Fructooligosaccharide synthesis by immobilized inulosucrase from lactobacillus reuteri 121 and improvement of product pattern using site-directed mutagenesis

Abstract

Inulin-type fructooligosaccharides (IFOS) are well-known prebiotics which can be produced from sucrose using bacterial inulosucrase (E.C. 2.4.1.9). However, the use of inulosucrase for IFOS synthesis has some limitations because it also synthesizes some polysaccharides and exhibits lower stability than the fungal enzymes. To overcome these limitations, improvement of inulosucrase was made by using enzyme immobilization and enzyme engineering. First, inulosucrase from Lactobacillus reuteri 121 was immobilized on a novel, high capacity core-shell chitosan beads using glutaraldehyde as a cross-linker. The results showed that both immobilized and free inulosucrase had the same optimum pH (5.5), while the optimum temperature was shifted from 50 to 60°C. In addition, the immobilized biocatalyst was more stable than the free enzyme, and could be reused for at least 12 cycles. The product pattern of inulosucrase was then improved by site-directed mutagenesis. Product characterization revealed that the mutation essentially affected the degree of polymerization (DP) of IFOS. Finally, the selected mutated inulosucrase (R483A) was immobilized bycross-linked enzyme aggregates (CLEAs). This CLEAs exhibited excellent pH and themostability compared to that of the free enzyme. This study not only provides the novel enzyme producing oligosaccharides, but also renders the information about structure-function relationship and product formation mechanism of inulosucrase.