Compositions and methods comprising the use of a bacillus agaradhaerens inulosucrase (inuo)

Abstract

Bacillus agaradhaerens strain WDG185 expresses an inulosucrase that efficiently synthesizes a broad range of IOS with a GF range of GF3-GF30. The isolated and/or purified inulosucrase, recombinantly engineered variants thereof, active fragments thereof, synthetic nucleic acids encoding the inulosucrase, its variants, or its active fragments, host cells comprising the synthetic nucleic acids, and compositions comprising the inulosucrase are provided. Methods of using the compositions include the manufacture of inulooligosaccharides.

What is claimed is: 
     
         1 . An isolated, recombinantly expressed inulosucrase from  Bacillus agaradhaerens  (INUO) comprising a polypeptide consisting of amino acids 32-453 of SEQ ID NO: 4, a recombinantly engineered variant thereof, or an active fragment thereof, wherein the variant is able to catalyze polymerization of inulin oligosaccharides containing β-(2→1) linkages, and wherein the variant has at least 60% sequence identity with amino acids 32-453 of the amino acid sequence of SEQ ID NO: 4. 
     
     
         2 . The inulosucrase of  claim 1 , wherein the polypeptide comprising the polypeptide consisting of amino acids 32-453 of SEQ ID NO: 4 comprises at least one amino acid not normally associated with naturally occurring INUO from  Bacillus agaradhaerens  strain WDG185 (SEQ ID NO: 4). 
     
     
         3 . The inulosucrase of  claim 1 , wherein the recombinantly engineered variant has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with amino acids 32-453 of the amino acid sequence of SEQ ID NO: 4. 
     
     
         4 . The inulosucrase of  claim 3 , wherein the recombinantly engineered variant has at least 99% or at least 99.5% sequence identity with amino acids 32-453 of the amino acid sequence of SEQ ID NO: 4. 
     
     
         5 . The inulosucrase of  claim 1 , wherein the amino acid residues of the recombinantly engineered variant that are not identical to amino acids 32-453 of SEQ ID NO: 4 are selected from conservative amino acid substitutions or deletions from either the C- or N-termini. 
     
     
         6 . The inulosucrase of  claim 1 , wherein the amino acid sequence of the recombinantly engineered variant comprises a sequence identical to amino acids 79-393 of SEQ ID NO: 4. 
     
     
         7 . A composition comprising the inulosucrase of  claim 1 . 
     
     
         8 . The composition of  claim 7 , wherein the inulosucrase is in a lyophilized powder form, an encapsulated form, a coated form, a granulated form, or a liquid formulation. 
     
     
         9 . The composition of  claim 7 , further comprising a diluent. 
     
     
         10 . A synthetic nucleic acid encoding the inulosucrase of  claim 1 . 
     
     
         11 . A vector comprising the synthetic nucleic acid of  claim 10 . 
     
     
         12 . The vector of  claim 10 , which is an expression vector. 
     
     
         13 . A host cell comprising the synthetic nucleic acid of  claim 10  or the vector of  claim 11 . 
     
     
         14 . A vector comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3. 
     
     
         15 . A host cell comprising the vector of  claim 14 . 
     
     
         16 . A host cell that is not  Bacillus agaradhaerens  comprising the nucleotide sequence of SEQ ID NO: 3. 
     
     
         17 . A composition comprising the host cell of  claim 13  and a food-grade, feed-grade, industrial-grade, or pharmacologically acceptable carrier, diluent, or excipient. 
     
     
         18 . A method of using the composition of  claim 17 , comprising administering the composition to an individual, wherein the composition is capable of acting as a probiotic in the individual. 
     
     
         19 . A method of producing an inulosaccharide (IOS) product, comprising contacting the inulosucrase of  claim 1  with a fructose source, and reacting the inulosucrase with the fructose source at pH 5-10 and at 40° C.-60° C. to produce the IOS product. 
     
     
         20 . The method of  claim 19 , wherein the fructose source is sucrose, stachyose, raffinose, inulin, or a fructooligosaccharide (FOS). 
     
     
         21 . The method of  claim 19 , wherein the IOS product has a GF range of GF3-GF100, GF3-GF30, or GF10-GF25. 
     
     
         22 . The method of  claim 19 , wherein the inulosucrase is provided in a composition comprising a host cell comprising a nucleic acid encoding recombinant INUO. 
     
     
         23 . The method of  claim 19 , further comprising chemical modification of the IOS product. 
     
     
         24 . A method of producing a tailored oligofructoside product, comprising contacting the inulosucrase of  claim 1  with a sucrose analogue having the glucose cap of sucrose substituted by another saccharide, and reacting the inulosucrase with the sucrose analogue at pH 5-10 and at 40° C.-60° C. to produce the tailored oligofructoside product. 
     
     
         25 . The method of  claim 24 , wherein the sucrose analogue has the glucose cap of sucrose substituted by a galactose, a mannose, a fucose, a xylose, or an allose. 
     
     
         26 . The method of  claim 24 , wherein the inulosucrase is provided in a composition comprising a host cell comprising a nucleic acid encoding recombinant INUO.