Directionally modulating the product chain length of an inulosucrase by semi-rational engineering for efficient production of 1-kestose.

Microbial inulosucrase as a transfructosylation tool has been used to produce inulin and inulin-type fructooligosaccharides with various polymerization degrees. Tailor-made oligosaccharides could be generated by inulosucrase via chain length modulation. In this study, a semi-rational design based on the modeled structure of Lactobacillus reuteri 121 inulosucrase was carried out to screen and construct variants. The residues Arg541 and Arg544 were determined to be significant to the product chain elongation of L. reuteri 121 inulosucrase. The variant R544W altered the product specificity of inulosucrase and produced short-chain fructooligosaccharides with 1-kestose as the main component. Molecular dynamic simulations verified an increased binding free energy of variant R544W with 1-kestose than the wild-type enzyme with 1-kestose. After optimization, 1-kestose and total short-chain fructooligosaccharides production reached approximately 206 g/L and 307 g/L, respectively. This study suggests the great potential of variant R544W in the biotransformation from sucrose to functional sugar.