WO2026044164A2 - Procédé de modification de gluco-oligosaccharides avec des kojibiose phosphorylases - Google Patents

Procédé de modification de gluco-oligosaccharides avec des kojibiose phosphorylases

Info

Publication number
WO2026044164A2
WO2026044164A2 PCT/US2025/043076 US2025043076W WO2026044164A2 WO 2026044164 A2 WO2026044164 A2 WO 2026044164A2 US 2025043076 W US2025043076 W US 2025043076W WO 2026044164 A2 WO2026044164 A2 WO 2026044164A2
Authority
WO
WIPO (PCT)
Prior art keywords
gluco
composition
glucopyranosyl
kojibiose
oligosaccharides
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/043076
Other languages
English (en)
Other versions
WO2026044164A3 (fr
Inventor
Marc DE DONCKER
Tom Desmet
Ronny Leontina Marcel Vercauteren
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cargill Inc
Original Assignee
Cargill Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cargill Inc filed Critical Cargill Inc
Publication of WO2026044164A2 publication Critical patent/WO2026044164A2/fr
Publication of WO2026044164A3 publication Critical patent/WO2026044164A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/0123—Kojibiose phosphorylase (2.4.1.230)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
    • A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres

Definitions

  • the present invention relates to an isolated polypeptide comprising kojibiose phosphorylase activity (E.C. 2.4.1.230), wherein the isolated polypeptide is capable of using glucose, kojibiose and maltose as an acceptor substrate and converting the acceptor substrate into kojibiose, kojitriose and alpha-1,2-glucosyl-maltose as a product respectively, and wherein the activity of the polypeptide towards kojibiose or maltose is higher as compared to its activity towards glucose as an acceptor substrate.
  • kojibiose phosphorylase activity E.C. 2.4.1.230
  • the isolated polypeptide is capable of using glucose, kojibiose and maltose as an acceptor substrate and converting the acceptor substrate into kojibiose, kojitriose and alpha-1,2-glucosyl-maltose as a product respectively, and wherein the activity of the polypeptide towards kojibiose or maltos
  • the present invention also relates to an enzymatic process for elongating gluco-oligosaccharides with a glucosyl moiety, wherein the enzymatic process uses the isolated polypeptide of the present invention.
  • the elongated gluco- oligosaccharides obtained by the process can be used in edible products, for instance as a dietary fiber or a (low calorie) bulking agent, a prebiotic, in sugar replacement or in calorie reduction.
  • the elongated gluco-oligosaccharides obtained by the process present a decreased digestibility compared to known gluco-oligosaccharides. BACKGROUND OF THE INVENTION [0004] Oligo- and polysaccharides are widely used in the food industry.
  • Hydrolysed plant- based starches are a source of glucose containing oligo- and polysaccharides. Hydrolysed starches include maltodextrins and glucose syrups.
  • Maltodextrins and glucose syrups may be used as a texturiser or as a full calorie ingredient providing bulking functionality. Maltodextrins and glucose syrups may also be used PT-2001-WO-PCT as a coating and for encapsulation. Maltodextrins and glucose syrups are easily digested and are fully and rapidly absorbed by the gastrointestinal tract. The fast digestibility results in a strong rise in blood glucose levels after consumption. Maltodextrins and glucose syrups are therefore products with a high glycemic index.
  • maltodextrins and glucose syrups are full calorie carbohydrates.
  • bulking ingredients and carbohydrates that have a lower or slower digestibility and that are lower in calories.
  • bulking ingredients and carbohydrates that decrease the glycemic impact of the oligo- or polysaccharides used for this purpose.
  • One suggestion has been to replace the existing polysaccharides and carbohydrates with naturally occurring alternatives with a lower calorie content.
  • Another alternative is to modify existing oligo- and polysaccharides.
  • the present inventors have found a process for modifying oligo- or polysaccharides such as maltodextrins or glucose syrups by means of an enzymatic process using an isolated polypeptide comprising kojibiose phosphorylase activity (E.C.2.4.1.230) wherein the isolated polypeptide is capable of using glucose, kojibiose and maltose as an acceptor substrate and converting the acceptor substrate into kojibiose, kojitriose, and alpha-1,2-glucosyl-maltose as a product respectively, and wherein the activity of the polypeptide towards kojibiose or maltose is higher as compared to its activity towards glucose as an acceptor substrate.
  • an isolated polypeptide comprising kojibiose phosphorylase activity (E.C.2.4.1.230) wherein the isolated polypeptide is capable of using glucose, kojibiose and maltose as an acceptor substrate and converting the acceptor substrate into kojibios
  • the present invention relates to an isolated polypeptide comprising kojibiose phosphorylase activity (E.C. 2.4.1.230), wherein the isolated polypeptide is capable of using glucose, kojibiose and maltose as an acceptor substrate and converting the acceptor substrate into kojibiose, kojitriose, and alpha-1,2-glucosyl-maltose as a product respectively, and wherein the activity of the polypeptide towards kojibiose or maltose is higher as compared to its activity towards glucose as an acceptor substrate.
  • kojibiose phosphorylase activity E.C. 2.4.1.230
  • the present invention also relates to an enzymatic process for elongating gluco-oligosaccharides with a glucosyl moiety, wherein the enzymatic process uses the isolated polypeptide of the present invention.
  • the elongated gluco- oligosaccharides obtained by the process can be used in edible products, for instance as a dietary fiber or a (low calorie) bulking agent, a prebiotic, in sugar replacement or in calorie PT-2001-WO-PCT reduction.
  • the elongated gluco-oligosaccharides obtained by the process present a decreased digestibility compared to known gluco-oligosaccharides.
  • the present invention provides in a first aspect an isolated polypeptide comprising kojibiose phosphorylase activity (E.C.2.4.1.230), wherein the isolated polypeptide is capable of using glucose, kojibiose and maltose as an acceptor substrate and converting the acceptor substrate into kojibiose, kojitriose, and alpha-1,2-glucosyl-maltose as a product respectively, and wherein the activity of the polypeptide towards kojibiose or maltose is higher as compared to its activity towards glucose as an acceptor substrate.
  • kojibiose phosphorylase activity E.C.2.4.1.230
  • the inventors found that use of an isolated polypeptide that is capable of using glucose, kojibiose and maltose as an acceptor substrate and converting the acceptor substrate into kojibiose, kojitriose, and alpha-1,2-glucosyl-maltose as a product respectively, wherein the activity of the isolated polypeptide towards kojibiose or maltose is higher as compared to its activity towards glucose as an acceptor substrate greatly increases the conversion rate of a polymer having a degree of polymerisation of three or more into higher fibers.
  • the invention relates to a process for elongating gluco- oligosaccharides with at least one ⁇ -(1,2)-terminally linked glucosyl moiety, comprising the steps of: a.
  • an acceptor composition comprising gluco-oligosaccharides having a degree of polymerisation of 2, 3, 4, 5 or 6 glucosyl moieties, wherein each gluco- oligosaccharide comprises at least one ⁇ -(1,4)-linkage and wherein the acceptor composition comprises at least two gluco-oligosaccharides having a different degree of polymerisation; and b. placing the acceptor composition in fluid contact with the above isolated polypeptide in the presence of a 1-glucosyl phosphate donor.
  • the process results in the addition of one or more glucosyl moieties to the acceptor gluco-oligosaccharide introducing an ⁇ -1,2-linkage between the glucosyl moiety and the gluco- oligosaccharide.
  • the glucosyl moiety is terminally linked to the gluco-oligosaccharide, i.e., it PT-2001-WO-PCT is bound to the non-reducing end of the gluco-oligosaccharide.
  • one or more glucosyl moieties are non-terminally ⁇ -1,2-linked to the acceptor gluco-oligosaccharide.
  • the inventors have advantageously found that using an acceptor composition which is a mixture of gluco-oligosaccharides having different degrees of polymerisation, in particular a hydrolysed starch, with newly identified isolated polypeptides having kojibiose phosphorylase activity, (wherein the activity of the polypeptide towards kojibiose or maltose is higher as compared to its activity towards glucose as an acceptor substrate), elongation of the gluco- oligosaccharides with (at least one) ⁇ -1,2-linked glucosyl moiety can be obtained, resulting in a gluco-oligosaccharide composition that has decreased digestibility (i.e.
  • the invention in a third aspect, relates to a composition comprising gluco- oligosaccharides elongated with at least one ⁇ -(1,2)-terminally linked glucosyl residue.
  • the invention in a fourth aspect, relates to an edible product comprising the composition of the invention.
  • An isolated polypeptide comprising kojibiose phosphorylase activity (E.C.2.4.1.230), PT-2001-WO-PCT
  • the isolated polypeptide is capable of using glucose, kojibiose and maltose as an acceptor substrate and converting the acceptor substrate into kojibiose, kojitriose, and alpha- 1,2-glucosyl-maltose as a product respectively, and wherein the activity of the polypeptide towards kojibiose or maltose is higher as compared to its activity towards glucose as an acceptor substrate.
  • the isolated polypeptide according to clause 3 wherein the isolated polypeptide comprises the amino acid residues 1-748 of SEQ ID NO:3, residues 1-746 of SEQ ID NO:4 and/or residues 1-704 of SEQ ID NO:5.
  • a process for elongating gluco-oligosaccharides with at least one ⁇ -(1,2)- terminally linked glucosyl moiety comprising the steps of: a.
  • an acceptor composition comprising gluco-oligosaccharides having a degree of polymerisation of 2, 3, 4, 5 or 6 glucosyl moieties, wherein each gluco- oligosaccharide comprises at least one ⁇ -(1,4)-linkage and wherein the acceptor composition comprises at least two gluco-oligosaccharides having a different degree of polymerisation; and b. placing the acceptor composition in fluid contact with an isolated polypeptide according to clauses 1 to 4 in the presence of a 1-glucosyl phosphate donor.
  • PT-2001-WO-PCT 6 The process according to clause 5, wherein the acceptor composition comprises at least 3, preferably at least 4 gluco-oligosaccharides having a different degree of polymerisation. 7.
  • the acceptor composition is a partially hydrolysed starch having a dextrose equivalent (DE) of less than 60, preferably less than 50, more preferably less than 45.
  • DE dextrose equivalent
  • the partially hydrolysed starch is a maltodextrin or glucose syrup.
  • the acceptor composition comprises at least two different gluco-oligosaccharides selected from the group consisting of maltose, maltotriose, maltotetraose, maltopentaose and maltohexaose. 10.
  • the polypeptide comprising maltose phosphorylase activity is a recombinant maltose phosphorylase, preferably obtained or obtainable from a bacterium selected from the group consisting of Lactobacillus acidophilus, Bacillus selenitireducens, Enterococcus faecalis, Lactobacillus brevis, Lactobacillus sanfranciscensis, Paenibacillus sp. And Bacillus sp. 13.
  • a composition comprising elongated gluco-oligosaccharides obtained or obtainable by the process according to any of clauses 5 to 12, comprising at least 40 wt.% of elongated gluco- oligosaccharides based on the total weight of the composition as dry substance.
  • a composition comprising at least 40 wt.% gluco-oligosaccharides elongated with at least one ⁇ -(1,2)-terminally linked glucosyl moiety based on the total weight of the composition as dry substance, wherein the gluco-oligosaccharides have a degree of polymerisation of 2, 3, 4, 5, 6 or 7; and wherein the composition comprises at least two gluco-oligosaccharides having a different degree of polymerisation and wherein each gluco-oligosaccharide comprises at least one ⁇ -(1,4)-linkage.
  • composition according to any of clauses 13 to 16, comprising at least two different elongated gluco-oligosaccharides selected from: 2-O- ⁇ -D-glucosyl-maltose ⁇ -D-Glucopyranosyl-(1 ⁇ 2)- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-D-glucopyranose PT-2001-WO-PCT 2-O- ⁇ -D-glucosyl-maltotriose ⁇ -D-Glucopyranosyl-(1 ⁇ 2)- ⁇ -D-glucopyranosyl-(1 ⁇ 4)- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-D- glucopyranose 2-O- ⁇ -D-glucosyl-maltotetraose ⁇ -D-Glucopyranosyl-(1 ⁇ 2)- ⁇ -D-glucopyranosyl-(1 ⁇ 4)- ⁇ -D-glucopyranosyl-(1 ⁇ 4)- ⁇ - D- D-glucopy
  • DE Dextrose equivalent
  • the approved method for determining DE is the Lane–Eynon titration method, which measures reduction of a copper sulfate solution. Unhydrolyzed starch has a DE value of zero, while the DE value of anhydrous D-glucose is 100. Glucose syrups range from 20 to 97 DE.
  • Elongated gluco-oligosaccharide refers to a gluco-oligosaccharide that comprises at least one ⁇ -(1,2)-terminally linked glucosyl moiety.
  • Fratose syrup refers to an aqueous solution of nutritive saccharides obtained from edible starch in which a portion of the dextrose (D-glucose) has been isomerized to fructose.
  • Gluco-oligosaccharides refers to oligosaccharides having two or more glucosyl moieties in the molecular chain.
  • glucosyl moieties can be bound to each other via ⁇ - (1,4)-linkages or via ⁇ -(1,6)-linkages.
  • the gluco-oligosaccharides in the acceptor composition comprise at least one ⁇ -(1,4)-linkage.
  • Glucose syrup refers to an aqueous solution of nutritive saccharides obtained from edible starch having a DE of 20 or more.
  • Kojibiose phosphorylase” or “KP” is a carbohydrate-processing phosphorolytic enzyme and is classified into the glycoside hydrolase family 65 (GH65).
  • Kojibiose phosphorylase is known by the IUBMB Enzyme Nomenclature number EC 2.4.1.230. Kojibiose phosphorylase can be used to catalyse the reaction between a saccharide such as maltose and inorganic phosphate to glucose and ⁇ -D-glucose-1-phosphate.
  • a saccharide such as maltose and inorganic phosphate
  • PT-2001-WO-PCT “Maltodextrin” refers to a dried product or aqueous solutions of saccharides obtained from edible starch having a DE of less than 20.
  • Polypeptide refers to a linear organic polymer consisting of a large number of amino- acid residues bonded together in a chain, forming part of or a whole a protein molecule.
  • Isolated polypeptide [0033] In one aspect, the present invention relates to an isolated polypeptide. In particular, the present invention relates to an isolated polypeptide comprising kojibiose phosphorylase activity (E.C.
  • the isolated polypeptide is capable of using glucose, kojibiose and maltose as an acceptor substrate and converting the acceptor substrate into kojibiose, kojitriose, and alpha-1,2-glucosyl-maltose as a product respectively, and wherein the activity of the polypeptide towards kojibiose or maltose is higher as compared to its activity towards glucose as an acceptor substrate.
  • the activity of the polypeptide towards kojibiose and maltose is higher as compared to its activity towards glucose as an acceptor substrate.
  • the isolated polypeptide comprising kojibiose phosphorylase activity is obtained or obtainable or derived or derivable from a microorganism selected from the group consisting of Thermococcus barophilus, Thermophilum pendens, and/or Palaeococcus pacificus.
  • the isolated polypeptide comprising kojibiose phosphorylase activity is obtained or obtainable or derived or derivable from a microorganism selected from the group consisting of Thermococcus barophilus, Thermophilum pendens Hrk 5, and/or Palaeococcus pacificus DY20341.
  • the isolated polypeptide comprising kojibiose phosphorylase activity is obtained or obtainable or derived or derivable from Thermococcus barophilus. In some examples of the present invention, the isolated polypeptide comprising kojibiose phosphorylase activity is obtained or obtainable or derived or derivable from Thermophilum pendens, optionally wherein the Thermophilum pendens is Thermophilum pendens Hrk 5.
  • the isolated polypeptide comprising kojibiose phosphorylase activity is obtained or obtainable or derived or derivable from Palaeococcus pacificus, optionally wherein the Palaeococcus pacificus is Palaeococcus pacificus DY20341.
  • the isolated polypeptide comprises residues selected from the group consisting of: residues 1-748 of SEQ ID NO:3, residues 1-746 of SEQ ID NO:4 and/or residues 1-704 of SEQ ID NO:5.
  • the isolated polypeptide comprises residues 1-748 of SEQ ID NO:3.
  • the isolated polypeptide comprises residues 1-746 of SEQ ID NO:4. In some examples of the present invention, the isolated polypeptide comprises residues 1-704 of SEQ ID NO:5. In some examples of the present invention, the isolated polypeptide comprises residues 1-748 of SEQ ID NO:3 or residues 1-746 of SEQ ID NO:4. In some examples of the present invention, the isolated polypeptide comprises residues 1-748 of SEQ ID NO:3 or residues 1-704 of SEQ ID NO:5. In some examples of the present invention, the isolated polypeptide comprises residues 1-746 of SEQ ID NO:4 or residues 1-704 of SEQ ID NO:5.
  • the isolated polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. In some examples of the present invention, the isolated polypeptide consists of the amino acid sequence set forth in SEQ ID NO:3. In some examples of the present invention, the isolated polypeptide comprises at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or, 80% identity to the amino acid sequence set forth in any one of SEQ ID NO: 3 and/or to the amino acid sequence of amino acid residues 1-748 of SEQ ID NO:3.
  • the isolated polypeptide consists of the amino acid sequence set forth in SEQ ID NO:4.
  • the isolated polypeptide comprises at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or, 80% identity to the amino acid sequence set forth in any one of SEQ ID NO:4 and/or to the amino acid sequence of amino acid residues 1-746 of SEQ ID NO:4.
  • the isolated polypeptide consists of the amino acid sequence set forth in SEQ ID NO:5.
  • the isolated polypeptide comprises at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or, 80% identity to the amino acid sequence set forth in any one of SEQ ID NO: 5 and/or to the amino acid sequence of amino acid residues 1-704 of SEQ ID NO:5.
  • TbarKP barophilus
  • a proline residue at an amino acid position corresponding to position 393 of TbarKP SEQ ID NO: 3
  • a leucine residue at an amino acid position corresponding to position 394 of TbarKP SEQ ID NO: 3
  • TbarKP barophilus
  • a proline residue at an amino acid position corresponding to position 393 of TbarKP SEQ ID NO: 3
  • a leucine residue at an amino acid position corresponding to position 394 of TbarKP SEQ ID NO: 3
  • have a much higher activity towards maltose compared to glucose when compared to kojibiose phosphorylases which contain, for example, other residues at amino acid positions corresponding to position 387, 393 and/or 394 of TbarKP (e.g.
  • residues E400, G405 and Y406 shown in SEQ ID NO:1, or residues E418, G423 and F424 shown in SEQ ID NO:2 which are the amino acid positions for the KPs derived from C. Saccharolyticus and T. Brockii, respectively that correspond to positions 387, 393 and 394 of TbarKP).
  • residues of a KP derived from P. pacificus (PpKP) (SEQ ID NO:4) corresponding to amino acids at position 387, 393 and 394 of TbarKP (SEQ ID NO:3) are A385, P391, and L392.
  • TpKP pendens
  • SEQ ID NO:5 corresponding to amino acids at position 387, 393 and 394 of TbarKP (SEQ ID NO:3) are A375, P381, and L382.
  • the residues corresponding to amino acids at position 387, 393 and 394 of TbarKP (SEQ ID NO:3) of a given sequence can be determined by routine methods in the art, e.g. using a sequence alignment tool such as a Clustal Omega with default parameters e.g. as outlined in Sievers et al (Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega) Molecular Systems Biology 7:539 (2011).
  • the isolated polypeptide comprises an alanine residue at the amino acid position corresponding to position 387 of Thermococcus barophilus kojibiose phosphorylase (SEQ ID NO: 3). In some examples of the present invention, the isolated polypeptide comprises a proline residue at the amino acid position corresponding to position 393 of Thermococcus barophilus kojibiose phosphorylase (SEQ ID NO: 3). In some examples of the present invention, the isolated polypeptide comprises a leucine residue at the amino acid position corresponding to position 394 of Thermococcus barophilus kojibiose phosphorylase (SEQ ID NO: 3).
  • the isolated polypeptide comprises an alanine residue at the amino acid position corresponding to position 387 of Thermococcus barophilus kojibiose phosphorylase (SEQ ID NO: 3), a proline residue at the amino acid position corresponding to position 393 of Thermococcus barophilus kojibiose phosphorylase PT-2001-WO-PCT (SEQ ID NO: 3) and a leucine residue at the amino acid position corresponding to position 394 of Thermococcus barophilus kojibiose phosphorylase (SEQ ID NO: 3).
  • the present invention relates to a process for elongating gluco- oligosaccharides with at least one ⁇ -(1,2)-terminally linked glucosyl moiety.
  • the process comprises the steps of: a. providing an acceptor composition comprising gluco-oligosaccharides having a degree of polymerisation of 2, 3, 4, 5 or 6 glucosyl moieties, wherein each gluco- oligosaccharide comprises at least one ⁇ -(1,4)-linkage and wherein the acceptor composition comprises at least two gluco-oligosaccharides having a different degree of polymerisation; and b.
  • the process of the invention has as a starting material an acceptor composition comprising gluco-oligosaccharides having a degree of polymerisation of 2, 3, 4, 5 or 6 glucosyl moieties, wherein each gluco-oligosaccharide comprises at least one ⁇ -(1,4)- linkage and wherein the acceptor composition comprises at least two gluco-oligosaccharides having a different degree of polymerisation.
  • the acceptor composition comprises at least two gluco-oligosaccharides having a different degree of polymerisation.
  • the acceptor composition may comprise a gluco-oligosaccharide having a degree of polymerisation of two (two glucosyl moieties) and a gluco-oligosaccharide having a degree of polymerisation of three (three glucosyl moieties).
  • the acceptor composition can be obtained by mixing 2, 3, 4 or more gluco- oligosaccharides having different degrees of polymerisation.
  • the acceptor composition of the invention can be obtained by partial hydrolysis of starch as will be described hereafter.
  • the acceptor composition comprises at least two different gluco- oligosaccharides selected from the group consisting of maltose, maltotriose, maltotetraose, maltopentaose and maltohexaose.
  • CAS numbers for these compounds are: Maltose: 69-79-4; PT-2001-WO-PCT Maltotriose 1109-28-0; Maltotetraose 34612-38-9; Maltopentaose 34620-76-3; Maltohexaose 34620-77-4.
  • the acceptor composition can comprise further oligo- or polysaccharides, for instance those present in an acceptor composition obtained by partial hydrolysis of starch.
  • the acceptor composition is preferably a partially hydrolysed starch having a dextrose equivalent (DE) of less than 60, more preferably less than 50, more preferably less than 45.
  • the acceptor composition is preferably a partially hydrolysed starch having a dextrose equivalent (DE) of at least 4, preferably at least 10.
  • the partially hydrolysed starch is maltodextrin or glucose syrup.
  • the partially hydrolysed starch is maltodextrin or glucose syrup having a DE of less than 45 and a DE of at least 10.
  • the partially hydrolysed starch may be obtained by submitting starch to enzymatic or acid hydrolysis or a combination thereof, such as acid hydrolysis followed by enzyme hydrolysis.
  • the acceptor composition may contain further gluco-oligosaccharides having ⁇ -1,6-linkages, such as the isoforms of maltotriose, maltotetraose, maltopentaose and maltohexaose and oligosaccharides containing mixed ⁇ -1,6 and ⁇ -1,4 linkages and oligosaccharides having glucopyranosyl branches through mixed ⁇ -1,6 and ⁇ -1,4 linkages.
  • the acceptor composition may also comprise gluco-oligosaccharides having a degree of polymerisation higher than 6, e.g., 7 or 8.
  • the isolated polypeptide is as described above under the heading “isolated polypeptide”.
  • Donor [0051]
  • the process of the present invention can comprise a step of preparing a donor.
  • the donor is 1-glucosyl phosphate.
  • the 1-glucosyl phosphate donor is ß-D-glucose-1-phosphate.
  • PT-2001-WO-PCT [0052]
  • the donor is prepared by placing an appropriate phosphorylase with a saccharide in the presence of a phosphate source in a process coupled reaction (described below).
  • ß-D-glucose-1-phosphate is commercially available and can be used as such.
  • the donor is prepared by placing an appropriate phosphorylase with a saccharide in the presence of a phosphate source.
  • the phosphate source is phosphoric acid and/or phosphate salts.
  • a 1-glucosyl phosphate donor can be prepared by placing maltose in fluid contact with a polypeptide having maltose phosphorylase catalytic activity and a phosphate source, wherein the phosphate source can be phosphoric acid and/or a salt thereof.
  • the acceptor composition may already contain the maltose, or the maltose is generated in situ during the ⁇ - 1,2-linkage-creating reactions (i.e., from glucose and ß-glucose phosphate by maltose phosphorylase).
  • the polypeptide comprising maltose phosphorylase activity is an enzyme that catalyses the reaction of maltose and inorganic phosphate to glucose and ⁇ -D-glucose-1- phosphate. It is classified under EC 2.4.1.8.
  • the polypeptide comprising maltose phosphorylase activity is a recombinant maltose phosphorylase, preferably a polypeptide obtainable from a Bacterium selected from the group consisting of Lactobacillus acidophilus, Bacillus selenitireducens, Enterococcus faecalis, Lactobacillus brevis, Lactobacillus sanfranciscensis, Paenibacillus sp. and Bacillus sp. [0057]
  • An example is a maltose phosphorylase from Lactobaciluus acidophilus, such as described by Nakai, H., et al.
  • the present invention comprises a step of preparing the 1-glucosyl phosphate donor by placing trehalose in fluid contact with a polypeptide having trehalose phosphorylase activity, phosphoric acid and/or a salt thereof.
  • the polypeptide having trehalose phosphorylase activity is a polypeptide that catalyses the reaction of trehalose and inorganic phosphate to glucose and ⁇ -D-glucose-1-phosphate. It is classified under (EC 2.4.1.64). See for instance TRENDS in Biotechnology Vol.20 No.10 October 2002.
  • the present invention comprises a step of preparing the 1-glucosyl phosphate donor by placing sucrose in fluid contact with a polypeptide having sucrose phosphorylase activity, phosphoric acid and/or a salt thereof.
  • Process conditions [0061] The process of the present invention is carried out by placing the acceptor composition, and donor in fluid contact with the isolated polypeptide. In general, this means that the process is carried out in an aqueous solution.
  • the isolated polypeptide can also be an immobilised isolated polypeptide.
  • Process conditions are those generally known in the field.
  • the concentration of the acceptor composition in the aqueous solution is from 5 to 60 wt.%
  • the pH of the reaction is from 4 to 7 (such as from 5 to 7)
  • the molar ratio acceptor composition to donor composition is from 1:1 to 1:10
  • the temperature is from 40 to 70 °C
  • the concentration of the isolated polypeptide is from 0.05 to 20 Units/mL.
  • further polypeptides are included in the reaction.
  • the further polypeptides can be included simultaneously or sequentially.
  • the polypeptides are alpha-amylase (including variations such as maltogenic alpha-amylase), beta-amylase, debranching enzymes (pullulanase or/and isoamylase), 4-alpha- glucanotransferase, transglucosidase, and/or, mixtures thereof.
  • the process can further comprise a step of purifying the composition obtained such as by filtration and/or microfiltration, decolorization/deodorization using active carbon or porous polymeric resins (like styrene divinyl benzene resins) and/or demineralization using ion exchange resins.
  • the present invention relates to a composition comprising elongated gluco-oligosaccharides obtained or obtainable by the processes described above.
  • the composition comprises at least 40 wt.% of elongated gluco-oligosaccharides based on the total weight of the composition as dry substance.
  • the composition comprises at least 40 wt.% gluco-oligosaccharides elongated with at least one ⁇ -(1,2)-terminally linked glucosyl moiety based on the total weight of the composition as dry substance, wherein the gluco- oligosaccharides have a degree of polymerisation of 2, 3, 4, 5, 6 or 7; and wherein the composition comprises at least two gluco-oligosaccharides having a different degree of polymerisation and wherein each gluco-oligosaccharide comprises at least one ⁇ -(1,4)-linkage.
  • the composition comprises at least two gluco-oligosaccharides having a different degree of polymerisation.
  • each gluco-oligosaccharide in the composition comprises at least one ⁇ -(1,4)-linkage.
  • the ⁇ -(1,2)-terminally linked glucosyl moiety is adjacent to an ⁇ -(1,4)-linkage.
  • the composition further comprises at least two different elongated gluco-oligosaccharides selected from:
  • PT-2001-WO-PCT 2-O- ⁇ -D-glucosyl-maltose ( ⁇ -D-Glucopyranosyl-(1 ⁇ 2)- ⁇ -D-glucopyranosyl-(1 ⁇ 4)- D-glucopyranose); and/or, 2-O- ⁇ -D-glucosyl-maltotriose ( ⁇ -D-Glucopyranosyl-(1 ⁇ 2)- ⁇ -D-glucopyranosyl- (1 ⁇ 4)- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-D-glucopyranose); and/or, PT-2001-WO-PCT 2-O- ⁇ -D-glucosyl-maltotetraose ( ⁇ -D-Glucopyranosyl-(1 ⁇ 2)- ⁇ -D- glucopyranosyl-(1 ⁇ 4)- ⁇ -D-glucopyranosyl-(1 ⁇ 4)- ⁇ -D-glucopyranosyl-(1 ⁇ 4)-D-glucopyra
  • composition of the invention is characterised by elongated gluco- oligosaccharides comprising saccharide linkages selected from ⁇ -(1,2)-; ⁇ -(1,4)- and other saccharide linkages; wherein the amount of ⁇ -(1,4)-linkages is more than 50% and the amount of ⁇ -(1,2)-linkages is less than 50% of all saccharide linkages, excluding linkages from kojibiose and kojioligosaccharides.
  • the ratio of ⁇ -(1,4)-linkages and ⁇ -(1,2)-linkages can be determined by high performance anion exchange chromatography (HPAEC), and/or NMR techniques and/or structural analysis by methylation and/or gas chromatography mass spectrometer (GC-MS).
  • HPAEC high performance anion exchange chromatography
  • GC-MS gas chromatography mass spectrometer
  • the composition may further contain kojibiose and kojioligosaccharides. These compounds also contribute to decreased digestibility of the composition of the invention.
  • Kojibiose and kojioligosaccharides are in particular characterised by the following formulas: PT-2001-WO-PCT Kojibiose ( ⁇ -D-Glucopyranosyl-(1 ⁇ 2)-D-glucopyranose) Kojitriose ( ⁇ -D-Glucopyranosyl-(1 ⁇ 2)- ⁇ -D-glucopyranosyl-(1 ⁇ 2)-D- glucopyranose) Kojitetraose ( ⁇ -D-Glucopyranosyl-(1 ⁇ 2)- ⁇ -D-glucopyranosyl-(1 ⁇ 2)- ⁇ -D- glucopyranosyl-(1 ⁇ 2)-D-glucopyranose) Kojipentaose ( ⁇ -D-Glucopyranosyl-(1 ⁇ 2)- ⁇ -D-glucopyranosyl-(1 ⁇ 2)- ⁇ -D- glucopyranosyl-(1 ⁇ 2)- ⁇ -
  • the composition of the invention comprises PT-2001-WO-PCT - one or more elongated gluco-oligosaccharides having a degree of polymerisation of 3, 4 or 5, wherein each elongated gluco-oligosaccharide comprises at least one ⁇ -1,2- terminally linked glucosyl moiety and at least one ⁇ -(1,4)-linkage, in an amount of from 25 wt.% or more, preferably from 30 wt.% or more, more preferably from 35 wt.% or more, even more preferably from 40 wt.% or more, even more preferably from 45 wt.% or more, most preferably from 50 wt.% or more of the total weight of dry substance of the composition; and/or - kojitriose,
  • the composition of the invention comprises - one or more elongated gluco-oligosaccharides having a degree of polymerisation of 3, 4 or 5, wherein each elongated gluco-oligosaccharide comprises at least one ⁇ -1,2- terminally linked glucosyl moiety and at least one ⁇ -(1,4)-linkage, in an amount of from 30 wt.% to 70 wt.%, preferably from 40 wt.% to 65 wt.%; most preferably from 50 wt.% to 60 wt.% of the total weight of dry substance of the composition; and/or - kojitriose, kojitetratose and kojipentaose in an amount of from 8 wt.% to 25 wt.%, preferably from 10 wt.% to 20 wt.%, most preferably from 12 wt.% to 15 wt.% of the total weight of dry substance of the composition; and
  • Digestibility of the composition can be shown by in-vitro digestion tests using in a first stage an alpha-amylase, followed by a digestion stage using a glucoamylase, an alpha- glucosidase or intestinal animal enzymes. Both stages can be combined to one digestion reaction using alpha-amylase and a glucoamylase or an alpha-glucosidase and intestinal animal enzymes.
  • PT-2001-WO-PCT [0080]
  • Other advantageous properties of the composition besides the lower calories compared to sucrose, are a high water solubility, viscosities comparable to commercial glucose syrups and maltodextrins and a lower relative sweetness than sucrose.
  • the composition can be used in the form of a syrup or it can be dried down to be used in the form of a powder.
  • the composition can be used in foodstuffs for human consumption. In particular it can be used as a sweetener, sugar replacer, (low calorie) bulking agent, fiber, low calorie carbohydrate composition, prebiotic composition, low glycemic carbohydrate composition, a starch retrograding inhibiting agent, a crystallization inhibiting agent, an osmotic pressure regulator or a water activity regulator. In particular it can be used for calorie and/or sugar reduction.
  • the composition can be used in foodstuffs in combination with other bulking agents, fillers, soluble fibers, resistant starch, polydextrose, dextrins, resistant maltodextrin, inulin, or insoluble fibers.
  • the combination can boost the fiber content of the foodstuff, enhance physiological benefits from consumption of the foodstuff, reduce the calorie content, reduce the sugars content, and/or enhance the nutritional profile of the foodstuff.
  • the composition can also be used in foodstuffs in combination with sweeteners, including high intensity sweeteners (such as sucralose, saccharin, aspartame, acesulfame K, brazzein, mogrosides (such as those extracted from Luo Han Guo fruit, including mogroside V) and steviol glycosides (such as those extracted from the stevia plant, including rebaudiosides and stevioside, or those obtained by fermentation or bioconversion), low calorie sweeteners (such as polyols, including erythritol, sorbitol, xylitol, lactitol, maltitol, mannitol, isomalt, etc.) and conventional caloric sweeteners (such as sucrose, glucose syrups, fructose syrups, dextrose, high fructose corn syrup, maltose, lactose, etc.).
  • sweeteners including high intensity sweeteners (such as sucralose, saccharin,
  • Foodstuffs in which the composition of the invention can be incorporated, are not limited and include: PT-2001-WO-PCT - bakery products, both yeast-raised (including donuts, sweet doughs, breads, brioche etc.) or chemically leavened (including cookies, cookie crisps, biscuits, muffins, cakes, brownies etc.); - breakfast cereals (including extruded cereals, puffed cereals etc.); - dairy products, for instance yogurt, yogurt drinks, dairy milk drinks, smoothies, ice cream, shakes, cottage cheese, dairy-based dressing, and dairy-based desserts (including mousse, puddings etc.) and the like; - confectionery products, for instance hard candies, fondants, nougats, marshmallows, gelatin jelly candies or gummies or jelly beans, jellies, chocolate, liquorice, chewing gum, caramels, toffees, chews, mints, tableted confections, and processed fruit snacks, chocolates, chocolate coatings, and icings
  • the composition can be used in feed or petfood (dry or moist) for animal consumption.
  • it can be used as prebiotic composition to improve animal digestive health and/or to reduce calories and/or sugar content.
  • the composition can be used in personal care products, in particular cosmetics (for instance skin creams) and oral care products (for instance toothpaste).
  • cosmetics for instance skin creams
  • oral care products for instance toothpaste
  • it can be used as personal care composition to improve the microbiome found on the skin.
  • it can be used in toothpaste or similar oral care compositions to improve the microbiome found in the mouth.
  • the composition may be utilized as a prebiotic and may also be coupled with a probiotic delivery system, for instance used in combination with one or more probiotics in a foodstuff.
  • prebiotic it is meant a food ingredient that beneficially affects the subject (human or animal) by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the gastro-intestinal tract, particularly the colon, and thus improves the digestive health of the host.
  • probiotic it is meant living microbiological dietary supplements that provide beneficial effects to the subject through their passage and function in the digestive tract.
  • the composition may be utilized as a prebiotic and may also be coupled with a postbiotic delivery system, for instance used in combination with one or more postbiotics in a foodstuff.
  • postbiotic also known as metabiotics, biogenics, or simply metabolites
  • soluble factors secreted by live bacteria, or released after bacterial lysis providing physiological benefits to the host.
  • These are typically bioactive compounds the probiotic bacteria produce when they consume prebiotics (such as dietary fiber).
  • Foodstuffs of the present invention can also be used to help control the blood glucose concentration (glycemia) in humans and animals, for instance that suffer from insulin resistance, pre-diabetes or diabetes.
  • the foodstuff can also be a nutraceutical, a dietary supplement, or a functional, fortified, and/or enriched food providing health benefits to the consumer.
  • Figure 1 provides an overview of the coupled reaction using maltose phosphorylase (MP) and kojibiose phosphorylase (KP).
  • MP maltose phosphorylase
  • KP kojibiose phosphorylase
  • PT-2001-WO-PCT Enzyme production Gene cloning and transformation
  • KP kojibiose phosphorylases
  • CsKP C. saccharolyticus
  • Tbrockii TbKP, UniProt identifier Q8L163
  • T. barophilus TbarKP, UniProt identifier F0LIH8
  • P. pacificus P. pacificus (PpKP, UniProt identifier A0A075LVI8), and T.
  • TpKP UniProt identifier A1RZC3
  • CAZy database of Carbohydrate-Active enZYmes.
  • the genes were codon optimized for E. coli and synthesised (Life Technologies, Merelbeke, Belgium). Sequences were subsequently subcloned into a pET21 vector at NheI and XhoI restriction sites, consequently introducing a C-terminal His 6 -tag.
  • the plasmid was transformed in E. coli BL21(DE3) electrocompetent cells.
  • Enzyme expression and recovery [0095] An overnight culture was inoculated (2%) in 500 mL LB-Lennox medium containing 100 ⁇ g/mL ampicillin in a 2-L shake flask and incubated at 37 °C with continuous shaking at 200 rpm. The cultures were grown to OD 600 0.6, and expression of both enzymes (in pET21a) was induced by adding isopropyl ⁇ -D-1-thio-galactopyranoside to a final concentration of 0.1 mM. Gene expression of all KPs took place for 16 hours at 30 °C. The cultures were then centrifuged (15 min, 9000 rpm), and the cell pellets were frozen at -20 °C for at least 4 hours.
  • the cell pellets were thawed and dissolved in 10 mL lysis buffer consisting of 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mg/mL lysozyme and 50 mM 2-morpholinoethanesulfonic acid (MES) pH 6.5. This suspension was incubated on ice for 30 min and sonicated three times for 3 min (Branson Sonifier 250, level 3, 50% duty cycle). [0097] Some of the enzymes were expressed as insoluble but active inclusion bodies, and therefore the crude cell lysates were not further purified. Potential background activity in the crude cell extract was inhibited by means of heat treatment.
  • PMSF phenylmethylsulfonyl fluoride
  • MES 2-morpholinoethanesulfonic acid
  • the glucose standard curve and glucose released by phosphorolysis of kojibiose and maltose was quantified with a colorimetric enzymatic coupled assay using glucose oxidase and peroxidase (GOD-POD).
  • Glucose Oxidase converts glucose into gluconolacton, producing hydrogen peroxide.
  • the peroxide is subsequently reduced by peroxidase (POD), using ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) as electron donor.
  • the oxidized ABTSH has a green colour, which can be measured at 420 nm.
  • Activity measurements were performed on 1 mL scale with a reaction mixture containing 100 mM kojibiose or maltose in 50 mM sodium phosphate (NaH2PO4/Na2HPO4) buffer, pH 6.5 at 55 ⁇ C and shaken at 800 rpm.25 ⁇ L samples were taken at set timepoints (0, 1, 2, 4, 6, 8 and 10 min), and inactivated in 25 microL 0.2 M NaOAc buffer pH 2.5.
  • NaH2PO4/Na2HPO4 sodium phosphate
  • Example 1 Relative acceptor activity of kojibiose phosphorylases
  • TbKP quantitative thin layer chromatography
  • Reactions were performed on a 1 mL scale, starting from 50 mM of the respective acceptor substrate, 200 mM ⁇ -G1P, in 50 mM MES buffer at pH 6.5, 55 ⁇ C and 800 rpm.
  • the enzyme was added in a sufficient amount to obtain a linear curve of product formation.
  • the chosen acceptor substrates were glucose (glc), kojibiose (K2), and maltose (M2).
  • Glucose and maltose (M2) were purchased from Sigma-Aldrich, kojibiose (Beerens, K. et al.
  • HPAEC high performance anion exchange chromatography
  • Table 1 The relative acceptor activity of known (TbKP, CsKP) and novel (TbarKP, PpKP, TpKP) KPs.
  • TbKP, CsKP novel acceptor activity of known KPs
  • TbarKP, PpKP, TpKP novel KPs.
  • PT-2001-WO-PCT EXAMPLE 2 Coupled reaction – comparison of with TbarKP [0107]
  • the performance of was evaluated in a coupled reaction using a commercial gluco-oligosaccharide mixture containing M2, M3 and M4, amongst others (30DE syrup:).
  • This 30DE syrup contains about 12 wt.% DP2, about 19 wt.% DP3, about 7 wt.% DP4, about 20 wt.% DP5, about 11 wt.% DP6 and about 21 wt.% DP11 (w/w % dry solids).
  • the remaining weight percentage to reach 100 wt.% on dry solids of the 30De syrup contains other saccharides as impurities.
  • Example 1 it was evaluated whether its beneficial acceptor preference as illustrated in Example 1 also leads to a more desired product composition from an industrially relevant substrate.
  • An identical reaction was performed with CsKP to enable their comparison.
  • maltose phosphorylase from Lactobacillus acidophilus (LaMP) was added.
  • MP uses inorganic phosphate to perform phosphorolysis of the maltose present in the reaction mixture, leading to the production of ⁇ - G1P (Hüwel et al., 1997).
  • KP can use the released ⁇ -G1P as a donor substrate to elongate the gluco-oligosaccharides present in the reaction mixture.
  • gluco-oligosaccharides comprising an a-(1,4)-linkage elongated with at least one ⁇ -(1,2)-terminally linked glucosyl moiety ( a1,2-MDx (DP3-5)) and kojioligosaccharides (K2-K5) in the reaction mixture was detected (see Table 1).
  • the PT-2001-WO-PCT remaining weight percentage to reach 100 wt.% on dry solids contains other saccharides such as glucose, maltose, and maltotriose.
  • SEQ ID NO: 1 Amino acid sequence of the kojibiose phosphorylase from Caldicellulosiruptor saccharolyticus (strain DSM 8903). (UniProt identifier A4XGP2). The C-terminal His6-tag and linker sequence are underlined.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

La présente invention concerne un polypeptide isolé comprenant une activité kojibiose phosphorylase (EC 2.4.1.230), le polypeptide isolé étant capable d'utiliser le glucose, le kojibiose et le maltose en tant que substrat accepteur et de convertir le substrat accepteur en kojibiose, kojitriose, et alpha-1,2-glucosyl-maltose en tant que produit respectivement. La présente invention concerne également un procédé enzymatique d'allongement de gluco-oligosaccharides avec une fraction glucosyle, le processus enzymatique utilisant le polypeptide isolé de la présente invention.
PCT/US2025/043076 2024-08-23 2025-08-22 Procédé de modification de gluco-oligosaccharides avec des kojibiose phosphorylases Pending WO2026044164A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP24196289.3 2024-08-23
EP24196289 2024-08-23

Publications (2)

Publication Number Publication Date
WO2026044164A2 true WO2026044164A2 (fr) 2026-02-26
WO2026044164A3 WO2026044164A3 (fr) 2026-04-09

Family

ID=92539031

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/043076 Pending WO2026044164A2 (fr) 2024-08-23 2025-08-22 Procédé de modification de gluco-oligosaccharides avec des kojibiose phosphorylases

Country Status (1)

Country Link
WO (1) WO2026044164A2 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6066477A (en) 1996-11-08 2000-05-23 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Method of producing glucosylated saccharides with kojibiose phosphorylase

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20250236898A1 (en) * 2021-10-12 2025-07-24 Cargill, Incorporated Process for modifying gluco-oligosaccharides

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6066477A (en) 1996-11-08 2000-05-23 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Method of producing glucosylated saccharides with kojibiose phosphorylase

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
BEERENS, K. ET AL.: "Biocatalytic synthesis of the rare sugar kojibiose: process scale-up and application testing", JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, 2017, pages 02258
CHAEN ET AL., JOURNAL OF BIOSCIENCE AND BIOENGINEERING, vol. 92, no. 2, 2001, pages 173 - 176
NAKADA, T. ET AL.: "Kojioligosaccharides: Application of Kojibiose Phosphorylase on the Formation of Various Kojioligosaccharides", OLIGOSACCHARIDES IN FOOD AND AGRICULTURE, 2003, pages 104 - 117
NAKADA, T. ET AL.: "Kojioligosaccharides: Application of Kojibiose Phosphorylase on the Formation of Various Kojioligosaccharides", OLIGOSACCHARIDES IN FOOD AND AGRICULTURE, pages 104 - 117
NAKAI, H. ET AL.: "The maltodextrin transport system and metabolism in Lactobacillus acidophilus NCFM and production of novel α-glucosides through reverse phosphorolysis by maltose phosphorylase", THE FEBS JOURNAL, vol. 276, 2009, pages 7353 - 7365, XP093301542, DOI: 10.1111/j.1742-4658.2009.07445.x
SIEVERS ET AL.: "Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega", MOLECULAR SYSTEMS BIOLOGY, vol. 7, 2011, pages 539
TRENDS IN BIOTECHNOLOGY, vol. 20, no. 10, October 2002 (2002-10-01)
VAN DER BORGHT, J.DESMET, T.SOETAERT, W.: "Enzymatic production of β-D-glucose-1-phosphate from trehalose", BIOTECHNOLOGY JOURNAL, vol. 5, 2010, pages 986 - 993, XP002791756, DOI: 10.1002/biot.201000203
YAMAMOTO, T. ET AL.: "Enzymatic Properties of Recombinant Kojibiose Phosphorylase from Caldicellulosiruptor saccharolyticus ATCC43494", BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY, vol. 75, no. 6, 2011, pages 1208 - 1210, XP093301543, DOI: 10.1271/bbb.110116

Also Published As

Publication number Publication date
WO2026044164A3 (fr) 2026-04-09

Similar Documents

Publication Publication Date Title
KR101540230B1 (ko) 분기 α-글루칸, 이를 생성하는 α-글루코실 전이 효소, 및 그의 제조 방법 및 용도
Goffin et al. Will isomalto-oligosaccharides, a well-established functional food in Asia, break through the European and American market? The status of knowledge on these prebiotics
Prapulla et al. Microbial production of oligosaccharides: a review
EP1545243B1 (fr) Utilisation d'edulcorants a faible indice glycemique dans des compositions d'aliment et de boisson
FI98336C (fi) Fruktoosia sisältävien oligosakkaridien käyttö vähäkalorisina makeutusaineina ja menetelmä ihmisen tai eläimen saaman kokonaisenergian määrän vähentämiseksi
AU2010237335B2 (en) Trehalulose-containing composition, its preparation and use
US20130216652A1 (en) Intrinsic sugar reduction of juices and ready to drink products
KR20190015334A (ko) 글리코실화 스테비올 배당체 조성물 및 글리코실화 스테비올 배당체 조성물의 제조 방법
KR20130014227A (ko) 신규한 α-글루코실 스테비오사이드 및 이의 제조 방법
JP2005531611A (ja) ガラクトシル・イソマルト、その製造方法及び使用
CN101044244B (zh) 异环麦芽寡糖和异环麦芽寡糖合酶以及其制备方法和用途
EP4416298B1 (fr) Procédé de modification de gluco-oligosaccharides
JP2018046762A (ja) 分岐α−グルカン混合物シラップとその用途
JP4224302B2 (ja) イソマルトースの製造方法並びに用途
WO2005021564A1 (fr) Maltosyle maltose cyclique, synthase de maltosyle maltose cyclique, procede de production et utilisation associes
Sorndech Isomaltooligosaccharides as prebiotics and their health benefits
KR101969040B1 (ko) 이소말토올리고당 함유 조성물 및 이의 제조방법
JP4238028B2 (ja) α−イソマルトシル転移酵素活性を有するポリペプチド
JP4164367B2 (ja) 分岐環状四糖とその製造方法ならびに用途
JP2003252895A (ja) 味質改善された羅漢果配糖体およびその製造方法
WO2002055708A1 (fr) Polypeptide presentant une activite de synthase d'$g(a)-isomaltosylglucosaccharide
Patel et al. Potential microbial bioresources for functional sugar molecules
TWI324635B (en) Process for producing isomaltitol and uses thereof
WO2026044164A2 (fr) Procédé de modification de gluco-oligosaccharides avec des kojibiose phosphorylases
Martins et al. Polysaccharide degradation for oligosaccharide production with nutraceutical potential for the food industry

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25771314

Country of ref document: EP

Kind code of ref document: A2