One-Pot Three-Enzyme System for Production of a Novel Prebiotic Mannosyl-β-(1 → 4)-Fructose Using a d-Mannose Isomerase from Xanthomonas phaseoli .

The present supply of prebiotics is entirely inadequate to meet their demand. To produce novel prebiotics, a d-mannose isomerase ( Xp MIaseA) from Xanthomonas phaseoli was first produced in Komagataella phaffii ( Pichia pastoris ). Xp MIaseA shared the highest amino acid sequence identity (58.0%) with the enzyme from Marinomonas mediterranea . Efficient secretory production of Xp MIaseA (282.0 U mL -1 ) was achieved using high cell density fermentation. The optimal conditions of Xp MIaseA were pH 7.5 and 55 °C. It showed a broad substrate specificity, which isomerized d-mannose, d-talose, mannobiose, epilactose, and mannotriose. Xp MIaseA was employed to construct a one-pot three-enzyme system for the production of mannosyl-β-(1 → 4)-fructose (MF) using mannan (5%, w/v) as the substrate. The equilibrium yield of MF was 58.2%. In in vitro fermentations, MF significantly stimulated (≤3.2-fold) the growth of 12 among 15 tested Bifidobacterium and Lactobacillus strains compared with fructo-oligosaccharides. Thus, the novel d-mannose isomerase provides a one-pot bioconversion strategy for efficiently producing novel prebiotics.