Modulation of fructooligosaccharide chain length and insight into the product binding motif of Lactobacillus reuteri 121 inulosucrase

Rational re-design of Lactobacillus reuteri 121 inulosucrase for product chain length control

“…From previous study, D479, S482, R483 and N543 residues were predicted to be the carbohydrate binding residues of Lactobacillus reuteri 121 inulosucrase. 13 Nevertheless, we noticed that the size of oligosaccharides synthesised by the variants did not correlate well with the distance between the mutated sites and catalytic sites. In the case of LrInu, for example, the N543A variant mainly produced FOSs with DP3-6, while the R483A variant, whose R483 was located close to N543, produced the FOSs up to DP12.…”

“…Previously, many researches demonstrated that a single mutation at some specic amino acid residues of LrInu affected the size of inulin oligosaccharide that could be produced. 13,16,17 The yield of short-chain oligosaccharides was signicantly improved when some amino acid residues of LrInu were mutated to be alanine, which may reduce the interactions between the enzyme and substrates with medium or long chain lengths. This nding has also been reported on other fructosyltransferases, such as Bacillus megaterium levansucrase 11 and Bacillus licheniformis levansucrase.…”

“…When this study was conducted, the crystal structure of Lactobacillus reuteri 121 inulosucrase was not available. Therefore, its homology model was created in the previous study 13 based on the crystal structure of Lactobacillus johnsonii inulosucrase (PDB ID: 2YFS) 15 and also used in this study. The sequence identity of these two enzymes are reasonable with the value of 74.17%.…”

“…12 Previously, we reported the amino acid residues of Lactobacillus reuteri 121 inulosucrase (LrInu) that play an essential role in FOS chain length determination. 13 Some variants of this enzyme, such as D479A, S482A, and R483A, can produce a broad range of oligosaccharides with a small amount of accumulating polymer, while N543A mainly produces the short chain FOSs (DP3-6, with traces of DP $ 7). Since FOSs show optimal prebiotic activity in the DP 2-8 range, 14 it is interesting to use the N543A variant of LrInu as a biocatalyst for the synthesis of such oligosaccharides.…”

“…The position of LrInu selected for mutagenesis was based on the oligosaccharide binding track of LrInu reported in previous study. 13 The Rosetta program was employed using the homology model of LrInu, built from crystal structure of Lactobacillus johnsonii inulosucrase (PDB ID: 2YFS), 15 to assess variants of LrInu to F, Y or W, to predict the substrate binding conformations and to compute the binding free energies (DG binding ) of the substrate/ enzyme complexes. The enzyme activities, biochemical properties and kinetic parameters of LrInu variants were determined and compared to that of the wild type.…”