasses.