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WO2013190529A1 - Disaccharides galactosyliques glycolysés, procédés de préparation et leur utilisation dans des produits consommables - Google Patents

Disaccharides galactosyliques glycolysés, procédés de préparation et leur utilisation dans des produits consommables Download PDF

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Publication number
WO2013190529A1
WO2013190529A1 PCT/IB2013/055179 IB2013055179W WO2013190529A1 WO 2013190529 A1 WO2013190529 A1 WO 2013190529A1 IB 2013055179 W IB2013055179 W IB 2013055179W WO 2013190529 A1 WO2013190529 A1 WO 2013190529A1
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group
trans
galactosyl
donor
mixture
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PCT/IB2013/055179
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Gyula Dekany
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Glycom A/S
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • 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
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/03—Organic compounds
    • 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
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7016—Disaccharides, e.g. lactose, lactulose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
    • C07H5/04—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to nitrogen
    • C07H5/06—Aminosugars
    • 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
    • C12P19/12—Disaccharides
    • 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
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • 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
    • 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
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • 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
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • 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
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides

Definitions

  • the present invention relates to a method for generating glycosylated galactosyl disaccharides with enhanced bifidogenic activity.
  • oligosaccharides human milk oligosaccharides, prebiotics which promote the colonization of microbiota like bifidobacteria and lactobacilli in the small intestine, thus establishing gut microflora with many health benefits, such as increased resistance to diarrhoea and infections, maturing the immune system and stimulating immune system activity.
  • the gut microflora of formula-fed infants differs from that of the breastfed infants.
  • the microbiota of breast-fed infants mainly contains bifidobacteria, while the microbiota of formula- fed infants is more diverse, with bifidobacteria often being the predominant species, but also containing other and less beneficial species in substantial amounts. This is presumably due to the lack of non-digestible human milk oligosaccharides in infant formulae, which act as prebiotics and thus contribute to the bifidogenic microbiota.
  • Prebiotics are non-digestible food ingredients that stimulate the growth and/or activity of bacteria in the digestive system thus conferring benefits upon host well-being and health.
  • the simpler compounds having prebiotic effect are disaccharides containing galactose like lactose, allolactose, Galppi-3Glc, Galppi-2Glc, Galppi-6Gal, Galppi-3Gal and lactulose.
  • HMOs are not available in bulk and their large-scale microbial, enzymatic or chemical synthesis in a cost-efficient way has not been solved yet, there is still a need for potential bifidogenic oligosaccharides having prebiotic properties similar to those of HMOs.
  • the invention relates to a method for modifying galactosyl disaccharides in order to enhance their bifidogenic effect, comprising: reacting at least one glycosyl donor, which is not a galactosyl donor, with a precursor galactosyl disaccharide, or a mixture of precursor galactosyl disaccharides, represented by the formula Gal- A, wherein A means a
  • the invention further relates to a use of a compound or mixture of compounds obtainable the the method mentioned above for enhancing the bifidogenic effect of a consumable product.
  • the invention relates to a consumable product, preferably a nutritional formulation, a pharmaceutical formulation or a food supplement, comprising a compound or a mixture of compounds obtainable the the method mentioned above and nutritionally and/or pharmaceutically acceptable carriers, preferably for enhancing bifidogenic effect.
  • galactosyl disaccharide means an oligosaccharide of formula Gal-A wherein a galactosyl moiety is linked to the group A, which group A means a monosaccharide unit, preferably glucose, galactose, fructose, N-acetylglucosamine or N-acetylgalactosamine and represents the reducing end.
  • group A means a monosaccharide unit, preferably glucose, galactose, fructose, N-acetylglucosamine or N-acetylgalactosamine and represents the reducing end.
  • the galactose is linked with ⁇ -linkage to group A.
  • GalpPl-6Glc (alio lactose), GalpPl-3Glc, GalpPl-2Glc, GalpPl-6Gal, GalpPl-3Gal, lactulose, N-acetyllactosamine and lacto-N-biose.
  • 'TSf-acetyl-glucosaminyl within the context of the present application means an N- acetyl-2-amino-2-deoxy-D-glucopyranosyl (GlcNAc) group linked with ⁇ -linkage:
  • phrases “fulnesssialyl” within the context of the present invention means the glycosyl residue of sialic acid (N-acetyl-neuraminic acid, Neu5Ac) linked with a-linkage:
  • optionally substituted phenoxy means a phenoxy group optionally substituted with 1 or 2 groups selected from nitro, halogen, alkyl, hydroxyalkyl, amino, formyl, carboxyl and alkoxycarbonyl, or two substituents in ortho position may form a methylenedioxy-group.
  • Preferred substituents are nitro (preferably in 2- and/or 4-position), halogen (preferably fluoro and chloro).
  • Especially preferred substituted phenoxy groups are selected from 4- nitrophenoxy, 2,4-dinitrophenoxy, 2-chloro-4-nitrophenoxy, 2-fluoro-4-nitrophenoxy, 3- fluoro-4-nitrophenoxy, 2-hydroxymethyl-4-nitrophenoxy, 3-hydroxymethyl-4-nitrophenoxy, 2-formyl-4-nitrophenoxy, 2-carboxy-4-nitrophenoxy, 2-methoxycarbonyl-4-nitrophenoxy, 5- fluoro-2-nitrophenoxy, 4-methoxycarbonyl-2-nitrophenoxy, 4-carboxy-2-nitrophenoxy, 2- aminophenoxy and 3,4-methylenedioxy-phenoxy.
  • pyridinyloxy means a pyridinyloxy, more preferably 2- or 4-pyridyloxy group, optionally substituted with 1 or 2 groups selected from nitro, halogen, alkyl and alkoxy.
  • Especially preferred pyridinyloxy groups are selected from 4-pyridinyloxy, 2-pyridinyloxy, 3-nitro-2-pyridinyloxy and 3-methoxy-2-pyridinyloxy.
  • the term "donor” is understood as a compound that provides or transfers a specific moiety in a chemical reaction, e.g. a nucleophilic or electrophilic substitution reaction, to a further compound, preferably an acceptor.
  • a “donor” is understood as a compound that provides or transfers a glycosyl residue to a further compound, preferably an acceptor, wherein the donor is not restricted to naturally occurring donors.
  • acceptor is understood as a compound that receives a specific moiety, preferably a glycosyl moiety, in a chemical reaction, e.g. nucleophilic or electrophilic substitution reaction, from a further compound, preferably a donor as defined above.
  • the present invention provides a method for enhancing the bifidogenic effect of galactosyl disaccharides, in which galactosyl disaccharides are modified in order to enhance their bifidogenic effect, characterized in that at least one glycosyl residue, wherein the glycosyl residue is not galactosyl, is coupled to a precursor galactosyl disaccharide defined above or mixture thereof via the anomeric carbon atom of said glycosyl residue under the catalysis of an enzyme capable of transferring said glycosyl moiety to said precursor galactosyl disaccharide.
  • the method comprises the steps of: a) providing at least one glycosyl donor, b) providing a precursor galactosyl disaccharide or mixture thereof, c) providing at least one enzyme comprising a trans-glycosidase or a glycosynthase activity; d) preparing a mixture of the components provided in steps a), b) and c); e) incubating the mixture prepared according to step d); f) optionally: repeating steps a), c), d) and e) with the mixture obtained according to step e).
  • step a) at least one glycosyl donor is provided, and the glycosyl donor is not a galactosyl donor.
  • Compounds for use as glycosyl donors in step a) may preferably be selected from the group consisting of: a sialyl donor, a fucosyl donor and an optionally galactosylated N-acetyl-glucosaminyl donor.
  • said sialyl donor, fucosyl donor and/or optionally galactosylated N-acetyl-glucosaminyl donor has a leaving group selected from the group consisting of: fluoro, azido and -OR group, wherein R can be a mono-, di- or oligosaccharide, glyco lipid, glycoprotein or glycopeptide, cyclic or acyclic aliphatic group, or aryl residue; or wherein the optionally galactosylated N-acetyl-glucosaminyl donor is an oxazoline.
  • said sialyl donor is characterized by formula 1
  • said fucosyl donor is characterized by formula 2
  • said optionally galactosylated N-acetyl-glucosaminyl donor is characterized by formulae 3 or 4
  • X independently, is selected from the group consisting of azide, fluoro, optionally substituted phenoxy, optionally substituted pyridinyloxy, lactose moiety, group A, group B, group C and group D
  • Ri and R 2 independently, is H or ⁇ -D-galactopyranosyl group with the proviso that at least one of the Ri and R 2 groups is H.
  • the donors are preferably selected with reference to the enzymes used during the method of the present invention.
  • the donors are selected depending on the enzyme's transglycosidase activity from compounds according to formulae 1 to 3, wherein X is optionally substituted 4-nitrophenoxy, lactose moiety, group A, group B, group C, or from compounds of formula 4.
  • the donors are selected depending on the enzyme's glycosynthase activity from compounds according to formulae 1 to 3, wherein X is azide or fluoro. Both selections may be carried out independently of each other or together.
  • compounds for use as donors in step a) may preferably be selected from compounds according to formulae 1 to 3, wherein X is lactose moiety, 4-nitrophenoxy, 2,4- dinitrophenoxy, 2-chloro-4-nitrophenoxy, 2,5-dimethyl-3-oxo-(2H)-furan-4-yloxy, 2-ethyl-5- methyl-3-oxo-(2H)-furan-4-yloxy, 5-ethyl-2-methyl-3-oxo-(2H)-furan-4-yloxy, 4,6- dimethoxy- 1 ,3,5-triazin-2-yloxy, 4,6-diethoxy- 1 ,3 ,5-triazin-2-yloxy, 4- methylumbelliferyloxy, or from compounds of formula 4 represented by formulae 5, 6 or 7.
  • step c) at least one enzyme comprising a transglycosidase activity and/or a glycosynthase activity is provided.
  • Enzymes suitable in step c) typically comprise at least one enzyme comprising a
  • transglycosidase activity and/or a glycosynthase activity preferably selected from enzymes having, e.g. a fucosidase, trans-fucosidase or fucosynthase activity, a sialidase
  • step c) may be selected from the group comprising wild type or mutated glycosidases or
  • transglycosidases preferably wild type or mutated glycosidases or transglycosidases having a fucosidase, trans-fucosidase or fucosynthase activity, a sialidase (neuraminidase) or trans- sialidase (transneuraminidase) activity, a N-acetylglucosaminidase or trans-N- acetylglucosaminidase activity, a lacto-N-biosidase or trans-lacto-N-biosidase activity and/or a N-acetyllactosaminidase or trans-N-acetyllactosaminidase activity, or preferably having a- trans-fucosidase, a-trans-sialidase, ⁇ -trans-N-acetylglucosaminidase, ⁇ -trans- lacto-N- biosidase and/
  • the source of the enzymes suitable in step c) furthermore may be selected from any genus known to a skilled person to express or secrete at least one enzyme as defined above, e.g. an enzyme having a transglycosidase activity and/or a glycosynthase activity, preferably an enzyme having a fucosidase, trans-fucosidase or fucosynthase activity, a sialidase
  • neurosaminidase or trans-sialidase (transneuraminidase) activity, a N-acetylglucosaminidase or trans-N-acetylglucosaminidase activity, a lacto-N-biosidase or trans-lacto-N-biosidase activity and/or a N-acetyllactosaminidase or trans-N-acetyllactosaminidase activity, or preferably having a-trans-fucosidase, a-trans-sialidase, ⁇ -trans-N-acetylglucosaminidase, ⁇ - trans-lacto-N-biosidase and/or ⁇ -trans-N-acetyllactosaminidase activity, or any further enzyme having such an activity.
  • the source of the enzymes suitable in step c) may be selected from non-pathogenic bacteria selected from Bacillus, Bifidobacterium, Lactobacillus, Leuconostoc, Lactococcus, Streptococcus, Streptomyces, Sulfolobus,
  • Thermotoga or Trypanosoma.
  • the source of the enzymes suitable in step c) is selected from the group comprising the non-pathogenic bacteria Bacillus circulans, lactic acid bacteria, such as Bifidobacterium bifidum JCM 1254, Bifidobacterium bifidum NCIMB 41171, Bifidobacterium bifidum NCIMB 41171, Bifidobacterium bifidum JCM 1254, Bifidobacterium bifidum
  • Bifidobacterium bifidum SI 7 Bifidobacterium bifidum SI 7, Bifidobacterium dentium Bdl, Bifidobacterium longum subsp. infantis ATCC 15697, Bifidobacterium longum subsp longum JDM 301, Bifidobacterium longum subsp. infantis JCM 1222, Lactobacillus casei BL23, Streptomyces sp., Sulfolobus solfataricus P2, Thermotoga maritima MSB8, and Trypanosoma cruzi.
  • Particularly preferred microorganisms in the above context comprise lactic acid bacteria.
  • Lactic acid bacteria, and more particularly non-pathogenic bacteria from the genus Bifidobacterium contain a series of glycosidases including a-2,3/6 sialidases (GH33), a- 1,2/3/4 fucosidases (GH29 and GH95), lacto-N-biosidases (GH20), ⁇ -galactosidases (GH2) and ⁇ - ⁇ - acetylhexosaminidases (GH18, GH20, GH56, GH84, GH85 and GH123) that are able to recognize prebiotics and/or (human) milk oligosaccharides.
  • these glycosidases are intra- or extracellular enzymes.
  • glycosidase and/or glycosynthases displaying a trans-fucosidase, trans-sialidase, trans-N- acetylglucosaminidase, trans-lacto-N-biosidase and/or trans-N-acetyllactosaminidase activity, preferably a a-trans-fucosidase, a-trans-sialidase, ⁇ -trans-N-acetylglucosaminidase, ⁇ -trans- lacto-N-biosidase and/or ⁇ -trans-N-acetyllactosaminidase activity, is a wild type or an engineered glycosidase, and most preferably a wild type glycosidase
  • a glycosidase and/or glycosynthase obtained from the group consisting of lactic acid bacteria is most preferably a glycosidase from Bifidobacterium, Lactobacillus, Lactococcus, Streptococcus or
  • a glycosidase selected from the genus Bifidobacterium is most preferably a glycosidase from Bifidobacterium longum subsp. Infantis, Bifidobacterium longum subsp. Longum, Bifidobacterium breve, Bifidobacterium bifidum and Bifidobacterium catenulatum.
  • engineered fucosidases from thermophilic organisms such as Sulfolobus solfataricus and Thermotoga maritima have recently been developed, which may be used in the method of the present invention.
  • These thermostable glycosidases have considerable potential for industrial applications since they can be used in biotechno logical processes at elevated temperatures, so facilitating the process, preventing risk of contamination, and increasing the solubility of the compounds used in the reaction.
  • the glycosidase and/or glycosynthase enzyme displaying a trans-fucosidase, trans-sialidase, trans-N-acetylglucosaminidase, trans- lacto-N-biosidase and/or trans-N-acetyllactosaminidase activity preferably an a-trans- fucosidase, a-trans-sialidase, ⁇ - trans-N-acetylglucosaminidase, ⁇ -trans-lacto-N-biosidase and/or ⁇ -trans-N-acetyllactosaminidase activity, is a wild type or an engineered glycosidase.
  • the wild type glycosidase is obtained from the group consisting of thermophilic organisms, which glycosidase is converted to a transglycosidase by rational engineering or/and directed evolution.
  • An a-L- fucosidase obtained from thermophilic organisms is most preferably an a-L-fucosidase from Thermotoga maritima and Sulfolobus solfataricus.
  • the at least one enzyme comprising a transglycosidase activity and/or a glycosynthase activity may be selected from an enzyme exhibiting a fucosidase, trans- fucosidase or fucosynthase activity, preferably as described below.
  • enzymes having a fucosidase, trans-fucosidase or fucosynthase activity, more preferably an a-trans- fucosidase activity are preferably selected from fucosidases in general, and more preferably from ⁇ -L-fucosidases, e.g. a-L-fucosidases as classified according to EC 3.2.1.38 and
  • a-L-Fucosidases are widely spread in living organisms such as mammals, plants, fungi and bacteria. These enzymes belong to the families 29 and 95 of the glycoside hydrolases (GH29 and GH95) as defined by the CAZY nomenclature (http://www.cazy.org). Fucosidases from GH29 are retaining enzymes (3D structure: ( ⁇ / ⁇ ) 8 ) whereas fucosidases from GH95 are inverting enzymes (3D structure: (a /a) 6 ). The substrate specificity of the GH29 family is broad whereas that of the GH95 family is strict to al,2-linked fucosyl residues.
  • the GH29 family seems to be divided into two subfamilies.
  • One subfamily typically has strict specificity towards al,3- and al,4-fucosidic linkages.
  • the members of a further subfamily have broader specificity, covering all a-fucosyl linkages.
  • ⁇ -L-fucosidases generally hydrolyse the terminal fucosyl residue from glycans. These enzymes are also capable of acting as catalysts for fucosylation reactions due to their transfucosylation activity and thus may be used in the context of the method of the present invention, preferably under kinetically controlled conditions.
  • Fucosidases which may be employed in the context of the present invention, may also comprise engineered fucosidases.
  • Such engineered fucosidases preferably comprise engineered ⁇ -L-fucosidases, preferably engineered fucosidases derived from fucosidases as described above, e.g. an engineered a-l,2-L-fucosynthase from Bifidobacterium bifidum, a-L- fucosynthases from Sulfolobus solfataricus and Thermotoga maritime, etc.
  • Such engineered fucosidases show an acceptor dependent regioselectivity and are devoid of product hydrolysis activity.
  • engineered fucosidases preferably comprise a-L-fucosidase from Thermotoga maritima, which has also been recently converted into an efficient a-L-trans- fucosidase by directed evolution (see Osanjo et al. Biochemistry 46, 1022 (2007)).
  • the at least one enzyme having a fucosidase and/or trans-fucosidase and/or fucosynthase activity may be selected from ⁇ -L-fucosidases derived from Thermotoga maritima MSB8, Sulfolobus solfataricus P2, Bifidobacterium bifidum JCM 1254,
  • Bifidobacterium bifidum JCM 1254 Bifidobacterium longum subsp. infantis ATCC 15697, Bifidobacterium longum subsp. infantis ATCC 15697, Bifidobacterium longum subsp. Infantis JCM 1222, Bifidobacterium bifidum PRL2010, Bifidobacterium bifidum SI 7, Bifidobacterium longum subsp longum JDM 301, Bifidobacterium dentium Bdl, or Lactobacillus casei BL23, etc.
  • the at least one enzyme having a fucosidase and/or trans-fucosidase and/or fucosynthase activity may be selected from following ⁇ -L-fucosidases as defined according to the following deposit numbers gi
  • infantis ATCC 15697
  • 213522629 Bifidobacterium longum subsp. infantis ATCC 15697
  • 213522799 Bifidobacterium longum subsp. infantis ATCC 15697
  • 213524646 Bifidobacterium longum subsp. infantis ATCC 15697
  • 320457227 Bifidobacterium longum subsp. infantis JCM 1222
  • 320457408 Bifidobacterium longum subsp. infantis JCM 1222
  • 320459369 Bifidobacterium longum subsp.
  • infantis JCM 1222 gi
  • Wild type or engineered fucosidases as defined above, displaying transfucosidase activity and showing a al-2, al-3 and/or al-4 regioselectivity, may be used in the present invention.
  • Such wild type or engineered fucosidases preferably display transfucosidase activity and catalyse the transfer of the fucosyl residue to: the galactose of the lacto-N-biosyl group with 1-2 interglycosidic linkage and/or the N-acetyl-glucosamine of the lacto-N-biosyl group with 1-4 mterglycosidic linkage and/or the N-acetyl-glucosamine of the N-acetyl-lactosaminyl group with 1-3 mterglycosidic linkage and/or a terminal galactosyl moiety of the galactosyl disaccharide with 1-2 or 1-3 mtergly
  • a-L-fucosidases or the wild types of engineered fucosidases with fucosidase/trans-fucosidase/fucosynthase activity are listed in the following Table 1 :
  • the at least one enzyme comprising a transglycosidase activity and/or a glycosynthase activity may be selected from an enzyme exhibiting a sialidase or trans- sialidase activity, preferably as described in the following.
  • enzymes having a sialidase or trans-sialidase activity are preferably selected from a sialidase or trans-sialidase as described in the following, e.g. sialidases (EC 3.2.1.18) and trans-sialidases (EC 2.4.1.-) as classified according to the the GH33 family. They are retaining enzymes. Sialidases and trans-sialidases are widely distributed in nature.
  • Trans-sialidases differ from sialidases since they can transfer sialic acids, preferably a-2,3-bonded sialic acids, from a donor molecule to an acceptor derivative, which is preferably a terminal galactose moiety with a ⁇ -interglycosidic linkage. As a result of this transfer, an a-glycosidic bond is formed between the sialic acid and the acceptor. However, if there is no suitable acceptor, the trans-sialidase hydrolyses the sialic acid.
  • TcTS trans-sialidase
  • Trypanosoma brucei gambiense Trypanosoma brucei rhodesiense
  • Trypanosoma brucei brucei Trypanosoma congolense.
  • the existence of trans-sialidases has been shown in Endotrypanum types, in Cory neb acterium diphtheriae and even in human plasma.
  • Sialidases can be classified into two different subgroups, endo- and exo-sialidases.
  • the endo- sialidases hydro lyse sialic acid linkages internal to macro molecules, while the exo-sialidases attack terminal sialic acid linkages, and desialylate glycoproteins, glycopeptides,
  • sialidases from Bifidobacterium bifidum and Bifidobacterium longum subsp. infantis have been identified, cloned and characterized. These sialidases can cleave and so recognize both a-2,3- and a-2,6- linked sialosides.
  • Sialidases from Bifidobacterium longum subsp. infantis have a consistent preference for a-2,6-linkage whereas sialidases from Bifidobacterium bifidum have a consistent preference for a-2,3-linkage.
  • These enzymes are also capable of acting as catalysts for sialylation reactions due to their trans-sialidase activity and thus may be used in the context of the method of the present invention, preferably under kinetically controlled conditions.
  • Sialidases which may be employed in the context of the present invention, may also comprise engineered sialidases. Based on sequence and structure comparisons, sialidase from
  • Trypanosoma rangeli may be mutated at six positions, wherein the resulting mutant is able to display a significant level of trans-sialidase activity (see Paris et al. J. Mol. Biol. 345, 923 (2005)).
  • truncation of a sialyl transferase form Photobacterium damsela resulted in an enzyme having a2-6-trans-sialidase activity (Cheng et al. Glycobiology 20, 260 (2010).
  • the at least one enzyme having a sialidase and/or trans-sialidase activity may be selected from sialidases or trans-sialidases derived from Bifidobacterium longum subsp. infantis ATCC 15697, Bifidobacterium bifidum JCM1254, Bifidobacterium bifidum S17, Bifidobacterium bifidum PRL2010, Bifidobacterium bifidum NCIMB 41171, Trypanosoma cruzi, etc.
  • the at least one enzyme having a sialidase and/or trans-sialidase activity may be selected from sialidases or trans-sialidases as defined according to the following deposit numbers: gi
  • siab2 (Bifidobacterium bifidum JCM1254), further sialidases or trans-sialidases from Bifidobacterium bifidum JCM1254), gi
  • wild type or engineered sialidases as defined above may be utilized herein, which display trans-sialidase activity and show a a,2-3 and/or a,2-6 regioselectivity. Such linkages are preferably targeted in the present invention.
  • Such wild type or engineered sialidases preferably display trans-sialidase activity and catalyse the transfer of the sialyl residue to: the galactose of the lacto-N-biosyl group with 2-3 interglycosidic linkage and/or the N-acetyl-glucosamine of the lacto-N-biosyl group with 2-6 interglycosidic linkage and/or the galactose of the N-acetyl-lactosaminyl group with 2-6 interglycosidic linkage and/or the terminal galactosyl moiety of the galactosylated disaccharide with 2-3 or 2-6 interglycosidic linkage, a N-acetylglucosaminyl moiety, preferably to a terminal N-acetylglucosaminyl moiety with 2-3 or 2-6 interglycosidic linkage.
  • sialidases or the wild types of engineered/truncated sialidases with sialidase/trans-sialidase activity are listed in the following Table 2:
  • the at least one enzyme comprising a transglycosidase activity and/or a glycosynthase activity may be preferably selected from an enzyme exhibiting a lacto-N- biosidase or trans-lacto-N-biosidase activity, preferably as described in the following.
  • enzymes having a lacto-N-biosidase or trans-lacto-N-biosidase activity are preferably selected from a lacto-N-biosidase or trans-lacto-N-biosidase as described in the following, e.g. lacto-N-biosidases (EC 3.2.1.140) as classified according to the GH20 family.
  • Lacto-N- biosidases typically proceed through a retaining mechanism. Only two lacto-N-biosidases from Streptomyces and Bifidobacterium bifidum have been described and characterized up to now, which may be utilized in the present invention as a lacto-N-biosidase or trans-lacto-N- biosidase (see Sano et al. Proc. Natl. Acad. Sci. USA 89, 8512 (1992); Sano et al. J. Biol. Chem. 268, 18560 (1993); Wada et al. Appl. Environ. Microbiol. 74, 3996 (2008)).
  • Lacto-N- biosidases specifically hydro lyse the terminal lacto-N-biosyl residue (P-D-Gal-(1 ⁇ 3)-D- GlcNAc) from the non-reducing end of oligosaccharides with the structure P-D-Gal-(1 ⁇ 3)-P- D-GlcNAc-(l ⁇ 3)-P-D-Gal-(l ⁇ R).
  • Wada et al. (supra) and Murata et al. (Glycoconj. J. 16, 189 (1999)) also demonstrated the ability of the lacto-N-biosidase from Bifidobacterium bifidum and Aureobacterium sp.
  • L-101 respectively, to catalyse the transglycosylation by incubating donor substrates (such as lacto-N-tetraose and / ⁇ - ⁇ - ⁇ , ⁇ ) with acceptors (such as various 1-alkanols and lactose).
  • donor substrates such as lacto-N-tetraose and / ⁇ - ⁇ - ⁇ , ⁇
  • acceptors such as various 1-alkanols and lactose
  • the at least one enzyme having a lacto-N-biosidase or trans-lacto-N- biosidase activity may be selected from lacto-N-biosidases or trans- lacto-N-biosidases derived from Bifidobacterium bifidum JCM1254, Bifidobacterium bifidum PRL2010, Bifidobacterium bifidum NCIMB 41171, Aureobacterium sp. L-101 or Streptomyces sp., etc.
  • the at least one enzyme having a lacto-N-biosidase or trans-lacto-N- biosidase activity may be selected from lacto-N-biosidases or trans-lacto-N-biosidases as defined according to the following deposit numbers: gi
  • PRL2010 gi
  • lacto-N-biosidases as defined above may be utilized herein, which display trans-lacto-N-biosidase activity and show preferably a ⁇ 1-3
  • Such linkages are preferably targeted in the present invention.
  • Such wild type or engineered lacto-N-biosidases preferably display trans-lacto-N-biosidase activity and catalyse the transfer of the lacto-N-biosyl residue to a galactosyl group with 1-3
  • interglycosidic linkage are targeted in the present invention.
  • lacto-N-biosidases with lacto-N-biosidase or trans-lacto-N-biosidase activity are listed in the following Table 3:
  • the at least one enzyme comprising a transglycosidase activity and/or a glycosynthase activity may be preferably selected from an enzyme exhibiting a N- acetyllactosaminidase or trans-N-acetyllactosaminidase activity, preferably as described in following.
  • enzymes having a N-acetyllactosaminidase or trans-N- acetyllactosaminidase activity are preferably selected from a N-acetyllactosaminidase or trans-N-acetyllactosaminidase as described in the following, e.g.
  • lacto-N-biosidases (EC 3.2.1.140) as classified according to the GH20 family.
  • chitinase from bacillus circulans more preferably chitinase Al from Bacillus Circulans WL-12 as deposited under gi
  • wild type or engineered glycosidases as defined above which display trans-N- acetyllactosaminidase activity and show a ⁇ 1-3 and/or ⁇ 1-6 regioselectivity, may be used in the present invention.
  • Such wild type or engineered glycosidases preferably display trans-N- acetyllactosaminidase activity and catalyse the transfer of the N-acetyl-lactosaminyl residue to a galactosyl group with 1-3 or 1-6 interglycosidic linkage.
  • N-acetyllactosaminidases or trans-N-acetyllactosaminidases are listed in the following Table 4:
  • the at least one enzyme comprising a transglycosidase activity and/or a glycosynthase activity may be preferably selected from an enzyme exhibiting a N- acetylhexosaminidase or trans-N-acetylhexosaminidase activity, preferably as described in the following.
  • enzymes having a N-acetylhexosaminidase or trans-N- acetylhexosamimdase activity are preferably selected from a N-acetylhexosaminidase or trans-N-acetylhexosaminidase as described in the following, e.g.
  • ⁇ - ⁇ -acetylhexosaminidases (systematic name 2-acetamido-2-deoxy ⁇ -D-hexopyranoside acetamidodeoxyhexohydrolases, EC 3.2.1.52 (exo-glycosidases) and 3.2.1.96 (endo-glycosidases)) as classified according to the GH3, GH18, GH20, GH56, GH84, GH85 and GH123 families.
  • ⁇ - ⁇ -acetylhexosaminidases are mainly found in GH3, GH18, GH20, GH84 and GH85 families.
  • ⁇ - ⁇ -Acetylhexosaminidases have been shown to be universally distributed among most types of living organisms, both prokaryotic and eukaryotic.
  • ⁇ - ⁇ -acetylhexosaminidases catalyse the hydrolysis of glycosidic linkages. When acting as exo-enzymes, they catalyse the cleavage of terminal ⁇ -D-GlcNAc and ⁇ -D-GalNAc residues in N-acetyl ⁇ -D-hexosaminides. In vitro they can catalyse the formation of a new glycosidic bond either by transglycosylation or by reverse hydrolysis (i.e. condensation, see review: Slamova et al, Biotechnology Advances 28, 682 (2010)).
  • wild type or engineered glycosidases as defined above may be utilized herein, which display trans-N-acetylglucosaminidase activity and show a ⁇ 1-3 and/or ⁇ 1-6 regioselectivity.
  • Such wild type or engineered glycosidases preferably display trans-N- acetylglucosaminidase activity and catalyse the transfer of the N-acetyl-glucosaminyl residue to a galactosyl group with 1-3 or 1-6 interglycosidic linkage.
  • N-acetylglucosaminidases or trans-N-acetylglucosaminidases are listed in the following Table 5, or a sequence exhibiting a sequence identity with one of the below mentioned enzyme sequences having a N-acetylglucosaminidase or trans-N- acetylglucosaminidase activity of at least 70 %, more preferably at least 80 %, more preferably at least 85 %, even more preferably at least 90 % and most preferably at least 95 % 97 %, 98 % or 99 % as compared to the entire wild type sequence on amino acid
  • proteins comprising a transglycosidase and/or a glycosynthase activity as defined above may also comprise engineered proteins comprising a transglycosidase and/or a glycosynthase activity.
  • wild type or mutated glycosidases displaying a transfucosidase, transsialidase, trans-N-acetylglucosaminidase, trans- lacto-N- biosidase and/or trans-N-acetyllactosaminidase activity, preferably a a-transfucosidase, a- transsialidase, ⁇ - trans-N-acetylglucosaminidase, ⁇ -trans-lacto-N-biosidase and/or ⁇ -trans-N- acetyllactosaminidase activity, can be used in the present invention to produce such oligosaccharides. Preparation of such enzymes is preferably carried out via site directed mutagenesis approaches or directed evolution.
  • mutants are created via site directed mutagenesis approaches, preferably by introduction of point mutations. This technique generally requires reliance on the static 3D protein structure. The mutations generally affect the active site of the enzymes such that they lose their ability to degrade their
  • a preferred strategy consists of the replacement of the catalytic nucleophile by a non-nucleophilic residue. This modification results in the formation of an inactive mutant or an altered enzyme with reduced
  • glycosynthase a mutant glycosynthase and their development represents one of the major advances in the use of glycosidases for synthetic purposes.
  • the glycosynthase concept can be applied to all GH specificities and offer a large panel of enzymes potentially able to synthesize various oligosaccharides with very high yields, up to 95%.
  • the second preferred technique is called directed evolution.
  • This strategy comprises random mutagenesis applied to the gene of the selected glycosidase, which thus generates a library of genetically diverse genes expressing glycosidase. Generation of sequence diversity can be performed using well-known methodologies, the most preferable being the error prone polymerase chain reaction (epCR) method.
  • epCR error prone polymerase chain reaction
  • This gene library may be inserted into suitable microorganisms such as E. coli or S. cerevisiae for producing recombinant variants with slightly altered properties. Clones expressing improved enzymes are then identified with a fast and reliable screening method, selected and brought into a next round of mutation process.
  • Proteins comprising a transglycosidase and/or a glycosynthase activity as defined above may also comprise fragments or variants of those protein sequences.
  • Such fragments or variants may typically comprise a sequence having a sequence identity with one of the above mentioned proteins sequences of at least 70 %, more preferably at least 80 %, equally more preferably at least 85 %, even more preferably at least 90 % and most preferably at least 95 % or even 97 %, 98 % or 99 % as compared to the entire wild type sequence on amino acid level.
  • “Fragments” of proteins or peptides in the context of the present invention may also comprise a sequence of a protein or peptide as defined herein, which is, with regard to its amino acid sequence N-terminally, C-terminally and/or intrasequentially truncated compared to the amino acid sequence of the original (native) protein. Such truncation may thus occur either on the amino acid level or correspondingly on the nucleic acid level.
  • a sequence identity with respect to such a fragment as defined herein may therefore preferably refer to the entire protein or peptide as defined herein or to the entire (coding) nucleic acid molecule of such a protein or peptide.
  • fragments of nucleic acids in the context of the present invention may comprise a sequence of a nucleic acid as defined herein, which is, with regard to its nucleic acid molecule 5'-, 3 '- and/or intrasequentially truncated compared to the nucleic acid molecule of the original (native) nucleic acid molecule.
  • a sequence identity with respect to such a fragment as defined herein may therefore preferably refer to the entire nucleic acid as defined herein.
  • “Variants” of proteins or peptides as defined in the context of the present invention may be encoded by the nucleic acid molecule of a polymeric carrier cargo complex.
  • a protein or peptide may be generated, having an amino acid sequence which differs from the original sequence in one or more mutation(s), such as one or more substituted, inserted and/or deleted amino acid(s).
  • these fragments and/or variants have the same biological function or specific activity compared to the full-length native protein, e.g. its specific antigenic property.
  • “Variants” of proteins or peptides as defined in the context of the present invention may also comprise conservative amino acid substitution(s) compared to their native, i.e. non-mutated physiological, sequence. Those amino acid sequences as well as their encoding nucleotide sequences in particular fall under the term variants as defined herein. Substitutions in which amino acids that originate from the same class are exchanged for one another are called conservative substitutions. In particular, these are amino acids having aliphatic side chains, positively or negatively charged side chains, aromatic groups in the side chains or amino acids having side chains that can enter into hydrogen bridges, e.g. side chains which have a hydroxyl function.
  • an amino acid having a polar side chain is replaced by another amino acid having a likewise polar side chain, or, for example, an amino acid characterized by a hydrophobic side chain is substituted by another amino acid having a likewise hydrophobic side chain (e.g. serine (threonine) by threonine (serine) or leucine (iso leucine) by iso leucine (leucine)).
  • an amino acid having a polar side chain is replaced by another amino acid having a likewise polar side chain
  • an amino acid characterized by a hydrophobic side chain is substituted by another amino acid having a likewise hydrophobic side chain
  • Insertions and substitutions are possible, in particular, at those sequence positions which cause no modification to the three-dimensional structure or do not affect the binding region.
  • Modifications to a three-dimensional structure by insertion(s) or deletion(s) can easily be determined e.g. using CD spectra (circular dichroism spectra) (Urry, 1985, Absorption,
  • Circular Dichroism and ORD of Polypeptides in: Modern Physical Methods in Biochemistry, Neuberger et al. (ed.), Elsevier, Amsterdam).
  • variants of proteins or peptides as defined herein may also comprise those sequences wherein nucleotides of the nucleic acid are exchanged according to the
  • the amino acid sequence or at least part thereof may not differ from the original sequence in one or more mutation(s) within the above meaning.
  • nucleic acid sequences or amino acid sequences as defined herein preferably the amino acid sequences encoded by a nucleic acid sequence of the polymeric carrier as defined herein or the amino acid sequences themselves
  • the sequences can be aligned in order to be subsequently compared to one another. Therefore, e.g. a position of a first sequence may be compared with the corresponding position of the second sequence. If a position in the first sequence is occupied by the same component as is the case at a position in the second sequence, the two sequences are identical at this position. If this is not the case, the sequences differ at this position.
  • the percentage to which two sequences are identical is then a function of the number of identical positions divided by the total number of positions including those positions which are only occupied in one sequence.
  • the percentage to which two sequences are identical can be determined using a mathematical algorithm.
  • a preferred, but not limiting, example of a mathematical algorithm which can be used is the algorithm of Karlin et al. PNAS USA 90, 5873 (1993) or Altschul et al. Nucleic Acids Res. 25, 3389 (1997). Such an algorithm is integrated in the BLAST program. Sequences which are identical to the sequences of the present invention to a certain extent can be identified by this program.
  • the proteins as added in step c) may be provided in a free form or alternatively be bound to or immobilized onto a surface.
  • the order of steps a), b) and c) is preferably inverted.
  • Binding to or immobilization onto a surface may be carried out e.g. via electrostatic bonds, van der Waals-bonds, covalent bonds, etc.
  • Binding to or immobilization onto a surface may be furthermore carried out using a covalent linker or a crosslinker, or a Tag, as known to a skilled person for purification of proteins.
  • Such tags comprise, inter alia, affinity tags or chromatography tags.
  • Affinity tags may include e.g.
  • CBP chitin binding protein
  • MBP maltose binding protein
  • GST glutathione-S-transferase
  • Strep-Tag Strep-Tag
  • the poly(His) tag is a widely-used protein tag that binds to metal matrices.
  • Chromatography tags are used to alter chromatographic properties of the protein to afford different resolution across a particular separation technique, and include e.g. polyanionic amino acids based tags, such as the FLAG-tag.
  • the surface may be the surface of a bioreactor, or any suitable reaction chamber.
  • a mixture is prepared from substances provided by steps a), b) and c).
  • a mixture according to step d) represents a mixture of one, two, three, four, five, one to five, three to ten, five to ten or even more different donors as defined according to step a), and one, two, three, four, five, two to five, two to ten, two to twenty, five to ten or even more different enzymes comprising transglycosidase activity and/or glycosynthase activity.
  • step e) the mixture containing at least one compound as defined according to step a), at least one compound as defined according to step b), and at least one enzyme as added according to step c), together forming a mixture according to step d), are incubated to allow generation of glycosylated galactosyl disaccharides via enzymatic means using the at least one enzyme comprising a transglycosidase activity and/or a glycosynthase activity as defined herein.
  • Such an incubation advantageously allows the generation of a multiplicity of different oligosaccharides.
  • step d Generation of such a multiplicity of different oligosaccharides is based on the use of enzymes with different activities provided in step c), but also on the use of diverse donors and acceptors according to steps a) and b), preferably as a mixture as already outlined in step d).
  • the method of the present invention advantageously allows variation of the possible number and type of oligosaccharides obtainable by the method in a simple and cost efficient manner.
  • the use of enzymes furthermore allows the preparation of various oligosaccharides to be carried out in a stereoselective manner.
  • modified oligosaccharides preferably occurs by transferring glycosyl moieties (e.g., a sialyl moiety, fucosyl moiety, N-acetylglucosaminyl moiety, N-acetyllactosaminyl moiety, or lacto-N-biosyl moiety) by forming new bonds at desired positions of the molecule, etc., in a well defined manner to obtain a mixture of various modified oligosaccharides.
  • glycosyl moieties e.g., a sialyl moiety, fucosyl moiety, N-acetylglucosaminyl moiety, N-acetyllactosaminyl moiety, or lacto-N-biosyl moiety
  • Incubation according to step e) preferably occurs with a concentration of (each of the) enzymes in a concentration of 1 mU/1 to 1,000 U/l, preferably 10 mU/1 to 100 U/l, when the activity capable of forming 1 ⁇ of specific product for a defined protein starting from a defined educt is defined as 1 unit (U), e.g. for a glycotransferase the production of a glycose- containing complex carbohydrate at 37 °C in 1 minute.
  • the activity of each enzyme as defined herein may be assessed with respect to its naturally occurring or engineered substrate.
  • the incubation according to step e) may be carried out in a reaction medium, preferably an aqueous medium, comprising the mixture obtained according to step d) of the method of the present invention and optionally water; a buffer such as a phosphate buffer, a carbonate buffer, an acetate buffer, a borate buffer, a citrate buffer and a tris buffer, or combinations thereof; alcohol, such as methanol and ethanol; ester such as ethyl acetate; ketone such as acetone; amide such as acetamide; and the like.
  • a buffer such as a phosphate buffer, a carbonate buffer, an acetate buffer, a borate buffer, a citrate buffer and a tris buffer, or combinations thereof
  • alcohol such as methanol and ethanol
  • ester such as ethyl acetate
  • ketone such as acetone
  • amide such as acetamide
  • step e) may be carried out in a reaction medium as defined above, wherein optionally a surfactant or an organic solvent may be added, if necessary.
  • a surfactant or an organic solvent may be added, if necessary.
  • Any surfactant capable of accelerating the formation of a complex carbohydrate as defined according to the present invention as a possible product of the invention can be used as the surfactant.
  • non-ionic surfactants such as polyoxy ethylene
  • octadecylamine e.g., Nymeen S-215, manufactured by Nippon Oil & Fats
  • cationic surfactants such as cetyltrimethylammonium bromide and alkyldimethyl
  • benzylammoniumchloride e.g., Cation F2-40E, manufactured by Nippon Oil & Fats
  • anionic surfactants such as lauroyl sarcosinate
  • tertiary amines such as alkyldimethylamine (e.g., Tertiary Amine FB, manufactured by Nippon Oil & Fats); and the like, which are used alone or as a mixture of two or more.
  • the surfactant may be used generally in a concentration of 0.1 to 50 g/1.
  • the organic solvent may include xylene, toluene, fatty acid alcohol, acetone, ethyl acetate, and the like, which may be used in a concentration of generally 0.1 to 50 ml/1.
  • the incubation according to step e) may be furthermore carried out in a reaction medium as defined above, preferably having a pH 3 to 10, pH 5 to 10, preferably pH 6 to 8.
  • the incubation according to step e) may be furthermore carried out at a temperature of about 0 °C to about 100 °C, preferably at a temperature of about 10 to about 50 °C, e.g. at a temperature of about 20 °C to about 50 °C.
  • inorganic salts such as MnCl 2 and MgCl 2 , may be added, if necessary.
  • the incubation according to step e) of the method of the present invention may be carried out in a bioreactor.
  • the bioreactor is preferably suitable for either a continuous mode or a discontinuous mode.
  • the incubation according to step e) may be carried out in a continuous or discontinuous mode.
  • the method preferably provides for a continuous flow of compounds and/or enzymes as necessary, preferably by continuously providing educts of the reaction to the reaction mixture and continuously removing products from the reaction mixture, while maintaining the concentration of all components, including enzymes at a predetermined level.
  • the enzymes used in a continuous mode may be added either in free form or as bound or immobilized to a surface.
  • the addition of donors and/or enzymes may be repeated according to an optional step f).
  • steps a), c), d) and e) may be repeated, preferably with the mixture obtained according to step e).
  • the at least one compound provided according to step a) is preferably different from that/those provided in the previous cycle
  • the at least one enzyme provided according to step c) is preferably different from that/those provided in the previous cycle.
  • Such a stepwise proceeding may allow within multiple rounds the rational generation of a defined set of compounds in a controllable manner. It may also provide for a rational exclusion of specific components.
  • the compounds and/or enzymes may be added simultaneously or sequentially, and preferably compounds and/or enzymes may be added simultaneously in one step and/or sequentially in different steps.
  • a mixture with at least one compound as defined herein for step a) and at least one compound as defined herein for step b) and at least one enzyme as defined herein according to step c) may be incubated in one step without repetition of one or more steps. Such a proceeding may be preferable in certain circumstances, as it may lead to the largest variety of possible products.
  • the method of the present invention as defined above preferably leads to generation of compounds on the basis of donors as provided in step a) and acceptors as provided in step b) upon adding enzymes according to step c) and incubating the mixture (step e)).
  • An optional repetition of these steps may be carried out as defined above.
  • the method as described above results in either a single modified galactosyl disaccharide or a mixture comprising two or more modified galactosyl disaccharides.
  • the at least one glycosyl residue comprised by the modified oligosaccharides according to the present invention can be linked to any of the
  • said at least one glycosyl moiety can be a monosaccharide unit such as sialyl, fucosyl and/or N-acetylglucosaminyl, or a disaccharide unit such as N-acetyllactosaminyl, lacto-N-biosyl or sialylated and/or fucosylated N- acetylglucosaminyl, or tri-, tetra- or polysaccharide unit such as N-acetyllactosaminyl and lacto-N-biosyl moieties further glycosylated with sialyl and/or fucosyl and/or N- acetyllactosaminyl and/or lacto-N-biosyl and/or N-acetylglucosaminyl
  • compounds obtained according to the method of the present invention as defined above are characterized by their linkages and modifications.
  • the compounds obtained by the method of the present invention after incubation step e) and/or a repetition of steps according to step f) are characterized in that one or more of the following are present: a N-acetyl-glucosaminyl group is attached to a galactosyl moiety of the galactosyl disaccharide with 1-3, 1-4 or 1-6 interglycosidic linkage, preferably with 1-3 or 1-6 linkage, a N-acetyl-glucosaminyl group is attached to the galactosyl moiety of a N-acetyl- lactosaminyl or lacto-N-biosyl group with 1-3 or 1-6 interglycosidic linkage, a N-acetyl-lactosaminyl group is attached to the galactosyl moiety of another N
  • a fucosyl residue is attached to a galactosyl moiety of galactosyl disaccharide with 1-2 or 1-3 interglycosidic linkage
  • a fucosyl residue is attached to a N-acetylglucosaminyl moiety, preferably to a terminal N-acetylglucosaminyl moiety with 1-3 or 1-4 interglycosidic linkage
  • a sialyl residue attached to the N-acetyl-lactosaminyl and/or the lacto-N-biosyl group is linked to
  • a sialyl residue is attached to a N-acetylglucosaminyl moiety, preferably to a terminal N- acetylglucosaminyl moiety with 2-3 or 2-6 interglycosidic linkage.
  • the interglycosidic linkages in the compounds of the present application are ⁇ , with the exception of fucosyl and sialyl residues which are attached via a- linkage.
  • the present invention provides a compound, preferably a mixture of compounds obtained or obtainable by the method of the present invention as described herein.
  • a mixture of compounds obtained or obtainable by the method of the present invention as described herein is preferably to be understood as a mixture of at least 2 to 10, 2 to 20, 2 to 100, 2 to 200, or even more different compounds as generally defined above.
  • Such compounds may be preferably selected without restriction from any of the compounds as defined above.
  • the present invention also provides or utilizes salts of herein defined compounds.
  • Such salts may be preferably selected from salts of the compounds defined above, which contain at least one sialyl residue: an associated ion pair consisting of the negatively charged acid residue of sialylated oligosaccharide and one or more cations in any stoichiometric proportion.
  • Cations as used in the present context, are atoms or molecules with positive charge. The cation may be inorganic or organic.
  • Preferred inorganic cations are ammonium ion, alkali metal, alkali earth metal and transition metal ions, more preferably Na + , K + , Ca 2+ , Mg 2+ , Ba 2+ , Fe 2+ , Zn 2+ , Mn 2+ and Cu , most preferably K , Ca , Mg , Ba , Fe and Zn .
  • Basic organic compounds in positively charged form may be relevant organic cations.
  • Such preferred positively charged counterparts are diethyl amine, triethyl amine, diisopropyl ethyl amine, ethanolamine, diethanolamine, triethanolamine, imidazole, piperidine, piperazine, morpholine, benzyl amine, ethylene diamine, meglumin, pyrrolidine, choline, tris-(hydroxymethyl)-methyl amine, N-(2-hydroxyethyl)-pyrrolidine, N-(2-hydroxyethyl)-piperidine, N-(2-hydroxyethyl)- piperazine, N-(2-hydroxyethyl)-morpholine, L-arginine, L-lysine, oligopeptides having L- arginine or L-lysine unit or oligopeptides having a free amino group on the N-terminal, etc., all in protonated form.
  • Such salt formations can be used to modify characteristics of the complex molecule as a whole, such as stability, compatibility to
  • compounds or a mixture of compounds obtained or obtainable by the method of the present invention as described herein may have enhanced bifidogenic activity compared to the precursor galactosyl disaccharide.
  • the compounds described above are particularly effective in the improvement and maturation of the immune system of neonatal infants, and has preventive effect against secondary infections following viral infections such as influenza.
  • prebiotic enhances the beneficial effects and efficiency of probiotics, such as Lactobacillus and Bifidobacterium species, in promoting the development of an early bifidogenic intestinal microbiota in infants, in reducing the risk of development or allergy and/or asthma in infants, and in preventing and treating pathogenic infections in such as diarrhoea in infants.
  • probiotics such as Lactobacillus and Bifidobacterium species
  • the present invention provides compounds or a mixture of compounds obtained or obtainable by the method of the present invention as described herein for use in enhancing the bifidogenic effect of consumable products.
  • the compounds described above are particularly effective in the improvement and maturation of the immune system of neonatal infants, and has preventive effect against secondary infections following viral infections such as influenza.
  • compounds or a mixture of compounds obtained or obtainable by the method of the present invention as described herein as prebiotics enhances the beneficial effects and efficiency of probiotics, such as Lactobacillus and Bifidobacterium species, in promoting the development of an early bifidogenic intestinal microbiota in infants, in reducing the risk of development or allergy and/or asthma in infants, and in preventing and treating pathogenic infections such as diarrhoea in infants.
  • probiotics such as Lactobacillus and Bifidobacterium species
  • compounds or a mixture of compounds obtained or obtainable by the method of the present invention as described herein may be used for the preparation of a consumable product, preferably for the preparation of a pharmaceutical composition, a nutritional formulation or a food supplement.
  • Such a compound or mixture of compounds obtained or obtainable by the method of the present invention as described herein is particularly effective in the improvement and maturation of the immune system of neonatal infants, and has preventive effect against secondary infections following viral infections such as influenza.
  • the use of compounds or a mixture of compounds obtained or obtainable by the method of the present invention as described herein as prebiotic enhances the beneficial effects and efficiency of probiotics, such as Lactobacillus and Bifidobacterium species, in promoting the development of an early bifidogenic intestinal microbiota in infants, in reducing the risk of development or allergy and/or asthma in infants, and in preventing and treating pathogenic infections such as diarrhoea in infants.
  • the present invention provides a pharmaceutical composition, comprising compounds or a mixture of compounds obtained or obtainable by the method of the present invention as described herein, and preferably a pharmaceutically acceptable carrier.
  • “Pharmaceutically acceptable carriers” include but are not limited to additives, adjuvants, excipients and diluents (water, gelatine, talc, sugars, starch, gum arabic, vegetable gums, vegetable oils, polyalkylene glycols, flavouring agents, preservatives, stabilizers, emulsifying agents, lubricants, colorants, fillers, wetting agents, etc.). Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field.
  • the dosage form for administration includes, for example, tablets, powders, granules, pills, suspensions, emulsions, infusions, capsules, injections, liquids, elixirs, extracts and tinctures.
  • nutritional formulations are provided such as foods or drinks, comprising compounds or a mixture of compounds obtained or obtainable by the method of the present invention as described herein.
  • the nutritional formulation may contain edible micronutrients, vitamins and minerals as well. The amounts of such ingredients may vary depending on whether the formulation is intended for use with normal, healthy infants, children, adults or subjects having specialized needs (e.g. suffering from metabolic disorders).
  • Micronutrients include, for example, edible oils, fats or fatty acids (such as coconut oil, soybean oil, monoglycerides, diglycerides, palm olein, sunflower oil, fish oil, linoleic acid, linolenic acid etc.), carbohydrates (such as glucose, fructose, sucrose, maltodextrin, starch, hydro lysed cornstarch, etc.) and proteins from casein, soy-bean, whey or skim milk, or hydro lysates of these proteins, but protein from other sources (either intact or hydrolysed) may be used.
  • edible oils, fats or fatty acids such as coconut oil, soybean oil, monoglycerides, diglycerides, palm olein, sunflower oil, fish oil, linoleic acid, linolenic acid etc.
  • carbohydrates such as glucose, fructose, sucrose, maltodextrin, starch, hydro lysed cornstarch, etc.
  • Vitamins may be chosen from the group consisting of vitamin A, Bl, B2, B5, B6, B12, C, D, E, H, K, folic acid, inositol and nicotinic acid.
  • the nutritional formula may contain the following minerals and trace elements: Ca, P, K, Na, CI, Mg, Mn, Fe, Cu, Zn, Se, Cr or I.
  • a nutritional formulation as defined above may further contain one or more probiotics, e.g.
  • lacto bacteriae Bifidobacterium species, prebiotics such as fructooligosaccharides and galactooligosaccharides, proteins from casein, soy-bean, whey or skim milk, carbohydrates such as lactose, saccharose, maltodextrin, starch or mixtures thereof, lipids (e.g. palm olein, sunflower oil, safflower oil) and vitamins and minerals essential in a daily diet.
  • Probiotics are preferably also contained in the nutritional formulation in an amount sufficient to achieve the desired effect in an individual, preferably in infants, children and/or adults.
  • the nutritional formulation as defined above is an infant formula.
  • infant formula preferably means a foodstuff intended for particular nutritional use by infants during the first 4-6 months or even 4 to 12 months of life and satisfying by itself the nutritional requirements of infants. It may contain one or more probiotic Bifidobacterium species, prebiotics such as fructooligosaccharides and galactooligosaccharides, proteins from casein, soy-bean, whey or skim milk, carbohydrates such as lactose, saccharose, maltodextrin, starch or mixtures thereof, lipids (e.g. palm olein, sunflower oil, safflower oil) and vitamins and minerals essential in a daily diet.
  • prebiotics such as fructooligosaccharides and galactooligosaccharides, proteins from casein, soy-bean, whey or skim milk
  • carbohydrates such as lactose, saccharose, maltodextrin, starch or mixtures thereof
  • lipids
  • a food supplement may be provided.
  • a food supplement contains ingredients as defined for nutritional food above, e.g. compounds or a mixture of compounds obtained or obtainable by the method of the present invention as described herein, vitamins, minerals, trace elements and other micronutrients, etc.
  • the food supplement may be for example in the form of tablets, capsules, pastilles or a liquid.
  • the supplement may contain conventional additives selected from but not limited to binders, coatings, emulsifiers, solubilising agents, encapsulating agents, film forming agents, adsorbents, carriers, fillers, dispersing agents, wetting agents, gellifying agents, gel forming agents, etc.
  • the food supplement is a digestive health functional food, as the administration of compounds or a mixture of compounds obtained or obtainable by the method of the present invention as described herein provides a beneficial effect on digestive health.
  • a digestive health functional food is preferably a processed food used with the intention of enhancing and preserving digestive health by utilizing compounds or a mixture of compounds obtained or obtainable by the method of the present invention as described herein, as physiologically functional ingredients or components in the form of a tablet, capsule, powder, etc.
  • Different terms such as dietary supplement, nutraceutical, designed food, or health product may also be used to refer to a digestive health functional food.
  • compounds or a mixture of compounds obtained or obtainable by the method of the present invention as described herein may be used for the preparation of nutritional formulations including foods, drinks and feeds, preferably infant formulae, food supplements and digestive health functional foods, preferably any of these as described above.
  • the nutritional formulation may be prepared in any usual manner.
  • glycosyl donor(s) such as derivatives of general formula 1 to 4 and GOS (10 mM -1M) is incubated in an incubation buffer at a pH range from 5.0 to 9.0 with recombinant glycosidase, a-transglycosidase or a-glycosynthase, such as a-fucosidase, a- transfucosidase, a-fucosynthase, a-sialidase, a-transsialidase, ⁇ - ⁇ -acetylglucosaminidase, ⁇ - trans-N-acetylglucosaminidase, ⁇ -lacto-N-biosidase, ⁇ -trans-lacto-N-biosidase, ⁇ - ⁇ - acetyllactosaminidase or ⁇ -trans-N-acetyllactosaminidase.
  • biogel chromatography P-2 Biogel, 16x900 mm

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Abstract

L'invention porte sur un procédé pour la modification de disaccharides galactosyliques afin d'accroître leur effet bifidogène, consistant à faire réagir au moins un donneur de glycosyl avec un disaccharide galactosylique précurseur ou un mélange de disaccharides galactosyliques précurseurs par catalyse enzymatique, sur des composés pouvant être obtenus au moyen dudit procédé et sur l'utilisation de tels composés dans des produits consommables.
PCT/IB2013/055179 2012-06-22 2013-06-24 Disaccharides galactosyliques glycolysés, procédés de préparation et leur utilisation dans des produits consommables WO2013190529A1 (fr)

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Citations (9)

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Publication number Priority date Publication date Assignee Title
WO1993024505A1 (fr) * 1992-05-26 1993-12-09 Alberta Research Council Reduction de l'inflammation par administration dependante dans le temps d'oligosaccharides glycosides relatifs a des determinants de groupes sanguins
WO1996032492A1 (fr) * 1995-04-11 1996-10-17 Cytel Corporation Synthese enzymatique amelioree d'oligosaccharides
US5716812A (en) * 1995-12-12 1998-02-10 The University Of British Columbia Methods and compositions for synthesis of oligosaccharides, and the products formed thereby
WO1998046784A1 (fr) * 1997-04-11 1998-10-22 The University Of British Columbia Synthese amelioree d'oligosaccharides au moyen de derives de glycosides actives
WO2007090894A1 (fr) * 2006-02-10 2007-08-16 Nestec S.A. Melange d'oligosaccharides
WO2009144137A1 (fr) * 2008-05-27 2009-12-03 Nestec S.A. Probiotiques destinés à améliorer le microbiote intestinal
EP2455387A1 (fr) * 2010-11-23 2012-05-23 Nestec S.A. Mélange oligosaccharide et produit alimentaire comportant ce mélange, spécialement une formule pour enfants
WO2012156897A1 (fr) * 2011-05-13 2012-11-22 Glycom A/S Procédé de préparation d'oligosaccharides du lait humain (hmo) ou de leurs précurseurs
WO2013110778A1 (fr) * 2012-01-25 2013-08-01 Arla Foods Amba Procédé de préparation d'une composition contenant des galacto-oligosaccharides

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993024505A1 (fr) * 1992-05-26 1993-12-09 Alberta Research Council Reduction de l'inflammation par administration dependante dans le temps d'oligosaccharides glycosides relatifs a des determinants de groupes sanguins
WO1996032492A1 (fr) * 1995-04-11 1996-10-17 Cytel Corporation Synthese enzymatique amelioree d'oligosaccharides
US5716812A (en) * 1995-12-12 1998-02-10 The University Of British Columbia Methods and compositions for synthesis of oligosaccharides, and the products formed thereby
WO1998046784A1 (fr) * 1997-04-11 1998-10-22 The University Of British Columbia Synthese amelioree d'oligosaccharides au moyen de derives de glycosides actives
WO2007090894A1 (fr) * 2006-02-10 2007-08-16 Nestec S.A. Melange d'oligosaccharides
WO2009144137A1 (fr) * 2008-05-27 2009-12-03 Nestec S.A. Probiotiques destinés à améliorer le microbiote intestinal
EP2455387A1 (fr) * 2010-11-23 2012-05-23 Nestec S.A. Mélange oligosaccharide et produit alimentaire comportant ce mélange, spécialement une formule pour enfants
WO2012156897A1 (fr) * 2011-05-13 2012-11-22 Glycom A/S Procédé de préparation d'oligosaccharides du lait humain (hmo) ou de leurs précurseurs
WO2013110778A1 (fr) * 2012-01-25 2013-08-01 Arla Foods Amba Procédé de préparation d'une composition contenant des galacto-oligosaccharides

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LEE, S.-G. ET AL.: "Production of sialyloligosaccharides by trans-sialidase catalyzed reaction using fetuin as a sialic acid donor", ENZYME AND MICROBIAL TECHNOLOGY, vol. 31, 2002, pages 742 - 746 *
VETERE, A. ET AL.: "Complete synthesis of 3'-sialyl-N-acetyllactosamine by regioselective transglycosylation", FEBS LETTERS, vol. 399, 1996, pages 203 - 206 *

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