# Dual-Enzyme Co-Catalysis Strategy for Fructooligosaccharides (FOS) Biocatalytic Synthesis for Valorization of Low-Cost Byproduct Sugarcane Molasses

## TL;DR

This study introduces a dual-enzyme system to efficiently produce prebiotic fructooligosaccharides (FOS) from sugarcane molasses, improving FOS yield significantly.

## Contribution

A novel dual-enzyme co-catalysis strategy that enhances FOS synthesis by reducing glucose inhibition and using low-cost molasses.

## Key findings

- The dual-enzyme system increased FOS synthesis rate from 42.31% to 55.51% (w/w).
- Optimal catalytic conditions were 45 °C, pH 5.5, and 1 mM Ba2+.
- Fermentation optimization achieved a 53.92% FOS yield, a 9.38% improvement over single-enzyme methods.

## Abstract

Fructooligosaccharides (FOS) represent a major source of prebiotic compounds. They are widely used in functional foods for their ability to modify intestinal microbiota in animals and humans. To address the significant issue of fructooligosaccharide production being influenced by glucose concentration, this study designed a dual-enzymatic co-catalysis system for glucose isomerase (GI) and a mutant FTase (FTase142P-242K). This system successfully increased the FOS synthesis rate (42.31 to 55.51%, w/w). Glucose isomerase catalyzes the isomerization of glucose to fructose, and the subsequent release of fructose from the active site permits the enzyme to re-enter its catalytic cycle. The optimal conditions for catalysis were found at 45 °C, pH 5.5, and 1 mM Ba2+. In contrast, the optimal fermentation process was established at 25 °C and induction with 1 mM IPTG. Finally, the efficient production of FOS using low-cost byproduct molasses was achieved. Fermentation optimization of the dual-enzyme system resulted in FOS yield of 53.92% (w/w), a significant increase (44.54%, w/w) from the yield obtained using single-enzyme catalysis. Based on the research, a novel and sustainable approach for high-yield synthesis of Fructooligosaccharides involves minimizing the inhibitory effect of glucose produced during sucrose transformation.