New enzymes for production of tailor made Fructo-oligosaccharides

Inulosucrases, are a class of bacterial fructosyltransferase enzymes that can produce inulin type fructan polymers from sucrose. Using various protein engineering tools, we have identified new protein variants that show high stability and activity following immobilization (level of activity not affected after two months), tolerance for high substrate concentrations (up to 2M sucrose with increased ki) and high catalytic efficiency (Kcat/Km =125,278). The most improved variant selected in this work, produces short chain FOS (DP3-DP7) in large quantities (0.5g fibers to mg of enzyme in 1h), from sucrose. To evaluate the effect of the enzymatically produced FOS in comparison to commercial FOS and sucrose on blood glucose levels we performed an OGTT test using c57BL6/J mice as a model. The results indicate that the reaction products have a significantly lower glycemic index than glucose (p = 0.0001), but there is no significant difference between the glycemic value of the treated sucrose (reaction products) and untreated sucrose (p = 0.99). This result does not negate the fact that although there is no difference in the glycemic value, the treated sucrose contains about 27% dietary fiber that contributes to its nutritional value. These findings suggest that the FOS generated in the enzymatic reaction using engineered enzyme can be used as a suitable substitute for sugar in food products, and that the improved enzyme created in this work can be used for their production as well as lowering the caloric value and increasing the nutritional value of the treated food product.