Bioproduction of oligosaccharides and derivatives from monosaccharides

Abstract

The present invention provides methods for glycosylation and compound preparation. The compounds include galactosylated, sialylated, fucosylated, and N-acetylglucosaminated compounds from simple oligosaccharides and sugars of animal, plant, or microbial origin. In certain embodiments, the invention provides for trinucleotide-free enzymatic production of oligosaccharides starting from simple sugars, including plant-based sugars. The present invention also provides methods of enzymatic production of fucosylated oligosaccharides and fucosylated antibody-glycan conjugates from ordinary enzymes. The production may be a cell-free, one-pot synthesis using an enzyme and, in some embodiments, an immobilized enzyme. The synthesis is a highly customizable and efficient cell-free manufacturing process. In some embodiments, lactose derivatives and human milk oligosaccharides (HMOs) are produced.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 430,271 (filed on December 5, 2022), U.S. Provisional Application No. 63 / 344,507 (filed on May 20, 2022), and U.S. Provisional Application No. 63 / 448,175 (filed on February 24, 2023), and each application is incorporated herein by reference in its entirety. Field of the Invention

[0003] The present invention provides methods for glycosylation and compound preparation. The compounds include galactosylation, sialylation, fucosylation and N-acetylglucosaminyl compounds from simple animal-derived, plant-derived or microbial-derived oligosaccharides and sugars. In certain embodiments, the present invention provides enzymatic production of oligosaccharides without trinucleotides starting from simple sugars including plant-based sugars. Production can be a cell-free, one-pot synthesis using enzymes and in some embodiments using immobilized enzymes. The synthesis is a highly customizable and efficient cell-free manufacturing process. In some embodiments, lactose derivatives and human milk oligosaccharides (HMOs) are produced.

[0004] The present invention also provides enzymatic production of fucosylated oligosaccharides and fucosylated antibody-glycan conjugates from common sugars. The production is a cell-free, one-pot synthesis using immobilized enzymes. Oligosaccharide synthesis is a highly customizable and efficient cell-free manufacturing process. In some embodiments, fucosylated human milk oligosaccharides (HMOs) are produced. Background of the Invention

[0006] Human milk oligosaccharides (HMOs) are particularly commercially interesting glycan models. HMOs are structurally diverse, soluble and unconjugated glycans composed of 3-22 sugars restricted to a set of only 5 monosaccharides: D-glucose, D-galactose, N-acetylglucosamine, L-fucose and N-acetylneuraminic acid (i.e. sialic acid), and are the third largest component of breast milk. Simple HMOs (3-4DP) (DP is the degree of polymerization) may serve as metabolic substrates for specific beneficial bacteria, thereby shaping the intestinal microbiota. However, more complex and branched HMOs (>4DP) may serve as soluble decoys for viral, bacterial or protozoan parasite adhesins, thereby preventing their attachment to infant mucosal surfaces. For example, studies have shown that fucosylated moieties in human milk inhibit colonization of Campylobacter jejuni, a leading cause of bacterial diarrhea that affects approximately 1 in 4,000 infants younger than 1 year of age, both ex vivo (human intestinal mucosa) and in rodents (in vivo). In addition to protection against Campylobacter, there is evidence that fucosylated HMOs may deploy a similar decoy mechanism to protect infants from other diarrheagenic pathogens, such as cholera and norovirus, which initiate infection by binding to cell surface receptors. These oligosaccharides may also provide protection against Shiga toxin-induced diarrhea by competing with the toxin for binding to its extracellular target. In addition, HMOs may modulate epithelial and immune cell responses, reducing excessive mucosal leukocyte infiltration and activation. These properties have been associated with a lower risk of developing necrotizing enterocolitis.

[0007] Although milk oligosaccharides form a diverse family of carbohydrates that are highly conserved in mammals, these features are not uniform across all species. They are typically composed of a lactose (gal+glu) core, extended by alternating N-acetylglucosamine and galactose units, and modified by sialylation and / or fucosylation. HMOs exhibit greater diversity than the oligosaccharides found in cow's milk, which is the basis of most commercial infant formulas. These compounds are trace components in cow's milk (0.05 g / L), compared to the 5-23 g / L concentrations found in human milk, where HMOs are comparable to the protein content (8 g / L). Approximately 200 HMO structures have been identified, with less than half that number found in cows.

[0008] The global human milk oligosaccharides (HMO) market size is expected to expand at a compound annual growth rate (CAGR) of 21.9% over the next eight years, reaching USD 71.2 billion by 2025. 38 The recent industrial-scale production of fucosylated lactose (2'FL) through recombinant microbial fermentation has opened the door to the HMO market. In terms of HMO product types, the fucosyllactose (2'FL) segment is currently the largest with a CAGR of 14.4%, while the sialyllactose segment has a CAGR of 15.1%. The functional food and beverage industry is also expected to experience considerable growth during this period as HMOs have many potential applications beyond infant formula.

[0009] The use of HMOs in infant formula requires improved methods for HMO synthesis. The major drivers for growth include rising consumer awareness and concern about infant health, increasing demand for nutritional foods for infants, projected population growth, and growing awareness of the health benefits of consuming HMOs, such as promoting beneficial microbiota in the digestive tract and enhancing brain and immune system development. On the other hand, scarce raw materials, expensive and complex chemical and enzymatic synthesis techniques, and lack of technical expertise are expected to continue to pose challenges.

[0010] While some simple probiotic HMOs (2'FL, 3DP) can now be efficiently produced by fermentation of infant formula, complex and branched HMOs are elusive and have been produced in small quantities at high cost by chemical catalysis. To make an HMO, the sugars need to be activated first so that they can be transferred sequentially into the growing structure. For example, activated fucose (L-fucose-1-GDP) and activated sialic acid (sialic acid-1-CMP) cost $35,000 and $8,000 per gram, respectively, while L-fucose and L-sialic acid cost $10 and $5.5 per gram, respectively, so the cost of any complex glycan is as high as hundreds of thousands of dollars per gram, for example, the defined pentasaccharide (5-carbon sugar) DP5 glycan costs $300,000 to $3,800,000 per gram. In addition to extraction from rare human milk, HMOs are produced in three main ways:

[0011] Chemical production: Chemical synthesis of HMOs is flexible but requires more steps, low yields, and specialized equipment, resulting in high production costs. In addition, complex HMOs require the synthesis of chemically protected monosaccharides, each of which requires laborious deprotection, typically requiring 20-50 chemical operations (lacto-N-tetraose synthesis). For these reasons, large-scale production of HMOs has not yet been achieved.

[0012] Microbial-based production: Fermentation is a cost-effective method for HMO production and is currently used to produce 2'-fucosyllactose (2'-FL) on a multi-ton industrial scale (Abbott Laboratories-Jennewein Biotechnologie GmbH, Glycom-Nestle, Inbiose-DuPont). However, microbial production is limited to simple unbranched and short-length (DP < 4) HMOs. More complex HMOs are out of reach because of the low yields of complex carbohydrates obtained by fermentation and the challenges of controlling glycan sequences in living organisms.

[0013] Enzyme-based production: Cell-free production circumvents this limitation by using glycosyltransferases that transfer nucleotide-activated donor sugars (e.g., GDP-L-fucose, CMP-sialic acid, etc.) to a variety of acceptor sugars with high specificity. Combining various enzymatic steps can generate large HMO libraries, which is the approach taken by commercial suppliers of HMOs (Chemily Glycoscience, Carbosynth, Santa Cruz Biotechnology). Chemical catalysis can be used to generate defined complex glucans due to fewer steps and stable enzymes, but the cost of production is still higher.

[0014] Biocatalysis is gaining popularity in chemical manufacturing as a green technology, rather than traditional expensive and inefficient processes. Its applications include the production of food ingredients, flavorings, fragrances, daily and fine chemicals, and active pharmaceuticals. However, when producing chemicals on an industrial scale, enzyme activity and loading drop drastically, resulting in significant performance degradation.

[0015] Continuous flow processing begins with pumping two or more different reactant streams at specific flow rates into a single unit. Reactions occur, and streams containing the resulting compounds are collected at the outlet. The solutions can also be directed to subsequent flow reactor loops to generate the final product. Continuous flow processing provides better reaction control and repeatability. It is a modular, customizable approach. High modularity allows one to configure the unit to meet the requirements of a specific reaction.

[0016] Glycans are complex carbohydrate structures that are major molecules on the cell surface, serving as the first contact point between cells, the extracellular matrix, and pathogens. Glycans are involved in many biological processes, such as cell-cell interactions. There is great interest in improving the accessibility and affordability of these molecules for research, preclinical, and commercial applications.

[0017] A recent CDC study found that approximately one in five newborns were born to mothers who never initiated breastfeeding, and the majority (>70%) did not reach the 6-month target, relying instead heavily or exclusively on commercially available infant formula. Nonadherence to breastfeeding recommendations is estimated to increase U.S. pediatric healthcare costs (directly) by $2.5 billion, with a total burden of morbidity and mortality totaling $13.8 billion. (Vera MK NG, “Implementation of Mother-friendly Workplace Policies in Hong Kong”, Hong Kong J Gynaecol Obstet. Midwifery 2015;15(1)11.

[0018] Human milk oligosaccharides (HMOs) are the third largest component of breast milk. They are of particular commercial interest. For example, they serve as metabolic substrates for specific beneficial bacteria and thus shape the intestinal microbiota. However, the more complex and branched HMOs (>4DP (i.e. degree of polymerisation or the number of monosaccharides linked together) appear to be mostly preventive. They act as soluble decoys for adhesins of viruses, bacteria or protozoan parasites, preventing them from attaching to mucosal surfaces in infants or adults.

[0019] In addition, HMOs can modulate epithelial and immune cell responses, reducing excessive mucosal leukocyte infiltration and activation. These properties have been associated with a lower risk of developing necrotizing enterocolitis and other infections, as well as autoimmune inflammation.

[0020] While some simple probiotic HMOs can be efficiently produced by fermentation for use in infant formula (2'FL, 3DP), complex and branched HMOs have been elusive. Microbial production is limited to simple unbranched and short-chain sugars, and bacteria secrete toxins that must be filtered out. Yeast fermentation does not require toxin removal and the overall process involves fewer steps, reducing production costs and leading to products that are easier to scale up. Engineered organisms are intensive and do not guarantee the ability to scale up.

[0021] Cell-free systems have been of great interest in biomanufacturing, but challenges exist (MP Cordoso Marque et al., Adv. Biochem. Eng. Biotechnol., https: / / doi.org / 10.1007 / 10_2020_160 and C. You, Adv. Biochem. Engl, DOI: 10.1007 / 10_2012_159 (2012)). Hokke, Cornelis H., et al. "One-pot enzymatic synthesis of the Galα1→3Galβ1→4GlcNAc sequence within situUDP-Gal regeneration." Glycoconjugate journal 13.4 (1996): 687-692. Zervosen, Astrid, and Lothar Elling. "A Novel Three-Enzyme Reaction Cycle for the Synthesis of N-Acetyllactosamine with in Situ Regeneration of Uridine 5'-Diphosphate Glucose and Uridine 5'-Diphosphate Galactose." Journal of the American Chemical Society 118.8 (1996): 1836-1840. The foregoing content is incorporated by reference in its entirety.

[0022] Therefore, the art seeks an economical and efficient method to produce glycans. Therefore, the art seeks an economical and efficient method to produce glycans, including HMOs, including but not limited to glycans of two saccharide units or larger, and glycans including 4 saccharide units or larger. This includes HMOs, including but not limited to glycans of two or four saccharide units or larger. This can overcome the major obstacles to advancing these glycans, galactosylated glycans and HMOs for probiotic, preventive and therapeutic purposes. This includes but is not limited to medical foods, food additives and supplements. Economical and efficient HMO production will be an important advancement in infant nutrition and disease prevention for infants of all ages around the world. Importantly, the present invention improves the efficiency of production of sugars and complex carbohydrates while reducing costs, thereby increasing the accessibility and affordability of these molecules. SUMMARY OF THE INVENTION

[0024] The present invention provides a method for preparing sugars starting from common sugar building blocks. This reduces the cost of sugar production. In certain embodiments, the sugar is of plant origin. The energy source is the hydrolysis of more complex sugars into simpler sugars. Activated sugars are starting materials and are generated in situ. Activated sugars are monophosphates or diphosphates present in catalytic amounts. Examples include nucleotide diphosphates and monophosphates UDP, GDP and CMP. Avoiding stoichiometric or excessive nucleotide reactants (including triphosphates such as ATP) avoids the need for cofactor recovery, reduces the amount of enzymes required, avoids reaction inhibition, and reduces the overall cost of the reaction relative to the current reaction. Nucleotide diphosphates and monophosphates are used in catalytic amounts. This is a significant improvement over prior art reactions that must use ATP in stoichiometric or excessive amounts.

[0025] The present invention provides methods with improved purification and yield. No acidification is required to achieve improved purification. In addition, no inorganic phosphate byproducts need to be purified from the reaction mixture. In certain embodiments, the sugar byproducts formed can be used as building blocks for various purposes. The enzyme will not be reactivated, thereby avoiding additional reactants and improving reaction efficiency. The present invention provides a cell-free biomanufacturing method for manufacturing human milk oligosaccharides and other animal-based oligosaccharides from cost-effective starting materials. In some embodiments, the reaction is carried out in a one-pot synthesis.

[0026] According to the present invention, any compound having at least one hydroxyl functional group can be glycosylated. This includes, but is not limited to, sugars, polysaccharides, carbohydrates and alcohols.

[0027] Therefore, in some embodiments, the present invention provides a method for producing a glycosylated major product, comprising the steps of:

[0028] a. contacting a catalytic amount of a sugar-nucleotide donor with a stoichiometric amount of an acceptor in the presence of a transferase to obtain a glycosylation primary product and a catalytic amount of a nucleotide; and

[0029] b. regenerating the nucleotide into a regenerated sugar-nucleotide donor by contacting a catalytic amount of the nucleotide with a stoichiometric amount of a sugar donor precursor in the presence of a transferase to obtain a regenerated sugar-nucleotide donor and a secondary product.

[0030] In some embodiments, the method further comprises:

[0031] a. contacting a catalytic amount of a regenerated sugar-nucleotide donor with a stoichiometric amount of an acceptor in the presence of a transferase to obtain a glycosylation primary product and a catalytic amount of a nucleotide; and

[0032] b. regenerating the nucleotide into a regenerated sugar-nucleotide donor by contacting a catalytic amount of the nucleotide with a stoichiometric amount of a sugar donor precursor in the presence of a transferase to obtain a regenerated sugar-nucleotide donor and a secondary product.

[0033] In other embodiments, the present invention provides a method for producing a glycosylated primary product, comprising the steps of adding a sugar donor precursor, an acceptor, a transferase, and a catalytic amount of nucleotides to obtain a glycosylated primary product and a secondary product. In certain embodiments, the sugar donor precursor and the nucleotides provide a sugar-nucleotide donor.

[0034] In certain embodiments, the sugar donor precursor and the nucleotide provide the sugar-nucleotide donor precursor and the auxiliary enzyme, and the sugar-nucleotide donor precursor provides the sugar-nucleotide donor.

[0035] In some embodiments, the sugar donor precursor is a sugar comprising 2 or more sugar units. In some embodiments, the sugar donor precursor is a sugar comprising 2, 3 or 4 sugar units. In some embodiments, the sugar donor precursor is a disaccharide. In some embodiments, the sugar donor precursor is a trisaccharide. In some embodiments, the sugar donor precursor includes galactose, sialic acid, fucose or N-acetylglucosamine. In some embodiments, galactose, sialic acid, fucose or N-acetylglucosamine is located at the terminal position of the sugar donor precursor.

[0036] In some embodiments, the sugar-nucleotide donor includes galactose, sialic acid, fucose or N-acetylglucosamine. In some embodiments, the sugar-nucleotide donor is galactose-nucleotide, sialic acid-nucleotide, fucose-nucleotide or N-acetylglucosamine-nucleotide. In some embodiments, the secondary product is a monosaccharide, a disaccharide or a trisaccharide.

[0037] Some embodiments of the present invention also include the step of contacting the secondary product with a processing enzyme to convert the secondary product into a secondary product derivative. In some embodiments, in the presence of a second processing enzyme, the secondary product derivative is converted into a modified secondary product derivative. Some embodiments of the present invention also include the step of contacting the primary product with a processing enzyme to convert the primary product into a primary product derivative. In some embodiments, the processing enzyme is an oxidase, an isomerase or a hydrolase.

[0038] In some embodiments, the acceptor is an organic compound containing a hydroxyl group. In some embodiments, the organic compound containing a hydroxyl group is a sugar. In some embodiments, the acceptor is obtained by converting a secondary product into an acceptor by a processing enzyme.

[0039] In some embodiments, the nucleotide is uridine diphosphate, cytidine monophosphate or guanosine diphosphate. In some embodiments, the amount of nucleotide relative to the sugar donor precursor or acceptor is 0.001 mol% to 10 mol%. In some embodiments, the amount of nucleotide relative to the sugar donor precursor or acceptor is 0.01 mol% to 1 mol%.

[0040] In certain embodiments, the transferase is any enzyme that transfers a sugar unit to an acceptor. In some embodiments, the transferase is a β-1,3-galactosyltransferase, a sucrase synthase, a β-1,4-galactosyltransferase, a sialyltransferase, a fucosyltransferase, or a glucosamine (N-acetyl) transferase 2. In some embodiments, the transferase includes a first transferase ( Figure 6 , 7 , GT1 in 13 or 14) and the second transferase ( Figure 6 , 7 , GT2 in 13 or 14). In some embodiments, the first transferase catalyzes the transfer of a sugar from a sugar-nucleotide donor to an acceptor to obtain a glycosylation primary product, and the second transferase catalyzes the reaction of a nucleotide with a sugar donor precursor to obtain a sugar-nucleotide donor and a secondary product.

[0041] In certain embodiments, the present invention provides a method for producing a galactosylated major product, comprising the steps of:

[0042] a. contacting a catalytic amount of a sugar-nucleotide donor with a stoichiometric amount of an acceptor to obtain a galactosylated primary product and a catalytic amount of a nucleotide; and

[0043] b. regenerating the nucleotide into a regenerated sugar-nucleotide donor by contacting a catalytic amount of the nucleotide with a stoichiometric amount of a sugar donor precursor to obtain a regenerated sugar-nucleotide donor and a secondary product.

[0044] In some embodiments, the method further comprises:

[0045] a. contacting a catalytic amount of a regenerated sugar-nucleotide donor with a stoichiometric amount of an acceptor to obtain a glycosylation primary product and a catalytic amount of nucleotide; and

[0046] b. regenerating the nucleotide into a regenerated sugar-nucleotide donor by contacting a catalytic amount of the nucleotide with a stoichiometric amount of a sugar donor precursor to obtain a regenerated sugar-nucleotide donor and a secondary product.

[0047] In some embodiments, the present invention provides a method for producing a galactosylation major product, comprising adding a sugar donor precursor, an acceptor, a transferase, and a catalytic amount of nucleotides to obtain a glycosylation major product and a minor product.

[0048] In some embodiments, the acceptor is lactose-N-triose II (LNTII), glucose or galacto-oligosaccharide (GOS), and the secondary product is fructose. Some embodiments include the step of contacting fructose in the presence of glucose isomerase to obtain glucose. Some embodiments include the step of obtaining LNTII, which contacts lactose and N-acetylglucosamine in the presence of β-N-acetylhexosaminidase (Bbh1) to obtain LNTII.

[0049] In some embodiments, the sugar donor precursor is sucrose. In some embodiments, the sugar donor precursor and nucleotide provide a sugar-nucleotide donor precursor, wherein the auxiliary enzyme and the sugar-nucleotide donor precursor provide a sugar-nucleotide donor. In some embodiments, the sugar-nucleotide donor precursor is a glucose nucleotide. In some embodiments, the secondary product is fructose. In some embodiments, the auxiliary enzyme is a galactose epimerase. In some embodiments, the nucleotide is uridine diphosphate.

[0050] In certain embodiments, the transferase is any enzyme that transfers a galactose unit to an acceptor. In some embodiments, the transferase comprises a first transferase ( Figure 6 , 7 , GT1 in 13 or 14) and the second transferase ( Figure 6 , 7 , GT2 in 13 or 14). In a specific embodiment, the first transferase catalyzes the transfer of sugar from a sugar-nucleotide donor to an acceptor to obtain a galactosylated primary product, and the second transferase catalyzes the reaction of a nucleotide and a sugar donor precursor to obtain a sugar-nucleotide donor and a secondary product. In some embodiments, the first transferase is a β-1,3-galactosyltransferase (Cvb3GalT) from Chromobacterium violaceum, a β-1,4-galactosyltransferase (NmLgtB) from Neisseria meningitidis, and the second transferase is a sucrose synthase (AtSuSy1) from Arabidopsis thaliana.

[0051] In some embodiments, the galactosylation main product is lactose-N-tetraose (LNT), lactose-N-neotetraose (LNnT), lactose or galacto-oligosaccharide (GOS). In some embodiments, the method includes contacting the secondary product or the main product with a processing enzyme to convert the secondary product into a secondary product derivative or the main product into a main product derivative. In some embodiments, the processing enzyme is an oxidase, an isomerase or a hydrolase. In some embodiments, the processing enzyme is an oxidase, and the galactosylation main product derivative is oxidized fructose or oxidized glucose.

[0052] In some embodiments, the present invention provides a method for producing a sialylated main product, comprising the steps of:

[0053] a. contacting a catalytic amount of a sugar-nucleotide donor with a stoichiometric amount of an acceptor to obtain a sialylated primary product and a catalytic amount of a nucleotide; and

[0054] b. regenerating the nucleotide into a regenerated sugar-nucleotide donor by contacting a catalytic amount of the nucleotide with a stoichiometric amount of a sugar donor precursor to obtain a regenerated sugar-nucleotide donor and a secondary product.

[0055] In some embodiments, the method further comprises:

[0056] a. contacting a catalytic amount of a regenerated sugar-nucleotide donor with a stoichiometric amount of an acceptor to obtain a glycosylation primary product and a catalytic amount of a nucleotide; and

[0057] b. regenerating the nucleotide into a regenerated sugar-nucleotide donor by contacting a catalytic amount of the nucleotide with a stoichiometric amount of a sugar donor precursor to obtain a regenerated sugar-nucleotide donor and a secondary product.

[0058] In some embodiments, the present invention provides a method for producing a sialylated major product, comprising the steps of adding a sugar donor precursor, an acceptor, a transferase, and a catalytic amount of nucleotides to obtain a sialylated major product and a minor product.

[0059] In some embodiments, the sugar donor precursor is 3'-sialyllactose. In some embodiments, the sugar-nucleotide donor is N-acetylneuraminic acid-nucleotide. In some embodiments, the secondary product is lactose. In some embodiments, the sialylated main product is sialyllactose-N-tetraose a (LSTa), sialyllactose-N-tetraose b (LSTb), sialyllactose-N-neotetraose c (LSTc), sialyllactose-N-neotetraose d (LSTd) or disialyllactose-N-tetraose (DSLNT). In some embodiments, the secondary product or the main product and the processing enzyme convert the secondary product into a secondary product derivative or convert the main product into a main product derivative. In some embodiments, the processing enzyme is an oxidase, an isomerase or a hydrolase. In some embodiments, in the presence of a processing enzyme, the secondary product is converted into a secondary product derivative. In some embodiments, the processing enzyme is lactase. In some embodiments, the secondary product derivative is glucose and galactose. In some embodiments, the processing enzyme is an enzyme that oxidizes lactose to lactobionic acid. In some embodiments, the processing enzyme is lactose oxidase. In some embodiments, in the presence of a second processing enzyme, glucose is converted into a modified secondary product derivative. In some embodiments, the second processing enzyme is D-galactose isomerase, and the modified secondary product derivative is tagatose. In some embodiments, galactose isomerase is derived from Geobacillus stearothermophilus (GsAI). In some embodiments, glucose oxidase and modified secondary product derivatives are gluconolactone. In some embodiments, the processing enzyme is an oxidase, an isomerase or a hydrolase, and the main product derivative is the main product of oxidation, the main product of isomerization or the main product of hydrolysis. In some embodiments, the hydrolase is α2-3 neuraminidase S. In some embodiments, α2-3 neuraminidase S and the main product DSLNT are converted to LSTb by neuraminidase.

[0060] In some embodiments, the main product derivative is an oxidized, isomerized or hydrolyzed LSTa, LSTb, LSTc, LSTd or DSLNT. In some embodiments, the nucleotide is cytidine monophosphate.

[0061] In certain embodiments, the transferase is any enzyme that transfers a sialic acid unit to an acceptor. In some embodiments, the transferase comprises a first transferase ( Figure 6 , 7 , GT1 in 13 or 14) and the second transferase ( Figure 6 , 7, GT2 in 13 or 14). In a specific embodiment, the first transferase catalyzes the transfer of sugar from a sugar-nucleotide donor to an acceptor to obtain a sialylated primary product, and the second transferase catalyzes the reaction of nucleotides and sugar donor precursors to obtain a sugar-nucleotide donor and a secondary product. In some embodiments, the first transferase is β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1), CMP-N-acetylneuraminic acid-β-1,4-galactoside α-2,3-sialyltransferase 3 (ST3GAL3) or α-N-acetylgalactosamine α-2,6-sialyltransferase 5 (ST6GALNAC5), and the second transferase is CMP-N-acetylneuraminic acid-β-galactosamide-α-2,3-sialyltransferase 4 (ST3GAL4). In some embodiments, the first transferase is ST3GAL3 and ST6GALNAC5.

[0062] In some embodiments, the present invention provides a method for producing a fucosylated major product, comprising the following steps:

[0063] a. contacting a catalytic amount of a sugar-nucleotide donor with a stoichiometric amount of an acceptor to obtain a fucosylated primary product and a catalytic amount of a nucleotide; and

[0064] b. regenerating the nucleotide into a regenerated sugar-nucleotide donor by contacting a catalytic amount of the nucleotide with a stoichiometric amount of a sugar donor precursor to obtain a regenerated sugar-nucleotide donor and a secondary product.

[0065] In some embodiments, the method further comprises:

[0066] a. contacting a catalytic amount of a regenerated sugar-nucleotide donor with a stoichiometric amount of an acceptor to obtain a glycosylation primary product and a catalytic amount of nucleotide; and

[0067] b. regenerating the nucleotide into a regenerated sugar-nucleotide donor by contacting a catalytic amount of the nucleotide with a stoichiometric amount of a sugar donor precursor in the presence of a transferase to obtain a regenerated sugar-nucleotide donor and a secondary product.

[0068] In some embodiments, the present invention provides a method for producing a fucosylated primary product, comprising the steps of adding a sugar donor precursor, an acceptor, a transferase, and a catalytic amount of nucleotides to obtain a fucosylated primary product and a secondary product. In some embodiments, the sugar donor precursor is 2'-fucosyllactose. In some embodiments, the sugar-nucleotide donor is a fucosyl nucleotide. In some embodiments, the secondary product is lactose.

[0069] In some embodiments, the major product is lactose-N-fucopentaose I (LNFPI), lactose-N-fucopentaose II (LNFPII), lactose-N-fucopentaose III (LNFPIII), difucosyllactose (DFL) or 3-fucosyllactose (3-FL).

[0070] In some embodiments, the method further comprises contacting the secondary product or the main product with a processing enzyme to convert the secondary product into a secondary product derivative or converting the main product into a main product derivative. In some embodiments, the processing enzyme is an oxidase, an isomerase or a hydrolase. In some embodiments, in the presence of a processing enzyme, the secondary product is converted into a secondary product derivative. In some embodiments, the processing enzyme is an enzyme that oxidizes lactose to lactobionic acid. In some embodiments, the processing enzyme is a lactose oxidase. In some embodiments, the processing enzyme is a lactase. In some embodiments, the secondary product derivative is glucose and galactose. In some embodiments, in the presence of a second processing enzyme, the secondary product derivative is converted into a modified secondary product derivative. In some embodiments, the second processing enzyme is a D-galactose isomerase, and the modified secondary product derivative is tagatose. In some embodiments, the D-galactose isomerase is derived from Geobacillus stearothermophilus (GsAI). In some embodiments, the second processing enzyme is a glucose oxidase, and the modified secondary product derivative is gluconolactone.

[0071] In some embodiments, in the presence of a processing enzyme, the main product is converted into a main product derivative. In some embodiments, the processing enzyme is an oxidase, an isomerase or a hydrolase, and the main product derivative is an oxidized main product, an isomerized main product or a hydrolyzed main product. In some embodiments, the main product derivative is an oxidized, isomerized or hydrolyzed LNFPI, LNFPII, LNFPIII, DFL or 3-FL. In some embodiments, the method includes a further step of oxidizing the fucosylated main product in the presence of an oxidase to provide a main product derivative. In some embodiments, the main product derivative is an oxidized LNFPI, LNFPII, LNFPIII, DFL or 3-FL. In some embodiments, the nucleotide is a guanosine diphosphate.

[0072] In certain embodiments, the transferase is any enzyme that transfers a fucose unit to an acceptor. In certain embodiments, the transferase is any enzyme that converts a sugar donor precursor into a sugar-nucleotide donor and a secondary product. In some embodiments, the transferase includes a first transferase ( Figure 6 , 7 , GT1 in 13 or 14) and the second transferase ( Figure 6 , 7, GT2 in 13 or 14). In a specific embodiment, the first transferase catalyzes the transfer of sugar from a sugar-nucleotide donor to an acceptor to obtain a fucosylated main product, and the second transferase catalyzes the reaction of a nucleotide with a sugar donor precursor to obtain a sugar-nucleotide donor and a secondary product. In some embodiments, the first transferase is α-1,2-fucosyltransferase (Te2FT) from Thermosynechococcus vestitus, α1-3 / 4-fucosyltransferase (Hp34FT) from Helicobacter pylori, fucosyltransferase 9 (FUT9) or fucosyltransferase 3 (FUT3), and the second transferase is α-1,2-fucosyltransferase (HmFucT) or fucosyltransferase (FUT1) from Helicobacter mustelae.

[0073] In some embodiments, the present invention provides a method for producing an N-acetylglucosaminylation main product, comprising the steps of:

[0074] a. contacting a catalytic amount of a sugar-nucleotide donor with a stoichiometric amount of an acceptor to obtain a major N-acetylglucosamine product and a catalytic amount of a nucleotide; and

[0075] b. regenerating the nucleotide into a regenerated sugar-nucleotide donor by contacting a catalytic amount of the nucleotide with a stoichiometric amount of a sugar donor precursor to obtain a regenerated sugar-nucleotide donor and a secondary product.

[0076] In some embodiments, the method further comprises:

[0077] a. contacting a catalytic amount of a regenerated sugar-nucleotide donor with a stoichiometric amount of an acceptor to obtain a glycosylation primary product and a catalytic amount of a nucleotide; and

[0078] b. regenerating the nucleotide into a regenerated sugar-nucleotide donor by contacting a catalytic amount of the nucleotide with a stoichiometric amount of a sugar donor precursor to obtain a regenerated sugar-nucleotide donor and a secondary product.

[0079] In some embodiments, the present invention provides a method for producing an N-acetylglucosaminylation main product, which comprises the steps of adding a sugar donor precursor, an acceptor, a transferase, and a catalytic amount of nucleotides to obtain an N-acetylglucosaminylation main product and a secondary product.

[0080] In some embodiments, the sugar donor precursor is lactose-N-disaccharide. In some embodiments, the sugar-nucleotide donor is N-acetylglucosamine nucleotide. In some embodiments, the secondary product is galactose. In some embodiments, the main product is LNTII or β-1,6-GlcNAc-LNnT. In some embodiments, the method also includes contacting the secondary product or the main product with a processing enzyme to convert the secondary product into a secondary product derivative or the main product into a main product derivative. In some embodiments, the processing enzyme is an oxidase, an isomerase or a hydrolase. In some embodiments, in the presence of a processing enzyme, the secondary product is converted into a secondary product derivative. In some embodiments, the processing enzyme is D-galactose isomerase, and the secondary product derivative is tagatose. In some embodiments, D-galactose isomerase is derived from Bacillus stearothermophilus (GsAI). In some embodiments, the method includes a further step of oxidizing the N-acetylglucosamine main product in the presence of an oxidase to provide a main product derivative. In some embodiments, the major product derivative is oxidized and the major product is LNTII or β-1,6-GlcNAc-LNnT.

[0081] In some embodiments, the nucleotide is uridine diphosphate.

[0082] In certain embodiments, the transferase is any enzyme that transfers an N-acetylglucosamine unit to an acceptor. In some embodiments, the transferase is β-1,3-N-acetyl-hexosaminyltransferase (NmLgtA) from Neisseria meningitidis. In some embodiments, the transferase includes a first transferase ( Figure 6 , 7 , GT1 in 13 or 14) and the second transferase ( Figure 6 , 7 , GT2 in 13 or 14). In a specific embodiment, the first transferase catalyzes the transfer of sugar from a sugar-nucleotide donor to an acceptor to obtain an N-acetylglucosaminyl main product, and the second transferase catalyzes the reaction of nucleotides and sugar donor precursors to obtain a sugar-nucleotide donor and a secondary product. In some embodiments, the first transferase is glucosaminyl (N-acetyl) transferase 2 (GCNT2), and the second transferase is β-1,3-N-acetyl-hexosaminyl transferase (NmLgtA) from Neisseria meningitidis. In some embodiments, a further step is to oxidize the N-acetylglucosaminyl main product in the presence of an oxidase to provide a main product derivative. In some embodiments, the main product derivative is an oxidized LNTII or an oxidized β-1,6-GlcNAc-LNnT. In some embodiments, the sugar is a naturally occurring isomeric form.

[0083] In some embodiments, glucose is D-glucose. In some embodiments, it is D-galactose. In some embodiments, fucose is L-fucose.

[0084] In some embodiments, the present invention provides compounds prepared by any of the methods herein. In some embodiments, the present invention provides glycosylated compounds prepared according to any of the methods herein. In some embodiments, the present invention provides galactosylated compounds prepared according to any of the methods herein. In some embodiments, the present invention provides sialylated compounds prepared according to any of the methods herein. In some embodiments, the present invention provides fucosylated compounds prepared according to any of the methods herein. In some embodiments, the present invention provides N-acetylglucosaminated compounds prepared according to any of the methods herein. In some embodiments, the present invention provides lactose derivatives prepared according to any of the methods herein. In some embodiments, the present invention provides lactose derivatives selected from lactobionic acid, lactitol, oligolacto-fructose, oligogalactose, lactulose and HMO according to any of the methods herein. In some embodiments, the present invention provides lactose, DSLNT, LNnT, LSTa, LSTb, LSTc or LSTd. In some embodiments, the compound is obtained from non-animal based plant materials.

[0085] In some embodiments, the invention provides a machine configured for the method of any embodiment. In certain embodiments, the method is in a single reaction vessel. The invention also provides a machine optimized for the method of the present invention. In certain embodiments, the machine is adjusted so that the method process is carried out in a single reaction vessel.

[0086] In some embodiments of the invention, one or more enzymes are immobilized. In other embodiments, each enzyme is immobilized. In other embodiments of the invention, the process is carried out in a single reaction vessel.

[0087] The present invention also provides compounds prepared by the methods of the present invention. In one embodiment, LNT is prepared by the methods of the present invention. In another embodiment, LNnT is prepared by the methods of the present invention. In another embodiment, lactose is prepared by the methods of the present invention. In another embodiment, galacto-oligosaccharides are prepared by the methods of the present invention. In another embodiment, LSTa is prepared by the methods of the present invention. In another embodiment, LSTb is prepared by the methods of the present invention. In another embodiment, LSTc is prepared by the methods of the present invention. In another embodiment, LSTd is prepared by the methods of the present invention. In another embodiment, DSLNT is prepared by the methods of the present invention. In another embodiment, LNFPI is prepared by the methods of the present invention. In another embodiment, LNFPII is prepared by the methods of the present invention. In another embodiment, LNFPIII is prepared by the methods of the present invention. In another embodiment, DFL is prepared by the methods of the present invention. In another embodiment, 3-FL is prepared by the methods of the present invention. In another embodiment, LNTII is prepared by the methods of the present invention. In another embodiment, branched HMOs are prepared by the methods of the present invention.

[0088] Any embodiment of the present invention may be employed to obtain compounds from non-animal based plant materials.

[0089] In another embodiment, glycosylation reactions employing catalytic nucleotides are performed in conjunction to provide a glycosylated primary product having two (2) or more sugar units that have been transferred to an acceptor by two (2) or more sugar-nucleotide donors of the invention.

[0090] Integrated fucosylation pathway

[0091] In another aspect, the present invention provides an integrated pathway for the synthesis of fucosylated sugars starting from common sugars using immobilized enzymes, including the one-pot synthesis of fucosylated sugars. A cell-free biomanufacturing method is included for making fucosylated human milk oligosaccharides and other fucosylated oligosaccharides from fructose, glucose, or a mixture of fructose and glucose. Starting from common sugars can reduce the production cost of fucosylated HMOs compared to using fucose as a starting material. Figure 15-18 An integrated fucosylation pathway of the present invention is depicted.

[0092] The present invention provides for the direct production of fucosylated HMOs starting from cost-effective glucose or fructose instead of fucose. The regeneration of cofactors in the enzyme reaction can improve efficiency and reduce costs. The production method allows the flexible production of all fucosylated HMOs (or oligosaccharides) by changing the final transferase and acceptor backbone (biorefining concept). The process does not require genetically modified organisms (non-GMO).

[0093] In some embodiments, the present invention provides a method for producing fucosylated oligosaccharides or fucosylated antibody-glycan conjugates, comprising the following steps:

[0094] a. contacting glucose or fructose in the presence of an enzyme that converts fructose or glucose into mannose;

[0095] b. contacting mannose with an enzyme that converts mannose into mannose-6-phosphate;

[0096] c. contacting mannose-6-phosphate with an enzyme that converts mannose-6-phosphate into mannose-1-phosphate;

[0097] d. contacting mannose-1-phosphate with an enzyme that converts mannose-1-phosphate into GDP-D-mannose;

[0098] e. contacting GDP-D-mannose with an enzyme that converts GDP-D-mannose into GDP-4-keto-6-deoxymannose;

[0099] f. contacting GDP-4-keto-6-deoxymannose with an enzyme that converts GDP-4-keto-6-deoxymannose into GDP-L-fucose;

[0100] g. contacting the GDP-L-fucose with a disaccharide, oligosaccharide or antibody-glycan conjugate and an enzyme that fucosylates the disaccharide, oligosaccharide or antibody-glycan conjugate; and

[0101] h. Obtaining fucosylated disaccharides, oligosaccharides or fucosylated antibody-glycan conjugates.

[0102] Each enzyme is immobilized and the process comprises a set of regenerating enzyme systems to convert ADP to ATP, PPi to Pi, GDP to GTP, and NADP+ to NADPH.

[0103] In certain embodiments, the method includes immobilized regeneration enzymes such as formate dehydrogenase, pyruvate oxidase, acetate kinase, catalase and inorganic pyrophosphatase. In certain embodiments, the immobilized regeneration enzymes are phosphite dehydrogenase, pyruvate oxidase, acetate kinase, catalase and inorganic pyrophosphatase. In certain embodiments, the immobilized regeneration enzymes are formate dehydrogenase, polyphosphate kinase and inorganic pyrophosphatase. In certain embodiments, the immobilized regeneration enzymes are phosphite dehydrogenase, polyphosphate kinase and inorganic pyrophosphatase.

[0104] In certain embodiments, the enzyme that converts glucose to mannose is an immobilized D-mannose isomerase. In certain embodiments, the enzyme that converts fructose to mannose is an immobilized N-acetyl-d-glucosamine-2-epimerase. In certain embodiments, the enzyme that converts mannose to mannose-6-phosphate is an immobilized hexokinase. In certain embodiments, the enzyme that converts mannose-6-phosphate to mannose-1-phosphate is an immobilized phosphomannose mutase. In certain embodiments, the enzyme that converts mannose-1-phosphate to GDP-D-mannose is an immobilized GDP-mannose pyrophosphorylase. In certain embodiments, the enzyme that converts GDP-D-Man to GDP-4-keto-6-deoxymannose is an immobilized GDP-mannose-4,6-dehydratase. In certain embodiments, the enzyme that converts GDP-4-keto-6-deoxymannose to GDP-L-fucose is an immobilized GDP-fucose synthase.

[0105] In some embodiments, the enzyme that fucosylate the disaccharide, oligosaccharide or antibody-glycan conjugate is an immobilized fucosyltransferase. In some embodiments, GDP-mannose is polymerized to mannan by a transferase. In certain embodiments, the oligosaccharide is 3'SL, LNTII, LNT, LNnT, LNFPI, LNFPII, LNFPIII, LSTa, LSTb, LSTc, LSTd and DSLNT, LNnH, 3"'3,3"'6-bis-O-α-Sia-LNnH 3"'3,3"'6-bis-O-α-Sia-(3"3,3"6-bis-O-α-Fuc)-LNnH, dilinear sialylated or fucosylated lactose-N-neohexaose and neoheptasaccharides, α-2,3-sialyllactose-N-neopentose, linear fucosyl and sialyllactose-N-neopentose, linear lactose-N-neopentose or dilinear lactose-N-neohexaose and heptasaccharides. In certain embodiments, the polysaccharide is 3'SL LNTII, LNT, LNnT, LNFPI, LNFPII, LNFPIII, LSTa, LSTb, LSTc, LSTd, DSLNT, 2'-FL, LNnH, DSLNnH or DSDFLNNH. In other embodiments, the polysaccharide is 3'SL, LNTII, 2'-FL, LNFPI, LSTa. In certain embodiments, the oligosaccharide is lactose, LNT, LNnT, LNnH or LNH. In certain embodiments, the fucosylated oligosaccharide is 2'FL or 3-FL. In certain embodiments, the d-fucosylated oligosaccharide is fucosylated HMO. In certain embodiments, the fucosylated oligosaccharide is fucosylated LDFT, fucosylated LNFP I, fucosylated LNFP II, fucosylated LNFP V, fucosylated LNDFH I, fucosylated LNDFH II, fucosylated DFLNHa, or fucosylated DFLNHc.

[0106] In certain embodiments, the process occurs in a single reaction vessel. In some embodiments, the invention provides a machine comprising an immobilized enzyme according to any embodiment of the invention, wherein the machine produces fucosylated disaccharides, fucosylated oligosaccharides or fucosylated antibody-glycan conjugates. In certain embodiments, the immobilized enzyme is contained within a single reaction vessel.

[0107] In some embodiments, the approach uses immobilized enzymes, such as enzymes immobilized in a bionanocatalyst (BNC), which in turn is embedded in a scaffold. The bionanocatalyst (BNC) according to the present invention includes an enzyme self-assembled with a magnetic nanoparticle (MNP). The BNC self-assembles with the scaffold. In some embodiments, each enzyme is immobilized in a BNC.

[0108] In certain embodiments, fucosylated human milk oligosaccharides (HMOs) are produced.

[0109] The one-pot integrated fucosylation synthesis can be performed in batch or flow. It should be understood that it is within the scope of the present invention to modify Examples 11-15 to a flow format. In a flow reactor (eg, a packed bed reactor), a reagent mixture flows through the flow reactor.

[0110] In certain embodiments, the scaffold BNC is located in a modular flow cell for flow manufacturing. In certain embodiments, the present invention provides a continuous flow process in which each step of the synthesis is carried out in a modular form. In production mode, these modules include a complete enzyme system (sugar activation and sugar transfer) for the specialized construction of polysaccharides. In some embodiments, polysaccharides are oligosaccharides.

[0111] In some embodiments, the scaffold comprises a magnetic metal oxide. In some embodiments, the scaffold is a highly magnetic and high porosity composite mixture of a thermoplastic containing magnetic particles forming a powder. They can be a single layer or multilayer material that accommodates BNCs. 3D printing can be used to produce objects of this design by sintering composite magnetic powders. In some embodiments, selective laser sintering (SLS) is used. Modular flow cells can be mixed and matched to achieve a highly customizable and efficient manufacturing process. In a preferred embodiment, human milk oligosaccharides (HMOs) are produced.

[0112] In certain embodiments, the immobilized enzyme is non-magnetic. In certain embodiments, the immobilized enzyme does not comprise nanoparticles.

[0113] Thus, the present invention provides for the cell-free production of defined glycans using combinatorial bionanocatalysts (BNCs) organized in sequential modules.

[0114] BRIEF DESCRIPTION OF THE DRAWINGS

[0115] Figure 1A An overview of galactosylation using sucrose to obtain LNT, LNnT or lactose is depicted and the legend associated with all figures herein ("2: Lac synthase" should be "1d) Lac synthase"). Figure 1B Derivation of LNT from LNTII is depicted. Figure 1C Depicted is the acquisition of LNnT from LNTII. Figure 1D Derivation of LNT from lactose is depicted. Figure 1D The acquisition of lactose from glucose is depicted. Figure 1F Derivation of GOS from galactose and sucrose is depicted.

[0116] Figures 2A-2D Sialylation using sialoside and lactose hydrolysis is depicted. Figure 2A The pathway to obtain LSTa from LNT is depicted. Figure 2B The pathway for obtaining LSTc from LNnT is depicted. Figure 2C The pathway for obtaining LSTd from LNnT is depicted. Figure 2D The pathway to obtain DSLNT from LNT is depicted.

[0117] Figures 3A-3C Fucosylation using fucoside and lactose hydrolysis is depicted. Figure 3A The pathway for obtaining LNFPI from LNT is depicted. Figure 3B The pathway for obtaining LNFPII from LNT is depicted. Figure 3C The pathway for LNFPII to be derived from LNnT is depicted.

[0118] Figure 4 Depicted is the addition of N-acetylglucosamine to prepare LNTII from lactose and chitin.

[0119] Figure 5 Lactose and its functional derivatives are depicted.

[0120] Figure 6 An overview of general embodiments of the invention and methods employed in the invention are depicted.

[0121] Figure 7 General embodiments and methods of the invention employing auxiliary enzymes are depicted.

[0122] Figure 8 Depicts a legend depicting the symbols used in Figures 9-12.

[0123] Fig.9A An overview of galactosylation using sucrose to obtain LNT, LNnT or lactose is depicted.

[0124] Fig. 9B The pathway for obtaining LNT from LNTII is depicted. Fig. 9C The pathway for obtaining LNnT from LNTII is depicted. Fig.9D The pathway for obtaining LNT from lactose is depicted. Fig.9E Depicted is the pathway for lactose to be derived from glucose. Fig.9F and Figure 9G Depicted is the pathway for lactose to be derived from glucose. Figure 9H The pathway to galacto-oligosaccharides from galactose and sucrose is depicted.

[0125] Figures 10A-10D Sialylation using sialoside and lactose hydrolysis is depicted. Fig. 10A The pathway to obtain LSTa from LNT is depicted. Fig. 10B The pathway for obtaining LSTc from LNnT is depicted. Fig. 10C The pathway for obtaining LSTd from LNnT is depicted. Fig. 10D The pathway to obtain DSLNT from LNT is depicted.

[0126] Figures 11A-11C Fucosylation using fucoside and lactose hydrolysis is depicted. Fig.11A The pathway for obtaining LNFPI from LNT is depicted. Fig. 11B The pathway for obtaining LNFPII from LNT is depicted. Fig. 11C The pathway for LNFPII to be derived from LNnT is depicted. Fig.11D The pathway for obtaining DFL from 2'-FL is depicted. Fig.11E The pathway for obtaining 3-FL from 2'-FL is depicted.

[0127] FIG. 12A depicts N-acetylglucosamination using lactose-N-biose as a GlcNAc donor. Fig. 12B Branched HMOs obtained from lactose-N-biose (LNB) and LNnT are depicted.

[0128] Fig.13 An overview of general embodiments of the invention and methods employed by the invention are described, which provide processing enzymes to convert a principal product into a principal product derivative.

[0129] Fig.14 General embodiments and methods of the invention are depicted wherein auxiliary enzymes and processing enzymes are employed to convert a primary product into a primary product derivative.

[0130] Figure 15-18 The present invention relates to an integrated fucosylation pathway.

[0131] Fig.15 In situ GDP-L-fucose production for 2'-FL synthesis or other fucosylation reactions (pyruvate-AcK / PyrOx+FDH regeneration) is depicted. See Example 12.

[0132] Fig.16 In situ GDP-L-fucose production for 2'-FL synthesis or other fucosylation reactions (pyruvate-AcK / PyrOx+PtxD regeneration) is depicted. See Example 13.

[0133] Fig.17 In situ GDP-L-fucose production for 2'-FL synthesis or other fucosylation reactions (PolyP-PPK+FDH regeneration) is depicted. See Example 14.

[0134] Fig.18 In situ GDP-L-fucose production for 2'-FL synthesis or other fucosylation reactions (PolyP-PPK+PtxD regeneration) is depicted. See Example 15.

[0135] Detailed description of the invention

[0136] The present invention provides an integrated streamlined approach to obtain sugar compounds, oligosaccharides and their derivatives using enzymes and activated sugars. In certain embodiments, the oligosaccharide is a complex oligosaccharide, including polysaccharides of 4 sugar units or larger, and includes polysaccharides.

[0137] In one aspect, the invention provides a cell-free de novo synthesis of glycans using activated sugar donors generated in situ and catalytic amounts of nucleotides. The integrated pathway of the enzymes leads to glycosylation (sugar-containing compounds) starting from sugars (including but not limited to plant sugars), including but not limited to carbohydrates, oligosaccharides or antibody-glycan conjugates. Advantages of the reactant and enzyme systems of this aspect of the invention include, consist essentially of, or consist of:

[0138] Avoid enzymes that recycle cofactors.

[0139] - Avoid stoichiometry or excess of nucleotide relative to sugar donor precursor or acceptor or both.

[0140] • Using nucleotide starting materials is less expensive than using nucleotide-sugar as starting materials.

[0141] • Ability to use less than 0.1 molar equivalents (<10%) of nucleotides compared to the acceptor molecule or limiting agent.

[0142] • Ability to use less than 0.1 molar equivalents (<50%) of nucleotide compared to sugar donor molecules or limiting reagents.

[0143] Avoid nucleotide triphosphates

[0144] Avoid inorganic phosphate by-products

[0145] - High reaction conversion rates are achieved by hydrolyzing high energy sugar donor precursors into products, which in some embodiments are enzymatically processed by processing enzymes to enhance conversion rates.

[0146] Avoid enzyme inhibition by organophosphates.

[0147] Avoid enzyme inactivation and reaction inhibition by using magnesium (Mg) instead of manganese (Mn).

[0148] Relatively low sucrose molar equivalent concentrations (1-4 equivalents) are required relative to the receptor.

[0149] Figure 6Describe the general embodiment of the present invention and the overview of the method adopted in the present invention.As used herein, "acceptor" is the part that accepts the transfer of sugar units from sugar-nucleotide donors.Transferase (designated as GT1 herein) catalyzes sugar transfer, thereby produces "primary product", i.e. glycosylation product provided by the present invention.Sugar-nucleotide donor is a compound comprising sugar unit and nucleotide, obtained by the reaction of sugar donor precursor and nucleotide in the presence of transferase (designated as GT2 herein).GT2 works in the opposite direction relative to GT1.GT1 transfers sugar unit to acceptor to form sugar acceptor compound.The effect of GT2 is to remove sugar unit.The amount of nucleotide present is less than stoichiometric, so sugar-nucleotide donor is regenerated and used for another reaction cycle.The reaction of sugar donor precursor and nucleotide in the presence of transferase also produces secondary product in addition to sugar-nucleotide donor.It is the energy source of the inventive method to decompose sugar donor precursor into smaller sugar units.In the presence of processing enzyme, secondary product can be converted into secondary product derivative.

[0150] Thus, in one embodiment, the present invention provides a method for obtaining a glycosylated compound (referred to herein as a "glycosylated primary product") by combining the following in a reaction vessel: a sugar donor precursor, an acceptor, a transferase, and a catalytic amount of nucleotides. The method involves iterative reaction cycles to produce a glycosylated primary product and secondary products. Without being bound by theory, once combined in a reaction mixture, the nucleotides react with the sugar donor precursor in the presence of a transferase to form a sugar-nucleotide donor. The sugar of the sugar-nucleotide donor is transferred to the acceptor to provide a glycosylated compound, a secondary product, and a regenerated nucleotide. Since the nucleotide is present in a catalytic amount, the sugar-nucleotide donor will be formed in approximately a catalytic amount. The reaction is then iterated through additional reaction cycles.

[0151] Figure 7 Depicted are general embodiments and methods of the invention employing sugar-nucleotide donor precursors. As shown, the reaction product between a nucleotide and a sugar donor precursor in the presence of a transferase provides a sugar-nucleotide donor precursor, which is further reacted in the presence of an auxiliary enzyme to obtain the sugar-nucleotide donor employed in the method of the invention.

[0152] Fig.13 and Fig.14 A general embodiment of the invention is depicted which employs a processing enzyme to convert a main product into a main product derivative. In Example 10, this method is employed to obtain LSTb from LNT. DSLNT is the main product converted to LSTb by neuraminidase.

[0153] Figures 1A-1FAn overview and illustration of the galactosylation reaction using sucrose are depicted. LNTII and glucose are the acceptor molecules that are galactosylated to provide LNT, LNnT or lactose, depending on the conditions (see ( Figure 1B-1D ).

[0154] Figure 1B Depicted is the approach of adding galactose to LNTII to obtain LNT. In the presence of sucrose synthase and UDP, UDP-glucose and fructose are obtained from sucrose. In the presence of GalE, UDP-galactose is obtained from UDP-glucose. In the presence of a transferase that forms a β3-type linkage (as shown, Cvb3GalT or B3GALT5), galactose from UDP-galactose is transferred to LNTII. See Example 2aA.

[0155] Figure 1C Depicted is the pathway for the addition of galactose to LNTII to obtain LNnT. The formation of UDP-galactose and fructose follows the same Figure 1B Same pathway as in . However, in this scheme there is a transferase that forms a β4-type linkage (as shown, NmLgtB or B4LAGT1). Galactose is transferred to LNTII to provide LNnT. See Example 2aB.

[0156] Figure 1D The pathway to obtain LNT from lactose is depicted. In the presence of DMC, triethylamine and water, N-acetylglucosamine is converted to oxazoline. N-acetylglucosamine is transferred to lactose via a β3-type linkage to provide LNTII. UDP-ga Figures 1A-1F An overview and illustration of the galactosylation reaction using sucrose are depicted. LNTII and glucose are the acceptor molecules that are galactosylated to provide LNT, LNnT or lactose, depending on the conditions (see ( Figure 1B-1D ).

[0157] Figure 1B Depicted is the approach of adding galactose to LNTII to obtain LNT. In the presence of sucrose synthase and UDP, UDP-glucose and fructose are obtained from sucrose. In the presence of GalE, UDP-galactose is obtained from UDP-glucose. In the presence of a transferase that forms a β3-type linkage (as shown, Cvb3GalT or B3GALT5), galactose from UDP-galactose is transferred to LNTII. See Example 2aA.

[0158] Figure 1C Depicted is the pathway for the addition of galactose to LNTII to obtain LNnT. The formation of UDP-galactose and fructose follows the same Figure 1BThe same pathway as in . However, in this scheme there is a transferase that forms a β4-type linkage (as shown, NmLgtB or B4LAGT1). Galactose is transferred to LNTII to provide LNnT. See Example 2aB.

[0159] Figure 1D A pathway for obtaining LNT from lactose is depicted. In the presence of DMC, triethylamine and water, N-acetylglucosamine is converted to oxazoline. N-acetylglucosamine is transferred to lactose via a β3 linkage to provide LNTII. UDP-galactose and fructose are obtained as described in Figure Ba. In the presence of a transferase (Cvb3GalT as shown in the figure), galactose is transferred to the acceptor LNTII to provide LNT.

[0160] Figure 1E The production of lactose from glucose is depicted. Figure 1B The method of obtaining UDP-galactose is described. In the presence of lactose synthase, galactose is transferred to glucose to obtain lactose. See Example 2aD.

[0161] Figure 1F The production of GOS from galactose and sucrose is depicted. UDP-galactose and fructose as Figure 1B Galactose is transferred to oligogalactose (where n is 3-15) to increase the galactose chain by 1. UDP-galactose Figure 1B See Example 2aE.

[0162] Figures 2A-2D Sialylation using sialoside and lactose hydrolysis is depicted. CMP-N-acetylneuraminic acid (Neu5Ac) is Figures 2A-2C Nucleotide-sugars used in each figure. Lactose was converted to galactose and glucose in the presence of lactase as shown in each figure. CMP-Neu5Ac was obtained in the presence of 3'-SL and transferase ST3GAL4.

[0163] Figure 2A The pathway for obtaining LSTa from LNT is depicted. In the presence of the transferase ST3GAL3, N-acetylneuraminic acid is transferred from CMP-Neu5Ac to LNT to obtain LSTc.

[0164] Figure 2B The pathway for obtaining LSTc from LNnT is depicted. In the presence of the transferase ST6GAL1, N-acetylneuraminic acid is transferred from CMP-Neu5Ac to LNnT to obtain LSTc.

[0165] Figure 2CThe pathway for obtaining LSTd from LNnT is depicted. In the presence of the transferase ST3GAL3, N-acetylneuraminic acid is transferred from CMP-Neu5Ac to LNnT to obtain LSTd.

[0166] exist Figure 2D In the presence of transferases ST6GAL3 and ST6GALNAC5, N-acetylneuraminic acid is transferred from CMP-Neu5Ac to LNT to obtain DSLNT.

[0167] Figures 3A-3C Fucosylation using fucoside and lactose hydrolysis is depicted. GDP-l-fucose is the nucleotide-sugar used in Figures 3a-3c. In the presence of transferase (HmFucT), lactose, lactase and GDP, 2'-FL provides GDP-l-fucose (GDP-Fuc), galactose and glucose.

[0168] Figure 3A The pathway for obtaining LNFPI from LNT is depicted. GDP-Fuc provides LNFPI in the presence of LNT and Te2FT.

[0169] Figure 3B The pathway for obtaining LNFPII from LNT is depicted. GDP-Fuc provides LNFPII in the presence of LNT and a transferase (Hp34FT).

[0170] Figure 3C The pathway for obtaining LNFPIII from LNnT is depicted. GDP-Fuc provides LNFPIII in the presence of LNnT and a transferase (FUT9).

[0171] Figure 4 Describe the pathway of adding N-acetylglucosamine to prepare LNTII from lactose and chitin. In the presence of chitin synthase and transferase (B3GNT2 or NmLgtA), UDP-N-acetylglucosamine (UDP-GlcNAc) is prepared from UDP, lactose, chitin, and GlcNAc is transferred to lactose to obtain LNTII. In the presence of transferase (Cvb3GalT as shown in the figure), galactose is transferred to acceptor LNTII to provide LNT.

[0172] Figure 5 Lactose and its functional derivatives are depicted.

[0173] Figure 8Legends for Figures 9-12 are depicted. N-acetylglucosamine (GlcNAc) is represented by a solid black square. L-fucose (fuc) is represented by a solid white triangle. Lactose is represented by a solid white circle connected to the shaded circle by a line, where 4 represents a bond. D-galactose (Gal) is represented by a solid circle. N-acetylneuraminic acid (Neu5Ac) is represented by a solid white diamond. D-glucose is represented by a shaded circle.

[0174] Figures 9A-9H An overview of the galactosylation reaction using sucrose is depicted. LNTII and glucose are the acceptor molecules that are galactosylated to provide LNT, LNnT or lactose, depending on the conditions (see ( Figures 9B-9D ).

[0175] Fig. 9B Depicted is the approach of adding galactose to LNTII to obtain LNT. In the presence of sucrose synthase and UDP, UDP-glucose and fructose are obtained from sucrose. In the presence of GalE, UDP-galactose is obtained from UDP-glucose. In the presence of a transferase that forms a β3-type linkage (as shown, Cvb3GalT), galactose from UDP-galactose is transferred to LNTII. See Example 6A.

[0176] Fig. 9C Depicted is the pathway for the addition of galactose to LNTII to obtain LNnT. The formation of UDP-galactose and fructose follows the same Fig. 9B Same pathway as in Example 6B. However, in this scheme there is a transferase that forms a β4-type linkage (NmLgtB as shown). Galactose is transferred to LNTII to provide LNnT. See Example 6B.

[0177] Fig.9D The pathway to obtain LNT from lactose is depicted. In the presence of DMC, triethylamine and water, N-acetylglucosamine is converted to oxazoline. In the presence of an amidase (here Bbh1), N-acetylglucosamine is transferred to lactose via a β3-type linkage to provide LNTII. Fig. 9B UDP-galactose and fructose are obtained as described. In the presence of a transferase (Cvb3GalT as shown in the figure), galactose is transferred to the acceptor LNTII to provide LNT.

[0178] Fig.9E The production of lactose from glucose is depicted. The production of UDP-galactose is shown in Fig. 9B As shown. In the presence of lactose synthase, galactose is transferred to glucose to obtain lactose. See Example 6C.

[0179] Fig.9FDepicted is the lactose obtained from glucose, sucrose and a catalytic amount of UDP using the four enzymes NmLgtB, AtSuSy1, GalE and glucose(xylose)isomerase (EC 5.3.1.5, D-xylose aldose-ketose isomerase). See Example 7.

[0180] Figure 9G Depicted is the lactose obtained from glucose, sucrose, fructose and a catalytic amount of UDP using the four enzymes NmLgtB, AtSuSy1, GalE and glucose(xylose)isomerase (EC 5.3.1.5, D-xylose aldose-ketose isomerase). See Example 8.

[0181] Figure 9H GOS obtained from galactose and sucrose are depicted. UDP-galactose and fructose are obtained as Fig. 9B The galactose is transferred to galacto-oligosaccharides (where n is 3-15) to increase the galactose chain by 1.

[0182] In some embodiments, the transferase is a galactosyltransferase, which catalyzes the transfer of activated UDP-galactose (UDP-Gal) to glucose, galactose, or GlcNAc.

[0183] In certain embodiments, the galactosyltransferase is a β-1,3-galactosyltransferase (EC 2.4.1.122). In specific embodiments, the β-1,3-galactosyltransferase is Cvβ3GalT from Chromobacterium violaceum, WbgO from Escherichia coli, CgtB from Campylobacter jejuni, B3GALT1 from Homo sapiens, B3GALT2 from Homo sapiens, B3GALT4 from Homo sapiens, or B3GALT5 from Homo sapiens.

[0184] In certain embodiments, the galactosyltransferase is a β-1,4-galactosyltransferase (EC2.4.1.22, EC2.4.1.38, EC2.4.1.133, EC2.4.1.275). In specific embodiments, the β-1,4-galactosyltransferase is NmLgtB from Neisseria meningitidis, NmLgtB-StGalE from Neisseria meningitidis and Streptococcus thermophilus, HpLgtB from Helicobacter pylori, B4GALT1 from Homo sapiens, B4GALT2 from Homo sapiens, B4GALT3 from Homo sapiens, B4GALT4 from Homo sapiens, B4GALT5 from Homo sapiens, B4GALT6 from Homo sapiens, B4GALT7 from Homo sapiens.

[0185] In certain embodiments, the galactosyltransferase is a β-1,6-galactosyltransferase. In specific embodiments, the β-1,6-galactosyltransferase is GalT29A from Arabidopsis thaliana.

[0186] In certain embodiments, the galactosyltransferase is an α-1,3-galactosyltransferase (EC 2.4.1.87, EC 2.4.1.37). In specific embodiments, the α-1,3-galactosyltransferase is the GTB (human protein) synthetic gene expressed in E. coli or WbnL from E. coli.

[0187] In certain embodiments, the galactosyltransferase is an alpha-1,6-galactosyltransferase (EC 2.4.1.241).

[0188] In certain embodiments, the galactosyltransferase is selected from the galactosyltransferases shown in the Figures.

[0189] In certain embodiments, an auxiliary enzyme is used in the pathway from a sugar donor precursor to a sugar-nucleotide donor. In those embodiments using an auxiliary enzyme, the sugar donor precursor is converted to a sugar-nucleotide donor precursor in the presence of a transferase. Then, in the presence of an auxiliary enzyme, the sugar-nucleotide donor precursor is converted to a sugar-nucleotide donor. In certain embodiments, the auxiliary enzyme is UDP-glucose 4-epimerase. Fig.9A In the galactosylation pathway shown in -H, the auxiliary enzyme is UDP-glucose 4-epimerase from Bifidobacterium longum.

[0190] In some embodiments, the accessory enzyme is an oxidase (UDP-Glc-6-dehydrogenase, EC 1.1.1.22) that catalyzes the conversion of UDP-Glc to UDP-GlcA (UDP-glucuronic acid).

[0191] In some embodiments, the auxiliary enzyme is an enzyme of isomerization activated sugar nucleotides. In certain embodiments, the enzyme of isomerization activated sugar nucleotides is an epimerase (UDP-Gal-4-epimerase, EC5.1.3.2) catalyzing the conversion of UDP-Glc into UDP-Gal. In a specific embodiment, the epimerase is EcGalE from Escherichia coli. In another specific embodiment, the epimerase is StGalE from Streptococcus thermophilus.

[0192] Figures 10A-10D Sialylation using low-cost sialoside and lactose hydrolysis is depicted. CMP-N-acetylneuraminic acid (Neu5Ac) is Figures 10A-10D Nucleotide-sugar used in each figure: As shown in each figure, in the presence of lactase, lactose is converted into galactose and glucose, and in the presence of 3'-SL and transferase ST3GAL4, CMP-Neu5Ac is obtained.

[0193] Fig. 10A The pathway for obtaining LSTa from LNT is depicted. In the presence of transferases ST3GAL4, ST3GAL3 and lactase, N-acetylneuraminic acid is transferred from CMP-Neu5Ac to LNT to obtain LSTa.

[0194] Fig. 10B The pathway for obtaining LSTc from LNnT is depicted. In the presence of transferases ST3GAL4, ST6GAL1 and lactase, N-acetylneuraminic acid is transferred from CMP-Neu5Ac to LNnT to obtain LSTc.

[0195] Fig. 10C The pathway for obtaining LSTd from LNnT is depicted. In the presence of transferases ST3GAL4, ST3GAL3 and lactase, N-acetylneuraminic acid is transferred from CMP-Neu5Ac to LNnT to obtain LSTd.

[0196] Fig. 10D The pathway for obtaining DSLNT from LNT is depicted. In the presence of transferases ST3GAL3, ST6GALNAC5, ST3GAL4 and lactase, N-acetylneuraminic acid is transferred from CMP-Neu5Ac to LNT to obtain DSLNT.

[0197] Some embodiments of the invention employ a sialyltransferase to catalyze the transfer of CMP-sialic acid (eg, CMP-Neu5Ac) to GlcNAc, galactose, or Neu5Ac. Figures 2A-2D and Fig. 10A The method shown in -D employs a sialyltransferase to transfer a sialic acid group from a sugar nucleotide donor to an acceptor.

[0198] In certain embodiments of the invention, the sialyltransferase is an α-2,3-sialyltransferase (EC2.4.99.4, EC2.4.99.6, EC2.4.99.7, EC2.4.99.9). In specific embodiments, the α-2,3-sialyltransferase is PmST1 (wild type and mutants) from Pasteurella multocida, NmST1-NmCSS fusion from Neisseria meningitidis, ST3GAL1 from Homo sapiens, ST3GAL2 from Homo sapiens, ST3GAL3 from Homo sapiens, ST3GAL4 from Homo sapiens, or ST3GAL5 from Homo sapiens, ST3GAL6 from Homo sapiens.

[0199] In certain embodiments of the invention, the sialyltransferase is an α-2,6-sialyltransferase (EC 2.4.99.1, EC 2.4.99.3). In specific embodiments, the α-2,6-sialyltransferase is Pd26ST from Photobacterium damsel, ST6GAL1 from Homo sapiens, ST6GAL2 from Homo sapiens, ST6GALNAC1 from Homo sapiens, ST6GALNAC2 from Homo sapiens, ST6GALNAC3 from Homo sapiens, ST6GALNAC4 from Homo sapiens, ST6GALNAC5 from Homo sapiens, or ST6GALNAC6 from Homo sapiens.

[0200] In certain embodiments of the invention, the sialyltransferase is an α-2,8-sialyltransferase (EC 2.4.99.8). In specific embodiments, the α-2,8-sialyltransferase is an α-2,3 / 8-sialyltransferase from Campylobacter jejuni, ST8SIA1 from Homo sapiens, ST8SIA2 from Homo sapiens, ST8SIA3 from Homo sapiens, ST8SIA4 from Homo sapiens, or ST8SIA5 from Homo sapiens.

[0201] In certain embodiments, the sialyltransferase is selected from the sialyltransferases shown in the accompanying figures.

[0202] Figures 11A-11E Fucosylation using fucoside and lactose hydrolysis is depicted. GDP-l-fucose is Figures 11A-11C Nucleotide-sugars used in.

[0203] Fig.11A The pathway for obtaining LNFPI from LNT is depicted. GDP-Fuc provides LNFPI in the presence of 2'-FL, LNT, Te2FT, HmFucT, lactase, and GsAI.

[0204] Fig. 11B The pathway for obtaining LNFPII from LNT is depicted. GDP-Fuc provides LNFPII in the presence of 2'-FL, LNT Hp34FT, HmFucT, lactase, and GsAI.

[0205] Fig. 11C The pathway for obtaining LNFPIII from LNnT is depicted. GDP-Fuc provides LNFPIII in the presence of 2'FL, LNnT FUT9, HmFucT, lactase and GsAI.

[0206] Fig.11D The pathway for obtaining DFL from 2'-FL is depicted. In the presence of 2'-FL, FUT1, FUT3, lactase, GsA1 and fucosidase, GDP-Fuc provides 3-FL, tagatose and glucose.

[0207] Fig.11E A pathway for obtaining 3-FL from 2'-FL is depicted. GDP-Fuc in the presence of 2'-FL, FUT1, FUT3, lactase, and GsAI. Lactase consumes lactose in this pathway. In addition to lactase, any other enzymatic or non-enzymatic method can be used to consume lactose. In addition, GsAI can be added to convert galactose to tagatose to enhance the reaction conversion. See Example 5a.D and Example 5a.E.

[0208] In some embodiments, the fucosyltransferase catalyzes the transfer of GDP-fucose (GDP-Fuc) to galactose, glucose, GlcNAc, or GalNAc. Figure 3A -C and Fig.11A The method shown in -E uses a fucosyltransferase to transfer a fucosyl group from a sugar-nucleotide donor to an acceptor.

[0209] In certain embodiments, the fucosyltransferase is an α-1,2-fucosyltransferase (EC 2.4.1.69). In specific embodiments, the α-1,2-fucosyltransferase is Te2FT from Thermosynechococcuse longatus, WbgL from Escherichia coli, HmFucT from Helicobacter pylori, FUT1 from Homo sapiens, or FUT2 from Homo sapiens.

[0210] In certain embodiments, the fucosyltransferase is an α-1,3-fucosyltransferase (EC2.4.1.152, EC2.4.1.214). In specific embodiments, the α-1,3-fucosyltransferase is HpFucT from Helicobacter pylori, Bf1,3FT from Bacteroides fragilis, Hp3 / 4FT from Helicobacter pylori, FUT3 from Homo sapiens, FUT4 from Homo sapiens, FUT5 from Homo sapiens, FUT6 from Homo sapiens, FUT7 from Homo sapiens, FUT9 from Homo sapiens, or FUT11 from Homo sapiens.

[0211] In certain embodiments, the fucosyltransferase is an alpha-1,4-fucosyltransferase (EC 2.4.1.65). In specific embodiments, the alpha-1,4-fucosyltransferase is Hp3 / 4FT from Helicobacter pylori or FUT2 from Homo sapiens.

[0212] In certain embodiments, the fucosyltransferase is an alpha-1,6-fucosyltransferase (EC 2.4.1.68). In specific embodiments, the alpha-1,6-fucosyltransferase is FUT8 from Homo sapiens.

[0213] In certain embodiments, the fucosyltransferase is selected from the fucosyltransferases shown in the accompanying figures.

[0214] Figure 12A and Fig. 12B Depicted is the N-acetylglucosaminylation pathway using lactose-N-biose (LNB) as a GlcNAc donor.

[0215] Figure 12A depicts the pathway for obtaining LNTII and tagatose from LNB and lactose. UDP-N-acetylglucosamine (UDP-GlcNAc) is prepared from UDP in the presence of NmLgtA and GsAI.

[0216] Fig. 12B A pathway for obtaining branched human milk oligosaccharides from LNB and LNnT in the presence of UDP, NmLgtA, GCNT2, and GsAI is depicted. β-2,6-GlcNAc-LNnT and tagatose are obtained. In the depicted pathway, GsAI is present to consume galactose. Any other enzymatic or non-enzymatic method can be used in place of GsAI to consume galactose.

[0217] In some embodiments, the N-acetylglucosamine transferase catalyzes the transfer of UDP-N-acetylglucosamine (UDP-GlcNAc) to galactose, mannose, or GlcNAc.

[0218] In certain embodiments, the N-acetylglucosaminyltransferase is a β-1,3-N-acetylglucosaminyltransferase (EC2.4.1.79, EC2.4.1.149, EC2.4.1.222). In specific embodiments, the β-1,3-N-acetylglucosaminyltransferase is HpLgtA from Helicobacter pylori, NmLgtA from Neisseria meningitidis, HP1105 from Helicobacter pylori, B3GNT2 from Homo sapiens, B3GNT3 from Homo sapiens, B3GNT4 from Homo sapiens, B3GNT7 from Homo sapiens, B3GNT8 from Homo sapiens, or B3GNT9 from Homo sapiens.

[0219] In certain embodiments, the N-acetylglucosaminyltransferase is an α-1,4-N-acetylglucosaminyltransferase (EC 2.4.1.223, EC 2.4.1.224).

[0220] In certain embodiments, the N-acetylglucosaminyltransferase is a β-1,2-N-acetylglucosaminyltransferase (EC 2.4.1.101, EC 2.4.1.143). In specific embodiments, the β-1,2-N-acetylglucosaminyltransferase is MGAT1 from Homo sapiens (GlcNAcT-I) or MGAT2 from Homo sapiens (GlcNAcT-II).

[0221] In certain embodiments, the N-acetylglucosaminyltransferase is a β-1,4-N-acetylglucosaminyltransferase (EC 2.4.1.144, EC 2.4.1.212). In specific embodiments, the β-1,4-N-acetylglucosaminyltransferase is MGAT3 from Homo sapiens (GlcNAcT-III), MGAT4A from Homo sapiens (GlcNAcT-IV), MGAT4B from Homo sapiens (GlcNAcT-IV), or MGAT4C from Homo sapiens (GlcNAcT-IV).

[0222] In certain embodiments, the N-acetylglucosaminyltransferase is a β-1,6-N-acetylglucosaminyltransferase (EC2.4.1.102, EC2.4.1.150, EC2.4.1.155). In specific embodiments, the β-1,6-N-acetylglucosaminyltransferase is GCNT2A from Homo sapiens, GCNT2B from Homo sapiens, GCNT2C from Homo sapiens, GCNT3 from Homo sapiens, GCNT4 from Homo sapiens, or MGAT5 from Homo sapiens (GlcNACT-V).

[0223] In certain embodiments, the N-acetylglucosaminyltransferase is selected from the N-acetylglucosaminyltransferases shown in the accompanying drawings.

[0224] In certain embodiments of the invention, the starting system of reactants and enzymes comprises, consists essentially of, or consists of those depicted in Figures 1-12.

[0225] The present invention uses sugar as an energy source. Therefore, activated sugar (sugar-nucleotide or sugar-nucleotide compound) is a starting material and an energy source. The energy source is the hydrolysis of more complex sugars to simpler sugars. This helps to drive the reaction of the present invention to completion. In one embodiment, sucrose is used for galactosylation, and the hydrolysis of lactose is used for sialylation and fucosylation. In another embodiment, lactose-N-disaccharide is used as a GlcNAc donor in the N-acetylglucosaminylation reaction. In another embodiment, the hydrolysis of 3'-SL is used in the sialylation reaction. In another embodiment, 2'-FL is used in the fucosylation reaction.

[0226] Energy is driven by the conversion of larger sugars into smaller sugars. For example, energy comes from the decomposition and hydrolysis of disaccharides into monosaccharides, such as the decomposition of sucrose into fructose and glucose. That is, sucrose and water are converted into fructose and glucose through hydrolysis.

[0227] The activated sugar is regenerated in the reaction mixture. This allows the nucleotide to be present in catalytic amounts. As shown in each figure, the sugar is transferred to the acceptor and then leads to the formation of free nucleotides. The nucleotide then reacts with the sugar to form the activated sugar. In Figure 1, the nucleotide is added to glucose to form UDP-glucose, in Figure 2, the nucleotide is added to N-acetylneuraminic acid to form CMP-Neu5Ac, and in Figure 3, the nucleotide is added to 2'-FL to form GDP-2'-FL.

[0228] Without being bound by theory, manganese (Mn) and high sucrose concentrations can lead to enzyme inactivation. In certain embodiments, the methods of the present invention employ magnesium (Mg) instead of manganese (Mn). For example, Mn catalyzes the decomposition of UDP into UMP, and UMP leads to the reduction inactivation of the cofactor NAD+ in GalE. In certain embodiments, the methods of the present invention employ magnesium (Mg) instead of manganese (Mn), and relatively low sucrose concentrations, such as 50-100 mM.

[0229] Manganese (Mn) and inorganic phosphate form insoluble byproducts, especially at higher industrially relevant concentrations, which makes them unsuitable for packed bed reactor applications and results in the removal of a key metal cofactor (Mn) from the solution phase, thereby reducing the activity of many glycosyltransferases. Embodiments of the present invention employ Mg, thus avoiding these disadvantages.

[0230] Sugar nucleotides with affinity for enzymes of the invention can be used in the methods disclosed herein. Enzymes useful in the invention include enzymes bioengineered for affinity. In certain embodiments, the activated sugar comprises, consists essentially of, or consists of galactose-UDP, galactose-ADP, sialic acid-CMP, and fucose-GDP. In certain embodiments, the activated sugar comprises, consists essentially of, or consists of galactose-UDP, sialic acid-CMP, and fucose-GDP.

[0231] In one embodiment, the present invention provides a biocatalytic method for preparing a sugar receptor, comprising the following steps:

[0232] a. contacting a sugar and a nucleotide to form a sugar-nucleotide, wherein the nucleotide is present in a catalytic amount; and

[0233] b. contacting the sugar-nucleotide and the acceptor in the presence of a transferase to form a sugar-acceptor.

[0234] In certain embodiments, the sugar is a monosaccharide. In certain embodiments, the monosaccharide is galactose, sialic acid, or l-fucose.

[0235] In another embodiment, the present invention provides a method for preparing a sugar compound, comprising the following steps:

[0236] a. contacting a monosaccharide selected from galactose, sialic acid and l-fucose with a catalytic amount of a nucleotide selected from UDP, ADP, CMP and GDP to form a sugar - nucleotide compound; and

[0237] b. contacting the sugar-nucleotide compound with an acceptor compound in the presence of a transferase to obtain the sugar.

[0238] In another embodiment, the present invention provides a method for preparing a sugar compound, comprising the following steps:

[0239] a. contacting a galactose sugar unit with a catalytic amount of a nucleotide selected from UDP and ADP or contacting a sialic acid or l-fucose sugar unit with a catalytic amount of a nucleotide selected from CMP and GDP to form a sugar-nucleotide compound.

[0240] b. contacting the sugar-nucleotide compound with an acceptor compound in the presence of a transferase to obtain the sugar.

[0241] In another embodiment, the present invention provides galactosylation of a receptor compound comprising the steps of:

[0242] a. contacting galactose with a nucleotide to form a galactose-nucleotide compound, wherein the nucleotide is present in a catalytic amount; and

[0243] b. contacting the galactose-nucleotide compound with the acceptor compound in the presence of a transferase to galactosylate the acceptor compound.

[0244] In certain embodiments, galactose is obtained in situ from sucrose.

[0245] In other embodiments, the method achieves high conversion rates by converting sucrose to Glc-UDP and fructose.

[0246] In another embodiment, the present invention provides a method for sialylation of a receptor compound comprising the steps of:

[0247] a. contacting sialic acid and a nucleotide to form a sialic acid-nucleotide compound, wherein the nucleotide is present in a catalytic amount; and

[0248] b. contacting the sialic acid-nucleotide compound and the compound with an acceptor compound in the presence of a transferase to sialylate the acceptor compound.

[0249] In certain embodiments, sialic acid is obtained in situ from 3'-SL or 6'-SL.

[0250] In other embodiments, the method achieves high conversion rates by converting lactose to galactose and glucose.

[0251] In another embodiment, the present invention provides a method for fucosylation of a receptor compound, comprising the steps of:

[0252] a. contacting fucose and a nucleotide to form a fucose-nucleotide compound, wherein the nucleotide is present in a catalytic amount; and

[0253] b. contacting the fucose-nucleotide compound and the acceptor compound in the presence of a transferase to fucosylate the acceptor compound.

[0254] In certain embodiments, the fucose is l-fucose and is obtained in situ from 2'-FL. In other embodiments, the method achieves high conversion rates by converting sucrose into Glc-UDP and fructose. In other embodiments of the invention, the acceptor compound is a monosaccharide, disaccharide, oligosaccharide or polysaccharide. In other embodiments of the invention, substantially no ATP is added to or produced by the method process.

[0255] In another embodiment, the invention provides a method for producing LNT and fructose, comprising contacting LNTII, sucrose, a transferase, a sucrose synthase, an epimerase, and UDP to produce LNT and fructose, wherein galactose-UDP is produced, and wherein UDP is present at about 0.01 mole percent to about 10 mole percent of galactose or sucrose. In certain embodiments, the transferase is Cvb3GalT. In other embodiments, the transferase is B3GALT5. In certain embodiments, the epimerase is GalE.

[0256] In another embodiment, the invention provides a method for producing LNnT and fructose, comprising contacting LNTII, sucrose, a glycosyltransferase, a sucrose synthase, an epimerase, and UDP to produce LNnT and fructose, wherein UDP is present at about 0.01 mole percent to about 10 mole percent of galactose or sucrose. In certain embodiments, the glycosyltransferase is LgtB. In other embodiments, LgtB is NmLgtB. In certain embodiments, the glycosyltransferase is B4GALT1. In certain embodiments, the glycosyltransferase is B4GALT1. In certain embodiments, the epimerase is GalE.

[0257] In another embodiment, the present invention provides a method for preparing LNT and fructose, comprising the steps of: contacting GlcNAc, 2-chloro-1,3-dimethylimidazolinium chloride, and a base to obtain a reaction mixture comprising GlcNAc-Oxa; and contacting a reaction mixture comprising GlcNAc-Oxa, lactose, sucrose, an amidase, a transferase, a sucrose synthase, and an epimerase to provide LNT and fructose, wherein UDP is present at about 0.01 mole percent to about 10 mole percent of lactose or sucrose.

[0258] In certain embodiments, the amidase is Bbhl. In certain embodiments, the transferase is Cvb3GalT. In certain embodiments, the epimerase is GalE. In certain embodiments, the base is triethylamine.

[0259] In another embodiment, the invention provides a method for preparing lactose and fructose, comprising contacting glucose, sucrose, lactose synthase, sucrose synthase, epimerase and UDP to provide lactose and fructose, wherein UDP is present in about 0.01 mole percent to about 10 mole percent of glucose and / or sucrose. In certain embodiments, the epimerase is GalE.

[0260] In another embodiment, the present invention provides a method for preparing LSTa, galactose and glucose, comprising contacting 3'-SL, LNT, CMP, sialyltransferase and lactase to provide LSTa, galactose and glucose, wherein CMP is present at about 0.01 mole percent to about 10 mole percent of 3'-SL. In certain embodiments, the sialyltransferase is ST3GAL3.

[0261] In another embodiment, the present invention provides a method for preparing LSTb, comprising contacting LNT, 3'-SL, and a catalytic amount of CMP in the presence of ST3GAL4, ST3GAL3, ST6GALNAC5, lactase, and α2-3 neuraminidase S, wherein the CMP is present at about 0.01 mole percent to about 10 mole percent of 3'-SL, wherein lactose is obtained and then processed into glucose and galactose by lactase, and wherein the α2-3 neuraminidase S converts DSLNT to LSTb.

[0262] In another embodiment, the present invention provides a method for preparing LSTc, galactose and glucose, comprising contacting 3'-SL, LNnT, CMP, a sialyltransferase and a lactase to provide LSTc, galactose and glucose, wherein CMP is present at about 0.01 mole percent to about 10 mole percent of 3'-SL. In certain embodiments, the sialyltransferase is ST3GAL3.

[0263] In another embodiment, the present invention provides a method for preparing LSTd, galactose and glucose, comprising contacting 3'-SL, LNnT, CMP, sialyltransferase and lactase to provide LSTd, galactose and glucose, wherein CMP is present at about 0.01 mole percent to about 10 mole percent of 3'-SL. In certain embodiments, the sialyltransferase is ST3GAL3.

[0264] In another embodiment, the present invention provides a method for preparing DSLNT, galactose and glucose, comprising contacting 3'-SL, LNT, CMP, sialyltransferase and lactase to provide DSLNT, galactose and glucose, wherein CMP is present at about 0.01 mole percent to about 10 mole percent of 3'-SL. In certain embodiments, the sialyltransferase is ST3GAL3 and ST6GALNAC5.

[0265] In another embodiment, the invention provides a method for preparing LNFPI, galactose and glucose, comprising contacting 2'-FL, LNT, GDP, Te2FT, HmFucT and lactase to provide LNFPI, galactose and glucose, wherein GDP is present at about 0.01 mole percent to about 10 mole percent of 2'-FL. In certain embodiments, the transferases are Te2FT and HmFucT.

[0266] In another embodiment, the invention provides a method for preparing LNFPII, galactose and glucose, comprising contacting 2'-FL, LNT, GDP, Hp34FT, HmFucT and lactase, wherein GDP is present at about 0.01 mole percent to about 10 mole percent of 2'-FL. In certain embodiments, the transferases are Hp34FT and HmFucT.

[0267] In another embodiment, the invention provides a method for preparing LNFPIII, galactose and glucose, comprising contacting 2'-FL, LNnT, GDP, FUT9, HmFucT and lactase to provide LNFPIII, wherein GDP is present at about 0.01 mole percent to about 10 mole percent of 2'-FL. In certain embodiments, the transferases are FUT and HmFucT.

[0268] In another embodiment, the present invention provides a method of preparing 3-FL, comprising contacting 2'-FL, GDP, FUT1, FUT3, lactase, GsAI, and fucosidase to provide 3-FL, tagatose, and glucose, wherein GDP is present at about 0.01 mole percent to about 10 mole percent of 2'-FL.

[0269] In another embodiment, the invention provides a method of preparing LNTII comprising contacting LNB, lactose, UDP, NmLgtA, and GsAI to provide 3-FL, tagatose, and glucose, wherein UDP is present at about 0.01 mole percent to about 10 mole percent of LNB.

[0270] In another embodiment, the invention provides a method of preparing LNTII comprising contacting LNB, lactose, UDP, NmLgtA, and GsAI to provide 3-FL, tagatose, and glucose, wherein UDP is present at about 0.01 mole percent to about 10 mole percent of LNB.

[0271] In another embodiment, the present invention provides a method for preparing β-1,6-GlcNAc-LNnT, comprising contacting LNB, LNnT, UDP, NmLgtA, GCNT2, and GsAI to provide β-1,6-GlcNAc-LNnT and tagatose, wherein UDP is present at about 0.01 mole percent to about 10 mole percent of LNB.

[0272] In certain embodiments, the sialic acid is N-acetylneuraminic acid (Neu5Ac).

[0273] In certain embodiments, sugar is its naturally occurring isomeric form. In some embodiments, glucose is D-glucose. In some embodiments, galactose is D-galactose. In some embodiments, fucose is L-fucose. In some embodiments, tagatose is D-tagatose. In some embodiments, N-acetylglucosamine (GlcNAc) is N-acetyl-D-glucosamine. In some embodiments, fructose is D-fructose.

[0274] In other embodiments, the nucleotide is present in a catalytic amount of about 0.01 to about 10 mole percent relative to the amount of sugar. The associated sugar is the starting sugar or the sugar that is generated and reacted with the nucleotide.

[0275] In some embodiments, the nucleotide monophosphates (or nucleotide monophosphates, both translated from "nucleotide monophosphate") and nucleotide diphosphates (or nucleotide diphosphates, both translated from "nucleotidediphosphate") used in the present invention are used in a catalytic amount relative to the molar concentration of the sugar bound to the monophosphate or diphosphate. In another embodiment, the nucleotide monophosphates and diphosphates used in the present invention are used in a catalytic amount relative to the molar concentration of the starting material sugar or other acceptor. These nucleotides include natural, artificial or synthetic nucleotides. As used herein, the "catalytic amount" of the nucleotide range is precisely, approximately, at most or less than, for example, 0.01% to 10% of the molar number of nucleotides to the molar number of sugars (sugar donors or acceptor molecules). As used herein, the "catalytic amount" of the nucleotide range is precisely, approximately, at most or less than, for example, 0.001% to 10% of the molar number of nucleotides to the molar number of sugar donors or acceptor molecules. In other embodiments, nucleotide monophosphates or nucleotide diphosphates are precisely, approximately, at most or less than, for example, 0.001% to 1% (molar percentage) amount. In certain embodiments, nucleotide monophosphates and nucleotide diphosphates are precisely, approximately, at most or less than, for example, 0.01% to 10% (molar percentage). In other embodiments, nucleotide monophosphates or nucleotide diphosphates are precisely, approximately, at most or less than, for example, 0.1% to 1% (molar percentage). In other embodiments, the addition amount of nucleotide monophosphates and nucleotide diphosphates is precisely, approximately, at most or less than, for example, 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08% or 0.09% (molar percentage). In other embodiments, the amount of nucleotide monophosphates and nucleotide diphosphates added is exactly, about, at most, or less than, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0% (molar percentage). In other embodiments, the amount of nucleotide monophosphates and nucleotide diphosphates added is exactly, about, at most, or less than, for example, the amount of monophosphates and diphosphates added is 0.01%, 0.1%, 0.3%, 1.0%, or 10% (molar percentage). In other embodiments, the amount of nucleotide monophosphates and nucleotide diphosphates added is exactly, about, at most, or less than, for example, the amount of monophosphates and diphosphates added is 0.1%, 0.3%, or 1.0% (molar percentage).In other embodiments, nucleotide monophosphates and nucleotide diphosphates are added in an amount of exactly or about 0.001%, 0.01%, 0.1%, 1.0%, or 10%. In other embodiments, monophosphates and diphosphates are added in an amount of exactly or about 0.1%, 1.0%, or 10%. In other embodiments, monophosphates and diphosphates are added in an amount of exactly or about 0.1%, 0.3%, or 1.0% (molar percentage). Figures 1-14 depict embodiments employing catalytic nucleotides.

[0276] In some embodiments, plant sugars are used to produce glycosylated compounds, including but not limited to galactosylated compounds, sialylated compounds, and fucosylated compounds, wherein galactose, sialic acid, and fucose are added to hydroxyl groups, including but not limited to hydroxyl groups of sugars. In another embodiment, lactose-N-disaccharide is used as a GlcNAc donor and is added to the hydroxyl groups of sugars in an N-acetylglucosaminylation reaction. Thus, plant sugars or simple HMOs produced by microbial fermentation or from animal sources are used to produce more complex sugars, including but not limited to oligosaccharides found in animals. In certain embodiments, compounds include but are not limited to LNT, LNnT, lactose, GOS, LSTa, LSTc, LSTd, DSLNT, LNFPI, LNFPII, LNFPIII. In other embodiments, compounds include but are not limited to LSTb and 3-FL. In other embodiments, plant sugars are used to synthesize lactose derivatives by a chemical enzymatic process to obtain lactose derivatives, including but not limited to lactobionic acid, lactitol, oligosaccharides, galacto-oligosaccharides (GOS), lactulose, and HMO (see Figure 5 ). MG Ganzle et al., International Dairy Journal, 18 (2008) 685-694.

[0277] like Figure 5 As shown, lactobionic acid is obtained from lactose by oxidation. Lactitol is obtained from lactose by reduction. Lactofructo-oligosaccharides are obtained from lactose by transfructosylation. Galacto-oligosaccharides (β-(1,4) or (β-(1,6) linkages) are obtained from lactose by transgalactosylation. Lactulose is obtained from lactose by isomerization.

[0278] In certain embodiments, the products obtained from the methods of the present invention are not of animal origin. Such products are advantageous in markets that require animal-free products.

[0279] This method eliminates the need for ATP and cofactor recovery. In certain embodiments, the activated sugar is activated galactose, activated Neu5Ac, activated fucose or activated glucose. In another embodiment, the activated sugar is N-acetylglucosamine (GlcNAc). These activated sugars are generated in situ. The reaction is designed to be free of nucleotide triphosphates, and reagents and cofactors are avoided to be recovered or regenerated. This reduces the number of enzymes required for producing each oligosaccharide and avoids acidification. This reduces costs and results in higher yields and higher purity products.

[0280] In certain embodiments, more than one sugar secondary product is produced in the reaction mixture. In certain embodiments, both glucose and galactose are produced as secondary products in a single reaction mixture.

[0281] In certain embodiments, use processing enzyme that secondary product is converted into secondary product derivative.In certain embodiments, this conversion helps promote reaction to complete and form primary product.In other embodiments, this conversion can be customized to obtain more desirable secondary product derivative for recovery and reuse.

[0282] In certain embodiments, a processing enzyme is used to convert the main product into a main product derivative. In certain embodiments, the conversion is beneficial to obtain a more desirable main product derivative for recovery and reuse.

[0283] In certain embodiments, more than one processing step can be used to further transform the main product derivative or secondary product derivative. The number of processing steps used depends on driving the reaction to completion and obtaining the desired final product. Some galactosylation methods of the present invention do not use or only use one main product or secondary product processing. In certain embodiments, the galactosylation method of the present invention does not use a processing step. Some sialylation, fucosylation and N-acetylglucosaminylation methods do not use, only use one or two main products or secondary product processing. In certain sialylation, fucosylation and N-acetylglucosaminylation embodiments, a processing step converts the main product into a main product derivative or converts a secondary product into a secondary product derivative.

[0284] Some embodiments of the invention utilize processing enzymes that hydrolyze, oxidize, or isomerize carbohydrates or oligosaccharides.

[0285] In certain embodiments, the processing enzyme is an enzyme that hydrolyzes lactose. In a specific embodiment, the enzyme that hydrolyzes lactose is lactase (β-galactosidase) (EC 3.2.1.23). In a more specific embodiment, the lactase is β-galactosidase from Aspergillus niger, β-galactosidase from Escherichia coli, β-galactosidase from Aspergillus oryzae, or β-galactosidase from Kluyveromyces lactis.

[0286] In certain embodiments, the processing enzyme is an enzyme that oxidizes lactose carbohydrates. In a specific embodiment, the enzyme that oxidizes lactose carbohydrates is glucose oxidase (EC1.1.3.4). In a more specific embodiment, the glucose oxidase is glucose oxidase from Aspergillus niger, glucose oxidase from Penicillium amagasakiense, glucose oxidase from Penicillium notatum (chrysogenum), glucose oxidase from Penicillium variabile, galactose oxidase (EC1.1.3.9), galactose oxidase from Dactylium dendroides, galactose oxidase from Polyporus circinatus, galactose oxidase from Fusarium graminearum, galactose oxidase from Drosophila melanogaster.

[0287] In a specific embodiment, the enzyme that oxidizes lactose carbohydrates is an enzyme that oxidizes lactose to lactobionic acid. In a more specific embodiment, the enzyme that oxidizes lactose carbohydrates is a carbohydrate oxidase from Microdochium nivale, a lactose oxidase from Myrmecridium flexuosum, a lactose oxidase from Sarocaldium oryzae, a lactose oxidase from a Paraconiothyrium species, a galactose oxidase (EC 1.1.3.9) from Polyporus annularis, a cellobiose dehydrogenase (EC 1.1.99.18) from Sclerotium rolfsii, a cellobiose quinone oxidoreductase (EC 1.1.5.1) from Phanerochaete chrysosporium, a cellobiose oxidase (EC 1.1.5.2) from Potoyrichum pulverolentum, the cellobioquinone oxidoreductase (EC 1.1.5.1), the carbohydrate oxidoreductase from Sarocladium oryzae, the carbohydrate oxidoreductase from Paraconiothyrium species, or the glucopyrano-oligosaccharide oxidase GOOX from Acremonium strictum.

[0288] In certain embodiments, the processing enzyme is an enzyme that isomerizes carbohydrates. In a specific embodiment, the enzyme that isomerizes carbohydrates is a glucose isomerase (xylose isomerase) (EC5.3.1.5). In a more specific embodiment, the glucose isomerase is a glucose isomerase from Streptomyces murinus, a glucose isomerase from Actinoplanes missouriensis, a glucose isomerase from Bacillus coagulans, a glucose isomerase from Arthrobacter, or a glucose isomerase from Xanthomonas campestris.

[0289] In a specific embodiment, the enzyme that isomerizes carbohydrates is an L-arabinose isomerase (galactose isomerase) (EC 5.3.1.4). In a more specific embodiment, the L-arabinose isomerase is an L-arabinose isomerase from Geobacillus stearothermophilus, an L-arabinose isomerase from Lactobacillus sakei, an L-arabinose isomerase from Bacillus subtilis, an L-arabinose isomerase from Escherichia coli, or an L-arabinose isomerase from Thermoanaerobacter mathranii.

[0290] In some embodiments, the processing enzyme is an enzyme that hydrolyzes carbohydrates. In certain embodiments, the enzyme that hydrolyzes carbohydrates is a fucosidase. A fucosidase is an enzyme that catalyzes the hydrolysis of fucose from a glycan, a carbohydrate, or a glycan-containing moiety. In specific embodiments, the fucosidase is an α-1,2-fucosidase (EC 3.2.1.63), an α-1,3-fucosidase (EC 3.2.1.111), or an α-1,4-fucosidase (EC unknown).

[0291] In certain embodiments, the enzyme that hydrolyzes carbohydrates is a sialidase (neuraminidase). Sialidase is an enzyme that catalyzes the hydrolysis of sialic acid from a glycan, a carbohydrate, or a glycan-containing moiety. In a specific embodiment, the sialidase is an xo-a-sialidase. In a more specific embodiment, the exo-a-sialidase is α2-3 neuraminidase S, α2-3,6,8,9 neuraminidase A, α2-3,6,8 neuraminidase.

[0292] In a specific embodiment, the sialidase is Endo-α-sialidase. In a more specific embodiment, the sialidase is α2,8 neuraminidase.

[0293] In certain embodiments, the processing enzyme is selected from the fucosyltransferases shown in the accompanying figures.

[0294] In certain embodiments of the galactosylation reaction exemplified herein, fructose is a secondary product produced that can be recovered or processed for further use. In certain embodiments of the sialylation and fucosylation reactions exemplified herein, galactose and glucose are secondary products produced that can be recovered or processed for further use. In certain embodiments of the acetylglucosaminylation reaction exemplified herein, lactose or galactose are secondary products produced that can be recovered or processed (e.g., processed into tagatose) for further use.

[0295] Nucleotides may also be recovered. In the embodiments exemplified herein, UDP or ADP may be recovered in a galactosylation reaction, CMP may be recovered in a sialylation reaction, and GDP may be recovered in a fucosylation reaction.

[0296] In certain embodiments, polysaccharides are obtained in the method of the present invention. Polysaccharides are carbohydrate-based compounds characterized in that one or more monosaccharides are connected by glycosidic bonds (including N-linked and O-linked bonds). Activated monosaccharides, oligosaccharides, polysaccharides, plant polysaccharides, animal polysaccharides and microbial polysaccharides are all within the scope of the present invention, and glycoconjugates, such as glycolipids, glycopeptides, glycoproteins and proteoglycans are also within the scope of the present invention. Polysaccharides also include humanized glycoproteins, humanized antibodies and glycoconjugate vaccines. Riley et al., "Glycosylation in health and disease" Nature Reviews Nephrology volume 15, pages 346-366 (2019). Rappuoli, "Glycoconjugate vaccines: Principles and mechanisms" Science Translational Medicine 29Aug 2018: Vol.10, Issue 456. The above document is incorporated by reference in its entirety.

[0297] According to the present invention, compounds that serve as receptors can be derivatized with glycosyl groups. Any organic compound containing at least one alcohol (hydroxyl) functional group can be a receptor and can therefore be glycosylated by the method of the present invention. Such compounds can include, but are not limited to, rare sugars, activated sugars, HMOs, polysaccharides with sugar modifications, glycosylated small molecules, polymerized fiber sugars, inulin, fructans, gluconic acid, invert sugars, flavors, and fragrances. Organic compounds with at least one alcohol functional group (organic hydroxyl functional group) that can be glycosylated according to the present invention include, but are not limited to, steviol, a class of compounds known as rebaudioside, phenolic compounds, proteins with O-glycosylation and N-glycosylation sites: serine, threonine, asparagine, and compounds related thereto.

[0298] Integrated fucosylation pathway

[0299] In another aspect, the present invention provides an integrated pathway for oligosaccharide synthesis using immobilized enzymes, including embodiments utilizing a one-pot synthesis of fucosylated oligosaccharides starting from fructose or glucose. This reduces the production cost of fucosylated HMOs compared to using fucose as a starting material. The polysaccharides to be fucosylated can be prepared separately for use in the one-pot synthesis of the present invention.

[0300] The present invention provides cell-free de novo synthesis of fucosylated glycans starting from simple sugars. The integrated pathway of immobilized enzymes performs one-pot fucosylation of oligosaccharides or antibody-glycan conjugates starting from fructose or glucose instead of fucose. The immobilized enzyme system comprises, consists essentially of, or consists of:

[0301] Enzymes that convert fructose and / or glucose to mannose

[0302] A group of enzymes that convert mannose to GDP-mannose

[0303] A group of enzymes that convert GDP-mannose to GDP-fucose

[0304] Enzymes that transfer fucose from GDP-fucose to the oligosaccharide backbone

[0305] A group of enzymes that recover cofactors from stoichiometric reagents

[0306] HMO 2'-FL can be produced from lactose. The last transferase in the enzyme cascade can be changed to fucosylate other oligosaccharide backbones. Switching to different fucosyltransferases constitutes the production of individual HMOs, also through a one-pot system. For example, one fucosyltransferase can be used to make 2'-FL, another fucosyltransferase can be used for LNFP1, another fucosyltransferase can be used for SM to make LNFPII, etc. Thus, modularity comes from changing the last enzyme of the system (i.e., the fucosyltransferase).

[0307] The method offers high volumetric production rates, is cost-effective, and is non-GMO labelled.

[0308] The immobilized enzyme can be coupled to a processing plant producing lactose, fructose or glucose feed for continuous flow manufacturing. The present invention can be integrated into a biorefinery to convert glucose or fructose into fucosylated HMOs.

[0309] The last transferase in the enzyme cascade can be altered to transfer GDP-fucose to other oligosaccharide backbones for fucosylation reactions.

[0310] The enzyme cascade can generate GDP-mannose, which can also be polymerized into mannan by transferases.

[0311] like Fig.15As shown, glucose and / or fructose are converted into GDP-L-fucose in an integrated synthesis. Enzyme E1 converts glucose into mannose. Alternatively, enzyme E1' converts fructose into mannose. Enzyme E2 then converts mannose into mannose-6-phosphate. Enzyme E3 converts mannose-6-phosphate into mannose-1-phosphate. Enzyme E4 converts mannose-1-phosphate into GDP-D-mannose. Enzyme E5 converts GDP-D-mannose into GDP-4-keto-6-deoxymannose. Enzyme E6 converts GDP-4-keto-6-deoxymannose into GDP-L-fucose. 2'FL is obtained from GDP-L-fucose, lactose and enzyme E7. All enzymes are immobilized.

[0312] Fig.15 Regeneration enzymes are also depicted. Enzymes E8, E9, E10, E11 and E12 regenerate reagents to allow integrated synthesis. The following reactions occur: GDP is converted to GTP (E10), inorganic diphosphate is converted to inorganic phosphate (E12), carbon dioxide and hydrogen peroxide are converted to oxygen and water (E11), ADP is converted to ATP (E10), acetate phosphate is converted to acetate (E10), inorganic phosphate and pyruvate are converted to acetate phosphate (E9), and formate is converted to carbon dioxide (E8).

[0313] In one embodiment, the enzymes used are Glk, RfbK, RfbM, Gmd and GFS, and the regeneration enzymes pyruvate-AcK, PyrOx and FDH. The enzyme converts glucose and / or fructose into mannose, which is then converted into mannose-6-phosphate, which is then converted into mannose-1-phosphate, which is then converted into GDP-D-mannose, which is then converted into GDP-4-keto-6-deoxymannose, and which is then converted into GDP-L-fucose. Further reactions involve the fucosylation of any sugar residue, including but not limited to oligosaccharides or fucosylated antibody-glycan conjugates. In the presence of fucosyltransferase and GDP-L-fucose, lactose is converted into 2'FL by fucosyltransferase. Similarly, oligosaccharides are converted into fucosylated oligosaccharides or antibody-glycan conjugates are converted into fucosylated antibody-glycan conjugates. The described fucosyltransferases of the enzyme cascade can be modified to transfer GDP-fucose to other oligosaccharide backbones for fucosylation reactions to provide fucosylated products.

[0314] For example, the enzyme AcK transfers phosphate from AcPi to GDP to generate GTP and the byproduct Ac (=acetate).PyrOx will use Pi from the PtxD step (in the example using phosphite) and the PmPpa step.

[0315] Lactose, fructose, pyruvic acid and phosphite are the main reactants in the integrated synthesis. Buffers including but not limited to Tris can be used. In some embodiments, the buffer maintains the pH between about pH 6.5 and about pH 8.5 or about pH 7.5.

[0316] Fig.16 Depicts different regeneration systems Fig.15 Here, the regeneration enzymes are E8', E9, E10, E11 and E12. Here, E8' converts phosphite into inorganic phosphate and NADP+ into NADPH. The enzymes that can be used are pyruvate-AcK, PyrOx and PtxD.

[0317] Fig.17 Depicts different regeneration systems using E8, E9' and E12 Fig.15 Here, the regeneration enzymes are PolyP-PPK and FDH.

[0318] Fig.18 Depicts different regeneration systems Fig.15 Here, the regeneration enzymes are E8', E9' and E12. E9' is involved in converting ADP into ATP and GDP into GTP, while E8' converts phosphite into Pi. E12 converts PPi into Pi and NADP+ into NADPH. The enzymes that can be used include PolyP-PPK and PtxD.

[0319] exist Figure 15-18 A feed solution is also used in the reactions shown.

[0320] For example, the feed solution contains lactose, fructose, formate, MgCl2, Tris, pyruvate, NADP+, GDP, and ATP ( Figure 15-18 ).

[0321] For example, the feed solution contains lactose, fructose, phosphite, MgCl2, a buffer (e.g., Tris buffer), GDP, NADP+, pyruvate, and ATP ( Fig.16 ).

[0322] Alternatively, the feed solution comprises lactose, fructose, formate, MgCl2, Tris, polyphosphate, a buffer (e.g., Tris buffer), GDP, NADP+, and ATP ( Fig.17 ).

[0323] Alternatively, the feed solution comprises lactose, fructose, phosphite, MgCl2, a buffer (e.g., Tris buffer), GDP, NADP+, polyphosphate, and ATP ( Fig.18 ).

[0324] Glucose or fructose can be interchanged, but the first enzyme must match the starting sugar. Thus, glucose and fructose can be interchanged, adding the appropriate enzyme E1 or E1', respectively.

[0325] In certain embodiments, compounds that can be fucosylated according to the methods of the present invention include, but are not limited to, rare sugars, activated sugars, HMOs, and glycans.

[0326] Without being bound by theory, manganese (Mn) combined with high sucrose concentrations leads to enzyme inactivation. In certain embodiments, the methods of the present invention employ magnesium (Mg) instead of manganese (Mn) and do not include UMP. Under these conditions, high sucrose does not lead to reductive inactivation of GalE.

[0327] In certain embodiments, the sugar is of plant origin. Thus, any embodiment of the present invention can be used to obtain compounds from non-animal based plant materials. In certain embodiments, the product obtained from the method of the present invention is not of animal origin. Such products are advantageous in markets that require animal-free products.

[0328] The polysaccharides synthesized as described herein can be simple or complex polysaccharides, which can be linear or branched. In certain embodiments, the polysaccharides to be fucosylated can be simple polysaccharides or complex polysaccharides, including linear or branched. Including polysaccharides with one or more sugar units. In certain embodiments, the polysaccharides have two, three, four or five sugar units or more units. In some embodiments, the polysaccharides have 1-18 units. In some embodiments, the polysaccharides have 1-10 units. In some embodiments, the polysaccharides have 1-5 units. In certain embodiments, the polysaccharides have five or more sugar units. In certain embodiments, the polysaccharides have 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 units. In certain embodiments, the polysaccharides have 1 unit, 2 units, 3 units, 4 units, 5 units or 6 units. In some embodiments, the polysaccharides are oligosaccharides. In some embodiments, the polysaccharides are straight or branched. In certain embodiments, the polysaccharides have 1-6 units and are straight. In other embodiments, the polysaccharide has 1-6 units and is branched. In certain embodiments, the polysaccharide has 1-5 units and is linear. In other embodiments, the polysaccharide has 1-5 units and is branched. Figure 1F In some embodiments, n is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0329] The compounds obtained according to the present invention (including but not limited to galactosylated, sialylated, fucosylated and N-acetylglucosaminated compounds) can be used as components for the synthesis of any polysaccharide-containing compound. In certain embodiments, the compounds obtained according to the present invention include but are not limited to galactosylated compounds. Materials functionalized with enzymes or enzyme systems can be used to produce drugs, biological products, nutritional products, cosmeceuticals and food ingredients. In certain embodiments, sugars and oligosaccharides are of non-animal origin.

[0330] In one embodiment, the fucosylated compounds obtained according to the present invention can be used as components in the synthesis of any polysaccharide-containing compound. With the ability to immobilize any enzyme for any process, materials functionalized with enzymes or enzyme systems can be used to produce pharmaceuticals, biologics, active nutraceuticals, active cosmeceuticals, and food ingredients.

[0331] In certain embodiments, the method provides a fucosylated oligosaccharide or a fucosylated antibody-glycan conjugate.In a preferred embodiment, fucosylated human milk oligosaccharides (HMOs) are produced.

[0332] In certain embodiments, the method provides a galactosylated oligosaccharide or a galactosylated antibody-glycan conjugate. In a preferred embodiment, galactosylated human milk oligosaccharides (HMOs) are produced.

[0333] In certain embodiments, the glycan to be reacted (e.g., galactosylated, sialylated, or fucosylated) is 3'SL, LNTII, LNT, LNnT, LNFPI, LNFPII, LNFPIII, LSTa, LSTb, LSTc, LSTd, and DSLNT, LNnH, 3"'3,3"'6-bis-O-α-Sia-LNnH 3"'3,3"'6-bis-O-α-Sia-(3"3,3"6-bis-O-α-Fuc)-LNnH, dilinear sialylated or fucosylated lactose-N-neohexaose and neoheptasaccharides, α-2,3-sialyllactose-N-neopentose, linear fucosyl- and sialyl-lactose-N-neopentaose, linear lactose-N-neopentaose, or dilinear lactose-N-neohexaose and heptasaccharides.

[0334] In certain embodiments, the glycan to be fucosylated is 3'SL, LNTII, LNT, LNnT, LNFPI, LNFPII, LNFPIII, LSTa, LSTb, LSTc, LSTd and DSLNT, LNnH, 3"'3,3"'6-bis-O-α-Sia-LNnH 3"'3,3"'6-bis-O-α-Sia-(3"3,3"6-bis-O-α-Fuc)-LNnH, dilinear sialylated or fucosylated lactose-N-neohexaose and neoheptasaccharides, α-2,3-sialyllactose-N-neopentose, linear fucosyl- and sialyl-lactose-N-neopentose, linear lactose-N-neopentose, or dilinear lactose-N-neohexaose and heptasaccharides. In certain embodiments, the glycan is 3'SL LNTII, LNT, LNnT, LNFPI, LNFPII, LNFPIII, LSTa, LSTb, LSTc, LSTd, DSLNT, 2'-FL, LNnH, DSLNnH or DSDFLNNH. In other embodiments, the glycan is 3'SL, LNTII, 2'-FL, LNFPI, LSTa.

[0335] In certain embodiments, the polysaccharide to be reacted (e.g., galactosylated) is 2'-FL, 3'SL, LNTII, LNT, LNnT, LNFPI, LNFPII, LNFPIII, LSTa, LSTb, LSTc, LSTd, DSLNT, LNnH, 3"'3,3"'6-bis-O-α-Sia-LNnH3"'3,3"'6-bis-O-α-Sia-(3"3,3"6-bis-O-α-Fuc)-LNnH, dilinear sialylated or fucosylated lactose-N-neohexaose and neoheptasaccharides, α-2,3-sialyllactose-N-neopentose, linear fucosyl- and sialyl-lactose-N-neopentose, linear lactose-N-neopentose, or dilinear lactose-N-neohexaose and heptasaccharides.

[0336] The enzyme used herein can be a natural or synthetic bioengineered enzyme, including a fusion enzyme. For example, the enzyme can be engineered to have affinity for an immobilized support (tag). The enzyme can also be engineered to improve kinetic properties (e.g., reduce Km).

[0337] In certain embodiments, the enzyme is immobilized. Enzyme immobilization can be performed by covalent fixation, embedding, adsorption, molecular labeling with affinity tags, protein affinity tags, non-covalent adsorption, non-covalent deposition, embedding, physical embedding, bioconjugation, chelation, cross-linking or disulfide bonds. See, for example, Basso & Serban Mol. Catal. 479 (2019) 110607; Xu et al., Frontiers in Bioeng & Biotech., published on June 30, 2020, publdoi: 10.3389 / fbioe.2020.00660; and Sheldon & van Pelt Chem. Soc. Rev., 2013, 42, 6223. The foregoing is incorporated herein by reference in its entirety.

[0338] In embodiments using immobilized enzymes, such enzymes include, but are not limited to, enzymes immobilized within a bionanocatalyst (BNC), which in turn is embedded within a scaffold. The bionanocatalyst (BNC) according to the present invention includes an enzyme self-assembled with magnetic nanoparticles (MNPs). The BNC self-assembles with the scaffold.

[0339] In some embodiments, the immobilized enzyme is non-magnetic. In certain embodiments, such as for oligosaccharides used in food ingredients, the immobilized enzyme does not comprise nanoparticles.

[0340] In other embodiments, immobilized enzymes involve permanent molecular encapsulation of enzymes in self-assembled nanoparticle (NP) clusters. Self-assembly is driven entirely by the electrostatic and magnetic interactions of the material. Ionic strength, buffer pH, and NP concentration are the main parameters that affect immobilization yield and optimized enzyme activity. The clusters are then magnetically templated onto a magnetic support or a formable magnetic support.

[0341] In these embodiments, the immobilized bio-nanocatalyst is a magnetic material with one or more enzymes that are immobilized and associated with a scaffold. In some embodiments, the scaffold is a composite mixture of a highly magnetic and highly porous metal oxide or a thermoplastic or thermosetting plastic including magnetic particles formed into a powder. In some embodiments, selective laser sintering (SLS) is used to design and produce objects by sintering composite magnetic powders 3D printing.

[0342] Certain immobilized enzymes, MNPs, macroporous powders, scaffolds, their structures, organizations, suitable enzymes and uses are described in WO2012 / 122437, WO2014 / 055853, WO2016 / 186879, WO2017 / 011292, WO2017 / 180383, WO2018 / 34877, WO2018 / 102319, WO2020 / 051159, WO2020 / 69227 and WO2022 / 119982 (U.S. Application Nos. 63 / 120,669), 63 / 285,082 and 63 / 430,271 and 63 / 448,218. The foregoing is incorporated herein by reference in its entirety.

[0343] The bionanocatalyst (BNC) includes an enzyme self-assembled with magnetic nanoparticles (MNPs). The self-assembled mesoporous aggregates of the magnetic nanoparticles include a glycan synthase, wherein the mesoporous aggregates are immobilized on a magnetic macroporous support. In one embodiment, the immobilized enzyme includes (i.) a glycan synthase self-assembled in magnetic nanoparticles, and (ii.) a magnetic support. The glycanase is immobilized on the nanoparticles, wherein the nanoparticles coat the support, and the enzyme is immobilized in or on the mesoporous structure formed by the nanoparticles.

[0344] In one embodiment, the nanoparticles include magnetite (Fe3O4) or hematite (Fe2O3). In another embodiment, the nanoparticles include products synthesized from FeCl2 and FeCl3, in particular products synthesized by continuous coprecipitation of FeCl2*4H2O and FeCl3.FeCl2*4H2O (iron (II) chloride tetrahydrate) and FeCl3*6H2O (iron (III) chloride hexahydrate). Therefore, in one embodiment, the nanoparticles include magnetite (Fe3O4).

[0345] Polysaccharide synthase is any enzyme that can be used for polysaccharide synthesis. Steps in polysaccharide synthesis can include activation sugar, transfer sugar units to extend sugar, cofactor recycling and balance shift. Polysaccharide synthase includes but is not limited to sugar activating enzyme, sugar elongating enzyme, reagent regeneration enzyme and sugar functionalization enzyme. In another embodiment, polysaccharide synthase includes but is not limited to sugar activating enzyme, sugar elongating enzyme, reagent regeneration enzyme, sugar functionalization enzyme, sugar support enzyme, sugar removal enzyme.

[0346] Some immobilized enzyme materials, particularly magnetic materials, for producing glycans use one or more enzymes immobilized in a bionanocatalyst (BNC), which in turn is embedded in a macroporous scaffold to provide a scaffolded bionanocatalyst (scaffolded BNC). The scaffolded BNC can be located within a modular flow cell for flow manufacturing. The modular flow cells can be mixed and matched for highly customizable and efficient manufacturing processes. The scaffolded BNC is used for reactions that synthesize glycans by the following steps: contacting a glycan subunit or substrate with the scaffolded BNC to produce a second glycan, contacting a first glycan subunit with a second glycan subunit to produce a glycan comprising a first and a second glycan subunit, or contacting a first glycan with the scaffolded BNC to produce a second glycan. This includes processes for modifying glycan subunits and linking glycans.

[0347] Magnetic enzyme immobilization involves embedding the enzyme in mesoporous magnetic clusters that self-assemble around the enzyme (level 1). The immobilization efficiency depends on many factors, including the initial concentrations of enzyme and nanoparticles, the properties of the enzyme surface, the electrostatic potential of the enzyme, the properties of the nanoparticle surface, and the contact time. Enzymes for industrial use in biocatalytic processes should be efficient, stable before and during the process, reusable in multiple biocatalytic cycles, and economical.

[0348] Mesoporous aggregates of magnetic nanoparticles can be incorporated into continuous or granular macroporous scaffolds (Level 2). The scaffold may or may not be magnetic. Such scaffolds are discussed in, for example, WO2014 / 055853, WO2017 / 180383, and Corgie et al., Chem. Today 34(5): 15-20 (2016), which are incorporated herein by reference in their entirety. Highly magnetic scaffolds are intended to immobilize, stabilize, and optimize any enzyme. This includes holoenzyme systems, with high loading and full activity, as well as for the production of, for example, small molecules.

[0349] Immobilized enzymes can be used in the method of the present invention (WO2022 / 119982). For example, immobilized enzymes can be prepared as follows. In the first step, strontium ferrite (SFE) scaffold material is coated with magnetic nanoparticles (MNP) by lowering the pH from 10.0 to 7.5. In the second step, polysaccharide synthase is added to the product of the first step to support BNC. A B-type scaffold BNC composition is made by this method.

[0350] Certain immobilized enzymes are prepared when a polysaccharide synthase is contacted with magnetic nanoparticles to form a bionanocatalyst ("BNC"), and then the BNC is contacted with a magnetic scaffold material. Type A scaffold BNC compositions are made by this method. In certain embodiments, the magnetic scaffold material is strontium ferrite. In one embodiment, the strontium ferrite is a spherical particle having a tight size distribution with an average particle size of 20 μm (S20) or 40 μm (S40W; wrinkled). Strontium ferrite according to the present invention is available upon request from Powdertech International.

[0351] Without being bound by theory, by combining the enzyme and the nanoparticles and then adding the combination to the scaffold, the enzyme is trapped (embedded) within the MNPs (Type A). By adding the enzyme to the nanoparticle-coated scaffold, a Type B composition is obtained, in which the enzyme is more exposed to the surface and not buried too much. As used herein, scaffold-MNP complex, scaffold-MNP matrix, and scaffold-MNP material each refer to a combination of a scaffold and MNPs according to the present invention, comprising, consisting essentially of, or consisting of magnetite nanoparticles and a strontium ferrite matrix.

[0352] In certain embodiments, the present invention employs enzymes immobilized using iron oxide materials, including but not limited to hematite, magnetite, and strontium ferrite. In certain embodiments, the immobilized enzyme is a Type A scaffold BNC or a Type B scaffold BNC.

[0353] Thus, the present invention provides a polysaccharide synthase scaffold BNC, which is made by contacting strontium ferrite with magnetite nanoparticles to form a scaffold-MNP complex and adding a polysaccharide synthase to the scaffold-MNP complex to form a scaffold BNC. Another embodiment provides a polysaccharide synthase scaffold BNC, which is made by combining magnetite nanoparticles and polysaccharide synthase to form a BNC and then contacting the BNC with a scaffold or matrix to form a scaffold BNC. Without being bound by theory, the bionanocatalyst is coated with a magnetic microporous scaffold material.

[0354] In one embodiment of the present invention, the scaffold BNC composition comprises magnetic nanoparticles and self-assembled mesoporous aggregates of polysaccharide synthase and magnetic microporous materials. The scaffold BNC according to the present invention comprises polysaccharide synthase immobilized on magnetic nanoparticles, wherein the magnetic nanoparticles are coated with magnetic macroporous materials. In certain embodiments, the scaffold BNC comprises a magnetic macroporous matrix material, which comprises self-assembled mesoporous aggregates of magnetic nanoparticles that magnetically capture immobilized polysaccharide synthase. In certain embodiments, the scaffold BNC consists of or is substantially composed of a magnetic macroporous matrix material, which comprises self-assembled mesoporous aggregates of magnetic nanoparticles that magnetically capture immobilized polysaccharide synthase. In any embodiment, the scaffold BNC comprises any basic enzyme module described herein or is located in a system module.

[0355] The scaffold BNC composition includes magnetic nanoparticles and self-assembled mesoporous aggregates of glycan synthase and magnetic microporous materials. The scaffold BNC that can be used in the method of the present invention includes glycan synthase immobilized on magnetic nanoparticles, wherein the magnetic nanoparticles are coated with magnetic macroporous materials. In certain embodiments, the scaffold BNC is composed of a magnetic macroporous matrix material, which includes self-assembled mesoporous aggregates of magnetic nanoparticles that magnetically capture immobilized glycan synthase. In certain embodiments, the scaffold BNC is used in the integrated approach disclosed herein.

[0356] The present invention provides the ability to synthesize in a one-pot reaction. One-pot synthesis can be performed in batches or in a flow, including but not limited to repeated batches or continuous flows. In a flow reactor (e.g., a packed bed reactor), a reagent mixture is passed through in a flowing manner. Therefore, in one embodiment, the present invention provides commercially applicable flow biocatalysis.

[0357] Immobilized enzymes provide a range of highly tunable materials and processes for universal enzyme immobilization based on magnetic metamaterials. The unique enzyme hierarchical immobilization platform provides optimal conditions for immobilizing individual and complete enzyme systems and allows finding and adjusting the optimal conditions for individual and complete enzyme systems. It provides enzyme stability, maximizes substrate (including cofactors) utilization and imparts modularity to flow processes. Therefore, certain methods of the present invention use a stabilized enzyme composition comprising a bionanocatalyst and a magnetic scaffold, wherein the bionanocatalyst includes a glycan synthase and magnetic nanoparticles, and the magnetic scaffold stabilizes the bionanocatalyst.

[0358] One embodiment provides a modular method for producing glycans, comprising a module which can be a flow cell, wherein: the module comprises a magnetic macroporous powder, the magnetic macroporous powder comprising magnetic microparticles, wherein the powder has immobilized thereon a preparation of self-assembled mesoporous aggregates of magnetic nanoparticles comprising a glycan synthase; wherein a substrate is introduced into the module (or passed through the flow cell) and the substrate is modified to provide glycans.

[0359] Continuous flow reactor includes but is not limited to packed bed and fixed bed reactor of tubular form, and it can be combined with discontinuous upstream and downstream process, so that whole process is semi-continuous.For example, the reaction feed of LNTII reaction (embodiment 3c-A) can be produced in continuous stirred tank reactor, and its product is continuously added in flow reactor.Microfluidic reactor also can be used in combination with the present invention.

[0360] Certain embodiments relate to methods comprising one or more modules. In methods comprising more than a first module, a first substrate is passed through a first modular flow cell to produce a modified substrate; wherein the modified substrate is a second substrate, which is passed through a second module to produce a second modified substrate. The present invention provides sets of modules to be combined, thereby allowing the synthesis of complex glycans.

[0361] In one embodiment, the present invention provides a method for producing glycans by immobilizing an enzyme with magnetic nanoparticles and contacting the immobilized enzyme with an appropriate synthesis reagent. The method can be performed in batch, flow, semi-continuous or continuous flow.

[0362] The scaffold according to the present invention is chemically inert, structurally adjustable to suit any process, and has high magnetic properties to ensure complete capture of the enzyme-containing clusters. In one embodiment, the magnetic macroporous material comprises a metal oxide or a metal oxide complex. In one embodiment, the scaffold comprises a metal oxide. In one embodiment, the metal oxide is strontium ferrite (SrFe 12 O 19 ). In certain embodiments, the magnetic macroporous material is a metal oxide and consists essentially of or consists of a metallic material or a ceramic and does not include a polymer. In certain embodiments, the scaffold is a metal oxide, rather than a nanoparticle.

[0363] The present invention also provides a method for preparing a scaffolded bionanocatalyst by combining magnetic nanoparticles and polysaccharide synthase to form a bionanocatalyst, and then contacting the bionanocatalyst with a scaffold to obtain a scaffolded bionanocatalyst, and a method for preparing a scaffolded bionanocatalyst by combining a scaffold and magnetic nanoparticles to form a scaffolded magnetic nanoparticle complex, and then contacting the scaffolded magnetic nanoparticle complex with polysaccharide synthase. A scaffolded BNC made by one of these methods is also provided.

[0364] The method for preparing the magnetic scaffold is flexible because the method used in the present invention is a convenient and flexible glycosylation reaction.

[0365] In one embodiment, the method for preparing the magnetic scaffold is flexible because the method used in the present invention is a convenient and flexible fucosylation reaction.

[0366] The method of preparing the magnetic scaffold is flexible and adjustable to fabricate objects using 3D designs of magnetically captured BNCs. The sintering process itself may produce a large surface area. It is also possible to recycle the material by removing the BNCs and then refunctionalizing them for reuse. See PCT / US19 / 53307, which is incorporated herein by reference in its entirety.

[0367] In some embodiments, the thermoplastic is polyethylene (PE) (different densities, such as LDPE, HDPE), polypropylene (PP), acrylic acid: polyacrylic acid (PAA), poly(methyl methacrylate) (PMMA), polyvinyl alcohol (and polyvinyl acetal), polyamide (nylon), polylactic acid (PLA), polycarbonate (PC), polyethersulfone (PES), polystyrene (PS), polyvinyl chloride (PVC), acrylonitrile butadiene styrene (ABS), polybenzimidazole (PBI), polyoxymethylene (POM), polyetheretherketone (PEEK), polyetherimide (PEI), polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE / Teflon), polyacrylonitrile (PAN)), which is mixed with a magnetic material (such as magnetite MMP) by melt / extrusion or by dissolving the plastic in a solvent to coat the magnetic material. In other embodiments, the powder is sintered by laser using SLS. Porosity may form during SLS.

[0368] Selective Laser Sintering (SLS) is an Additive Manufacturing (AM) technology that uses a laser as a power source to sinter powdered materials such as plastics, metals, ceramics, glass powders, nylon or polyamide. The laser automatically targets points in space defined by a 3D model (such as an additive manufacturing file, AMF or CAD file), binding the materials together to create a solid structure. After each cross-section is scanned, the powder bed is lowered by one layer of thickness, a new layer of material is applied on top, and the process is repeated until the part is complete. There are many different techniques, such as stereolithography (SLA) or fused deposition modeling (FDM).

[0369] SLS is similar to Direct Metal Laser Sintering (DMLS), but differs in technical details. DSLM uses a similar concept, but in DSLM, the material is completely melted instead of sintered. This allows the manufacture of materials with different properties (such as crystal structure and porosity). SLS is a relatively new technology that can be expanded into a commercial-scale manufacturing process.

[0370] In one embodiment, the polypropylene-magnetite material can be 3D printed via SLS in any shape and form.

[0371] In other embodiments, the size of the extruded composite is reduced by cryogenic grinding or another form of grinding. In other embodiments, the composite powder is sieved to the desired particle size. In a preferred embodiment, the particle size is 60 + / - 20 μm.

[0372] The powder or 3D printed object can be functionalized with BNCs containing one or more enzymes or enzyme systems. The BNCs are magnetically captured on the surface of the powder or 3D printed object.

[0373] They can be single or multiple layers of material that house the BNCs. Objects of this design can be produced using 3D printing by sintering composite magnetic powders. In some embodiments, selective laser sintering (SLS) is used. Modular flow cells can be mixed and matched to achieve a highly customizable and efficient manufacturing process.

[0374] Composite powders can also be optimized for flowability. In some embodiments, 3D objects can be printed to optimize flow within them for use in flow reactors.

[0375] In some embodiments, the 3D objects and composite powders can be washed away from the BNCs by acid washing, rinsed with water, and then refunctionalized with fresh BNCs.

[0376] The highly magnetic scaffold (Macroporous Magnetic Scaffold or MMP) is designed to immobilize, stabilize and optimize any BNC containing enzymes. This includes complete enzyme systems with high loading and full activity for the production of small molecules. By combining natural or engineered enzymes, and in some embodiments, cofactor recovery systems, the scaffold allows one to scale up biocatalysis to production scale and generate innovations.

[0377] A highly magnetic and highly porous composite mixture of thermoplastics and magnetic particles is included to form a powder that can be a single layer or multilayer material that can accommodate BNCs. Objects of this design can be produced by sintering the composite magnetic powder using 3D printing. In some embodiments, selective laser sintering (SLS) is used. Modular flow cells can be mixed and matched for a highly customizable and efficient manufacturing process.

[0378] The MMPs made of thermoplastic and magnetic materials of the present invention can take the form of magnetic powders suitable for flow chemistry applications. These powders can be 3D printed into structures, functional objects or flow cells or plate reactors by SLS. The high surface area allows for maximum enzyme loading, and flow can be engineered within the material to maximize biocatalysis productivity.

[0379] SLS can be used to process almost any type of material, including metals, ceramics, plastics, and combinations thereof, for custom composites. However, it is critical that the material be provided in a fine powder form and that the powder particles be able to fuse when exposed to heat (Kruth et al., Assembly Automation 23(4):357-371 (2003), the entire contents of which are incorporated herein by reference).

[0380] When a material lacks these characteristics or is susceptible to phase changes within the temperature range or conditions of the sintering process, the addition of a sacrificial binder can allow the process to remain viable for that material. Typically, polymers are used as sacrificial binders to expand the range of materials suitable for this technology. After sintering, the sacrificial binder can be removed by thermal decomposition or remain as part of the composition.

[0381] This concept is applicable to magnetite, which loses its permanent magnetism above 585°C. This is significantly lower than its melting temperature (1538°C). Another advantage of using a polymer matrix to incorporate magnetite particles is that the former can act as a protective barrier against oxidation and corrosion and help disperse the magnetite particles. In addition, magnetite can mechanically reinforce the polymer. (Shishkovsky et al., Microelectronic Engineering 146:85-91 (2015), incorporated herein by reference in its entirety).

[0382] Laser sintering of plastic parts is one of two additive manufacturing processes used for rapid manufacturing (Wegner, Physics Procedia 83: 1003-1012 (2016), incorporated herein by reference in its entirety). There are several polymer properties that determine their ability to sinter and produce high-quality 3D objects. These include structural properties such as crystal structure (i.e., thermal properties, such as Tm, Tg, and Tc), mechanical properties (Young's modulus and elongation at break, etc.), density, particle size, and shape.

[0383] In SLS, the temperature processing window is determined by the difference between the melting temperature and the crystallization temperature of the polymer. For example, nylon 12 (PA12) has one of the highest operating windows and is therefore a widely used SLS material. In theory, the higher the value, the easier the material is to sinter. However, in practice, there are still more parameters that make the process difficult for any particular polymer (Shishkovsky et al., Microelectronic Engineering 146: 85-91 (2015), incorporated herein by reference in its entirety). In order to prevent the sintered parts from curling, a low polymer crystallization rate and a melt index that provides suitable rheology and surface tension are required.

[0384] In addition, the bulk density, particle shape and size distribution of the powder are key factors (Wegner, Physics Procedia 83: 1003-1012 (2016), incorporated herein by reference in its entirety). It has been determined that in certain embodiments, the optimal particle size range is about 40 to about 90 microns. Smaller particles hinder fluidity, and their rapid vaporization is harmful to the optical sensors of the sintering device. This can fog the device and lead to inaccurate sintered parts (Goodridge et al., Materials Science 57: 229-267 (2012), incorporated herein by reference in its entirety). The powder should have good flow properties and preferably have a particle shape that is approximately round. This allows the powder to be spread well during the process. At the wavelength of the CO2 laser beam (10.6 microns), the material is required to have high thermal conductivity. This is not the case for most polymers. The last two requirements can be met by adding additives (e.g., high energy absorbing materials, such as carbon black, to increase the heat absorption rate) and fumed silica nanoparticles (talcum powder) (to help the particle fluidity of irregularly shaped particles).

[0385] Additive manufacturing (AM), also known as 3D printing, involves manufacturing parts by depositing material layer by layer. This is in contrast to traditional processes such as subtractive processes (i.e. milling or drilling), forming processes (i.e. casting or forging), and joining processes (i.e. welding or fastening). Fast production times, low prototype costs, and design flexibility make 3D printing a valuable tool for prototyping and industrial manufacturing. The three most common types of 3D printers are fused filament fabrication, stereolithography, and selective laser sintering.

[0386] Fused filament fabrication (FFF) melts a thermoplastic continuous filament and builds the object layer by layer until the print is complete. While alternative materials exist, the two most popular filament materials are polylactic acid (PLA) and acrylonitrile butadiene styrene (ABS). FFF printers and materials are among the cheapest on the market, but currently offer lower print resolution and build quality.

[0387] Stereolithography (SLA) uses lasers to polymerize photosensitive resins. Uncured liquid resin is placed in a vat where a laser is used to cure the resin into solid plastic and build the object layer by layer. Due to the fine spot size of the laser, SLA printers have much higher resolution than FFF printers, so intricate features and complex shapes can be printed. However, resins are more expensive than filaments, and finished prints currently require post-treatment with solvents to optimize surface finish and material properties.

[0388] Selective laser sintering (SLS) is a powder-based layer additive manufacturing process commonly used for rapid prototyping and rapid machining. A laser beam in continuous or pulsed mode is used as a heat source to scan and connect powder in layers of predetermined size and shape. The geometry of the scanned layer corresponds to the individual cross-sections of the computer-aided design (CAD) model or stereolithography (STL) file of the object. After scanning the first layer, a second layer of loose powder is deposited on it, and the process is repeated from bottom to top until the workpiece (3D object) is completed. Kumar, JOM, 55 (10), 43-47 (2003), incorporated herein by reference in its entirety.

[0389] SLS offers advantages for printing objects with magnetic properties that can be used to immobilize BNCs. This is because the printing process creates porosity and high surface area. The surrounding unsintered powder acts as a natural support, eliminating the need for dedicated support structures. The lack of support structures allows for complex geometries that cannot be manufactured by alternative 3D printing methods. In addition, the nature of sintering itself creates macro- and microporous volumes. During the printing process, the laser flashes between the glass transition temperature and melting temperature of a thermoplastic crystalline thermoplastic powder (e.g., polypropylene, polystyrene) to produce a rigid part. By using fast laser scanning speeds (>100mm / s) to avoid amorphous behavior, the powders are sintered in place to form small bonds between them. The low-density powder traps air in its structure, creating significant porosity and surface area in three dimensions. These pores increase the surface area for enzyme immobilization.

[0390] In recent years, the industrial application of enzymes has attracted widespread attention due to a wide range of potential manufacturing applications. The use of enzymes in industrial processes has multiple advantages over traditional chemical methods. This includes high catalytic activity, the ability to perform complex reactions, and promoting greener chemical reactions by reducing by-products and the need for toxic chemicals (Singh et al., Microbialenzymes: industrial progress in 21st century. 3Biotech. 6 (2): 174 (2016), incorporated herein by reference in its entirety).

[0391] One of the biggest obstacles for biocatalysis to be widely used in industrial production is low enzyme stability. This is further hindered by relatively harsh process conditions, which may make the enzyme unstable and shorten its life span (Mohamad et al., Biotechnology, Biotechnological Equipment 29 (2): 205-220 (2015), incorporated herein by reference in its entirety). In addition, the free enzymes used in these processes are usually lost from the system as waste products, thus becoming expensive operating costs. The main method to solve these problems is to fix the enzyme on a support to enhance its operational stability and catalytic activity. Enzyme immobilization also provides a method for enzyme recovery, making the biocatalytic process more economically feasible.

[0392] At present, the biocatalytic process for industrial production is usually carried out in a batch reactor because they are simple and easy to operate. Although there are benefits to using a batch reactor, a continuous flow system can achieve higher production rates and better process control (Wiles C et al., Green Chem. (14): 38-54 (2012)). The rapid development of flow chemistry in biocatalytic processes is mainly driven by people's growing interest in process intensification and green chemistry. Continuous flow systems promote process intensification by reducing residence time (usually from a few hours to a few minutes), reducing the size of required equipment and achieving output enhancement (Tamborini et al., Cell. 36 (1): 73-78 (2018)). From the perspective of green chemistry, due to thermal management and mixing control, these systems provide significant improvements in safety, waste generation and energy efficiency (Newman and Jensen, Green Chem. (15): 1456-1472 (2013)). The foregoing is incorporated herein by reference in its entirety.

[0393] The present invention has many advantages over the prior art. It enables efficient and economical production of glycans, such as complex polysaccharides, including but not limited to HMOs, using enzymes captured in modular flow processing units. The flow cell can contain materials with large pores or high magnetic surface areas for BNC immobilization. The flexible composition of the magnetic support for sintering can be made with any fusible thermoplastic and magnetic material composition. The flow cell can have one or more enzyme systems that can be pieced together for a specific sugar manufacturing process.

[0394] A solution to combining biocatalysis and continuous flow systems is to use functionalized flow cells. Biocatalytic flow cells are scaffolds containing immobilized enzymes for reactors such as continuous stirred tank reactors (CSTRs) and packed bed reactors (PBRs). Both types of reactors are known in the art, but are selected primarily based on the type of immobilization used. The total market value of immobilized enzymes in 2010 was $5.8 billion, and they are used in a variety of large-scale processes, including high fructose corn syrup production (10 7 tons / year), edible oil transesterification (10 5 tons / year), biodiesel synthesis (10 4 tons / year) and chiral separation of alcohols and amines (10 3 Tons / year) (DiCosimo et al., Chem. Soc. Rev. (42): 6437-6474 (2013), incorporated herein by reference in its entirety). Compared to batch reactors, these systems allow for improved downstream process management of enzyme systems in terms of online control, enzyme reuse, and production scalability.

[0395] For the reasons stated above, the methods of the present invention employ a biocatalytic system for small to large scale manufacturing using BNCs in scaffolds formed by 3D printing. In some embodiments, the biocatalytic system is continuous flow.

[0396] The support can include cross-linked water-insoluble polymers and roughly uniformly distributed embedded magnetic microparticles (MMP). The support can include thermosetting resins, including epoxy resins, polyesters, polyurethanes, melamine resins, vinyl esters, silicones (polysiloxanes), furan resins, polyureas, phenolic resins, phenol formaldehyde, urea-formaldehyde, diallyl phthalate (DAP), benzoxazine, polyimide and bismaleimide, and cyanate esters can be used. By combining natural or engineered enzymes, and in some embodiments combining cofactors and cofactor recovery systems, the support technology disclosed herein allows the innovation of biocatalysis to be quickly converted into production innovations in batches and flow processes. Magnetic powders are suitable for flow chemistry applications, such as packed bed reactors.

[0397] Self-assembled mesoporous nanoclusters containing entrapped enzymes are highly active and robust. This technology is a powerful fusion of biochemistry, nanotechnology, and bioengineering at three integrated organizational levels: Level 1 is the self-assembly of enzymes with magnetic nanoparticles (MNPs) for the synthesis of magnetic mesoporous nanoclusters. This level uses a molecular self-entrapment mechanism to immobilize and stabilize enzymes. Level 2 is the stabilization of MNPs into other matrices. Level 3 is product conditioning and packaging for level 1+2 delivery. The assembly of magnetic nanoparticles adsorbed to enzymes is also referred to herein as "bionanocatalysts" (BNCs).

[0398] MNPs allow for a wider range of operating conditions, such as temperature, ionic strength, and pH. The size and magnetization of the MNPs affect the formation and structure of the NPs, all of which have a significant impact on the activity of the entrapped enzymes. Due to their surprising flexibility under a variety of reaction conditions, MNPs can be used as improved enzymes or catalysts over other such agents currently used. In addition, they can be used in other applications for which enzymes have not yet been considered or discovered.

[0399] The BNCs contain mesopores, ie, gaps between the magnetic nanoparticles. The enzyme is preferably embedded or immobilized within at least a portion of the mesopores of the BNCs. As used herein, the term "magnetic" encompasses all types of useful magnetic properties, including permanent magnetism, superparamagnetism, paramagnetism, ferromagnetism, and ferrimagnetism.

[0400] Magnetic nanoparticles or BNCs have nanoscale dimensions, i.e., typically no greater than 500 nm. As used herein, the term "size" may refer to the diameter of a magnetic nanoparticle when it is approximately or substantially spherical. In the case where the magnetic nanoparticles are not approximately or substantially spherical (e.g., substantially oval or irregularly shaped), the term "size" may refer to the longest dimension of the magnetic nanoparticle or the average of three dimensions. The term "size" may also refer to the size average (i.e., "average size") of a population of magnetic nanoparticles.

[0401] In various embodiments, the size of the magnetic nanoparticles is exactly, about, at most, or less than, for example, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, 50 nm, 40 nm, 30 nm, 25 nm, 20 nm, 15 nm, 10 nm, 5 nm, 4 nm, 3 nm, 2 nm, or 1 nm, or a size within a range defined by any two of the above exemplary sizes.

[0402] In BNC, a single magnetic nanoparticle can be considered as a primary nanoparticle (i.e., primary crystal) having any of the above sizes. The size of the aggregates of the nanoparticles in the BNC is larger than the nanoparticles, and generally has a size of at least about 5 nm (i.e., secondary size). In various embodiments, the size of the aggregates is precisely, approximately, at least, above, at most, or less than, for example, 5 nm, 8 nm, 10 nm, 12 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, or 800 nm, or a size within the range defined by any two of the aforementioned exemplary sizes.

[0403] Typically, primary and / or aggregated magnetic nanoparticles or their BNCs have a size distribution, i.e., their sizes are typically dispersed, narrowly dispersed or broadly dispersed. In various embodiments, any range of primary or aggregate sizes may constitute a major or minor proportion of the total range of primary or aggregate sizes. For example, in some embodiments, a specific range of primary particle sizes (e.g., at least about 1, 2, 3, 5, or 10 nm, at most about 15, 20, 25, 30, 35, 40, 45, or 50 nm) or a specific range of aggregate particle sizes (e.g., at least about 5, 10, 15, or 20 nm, at most about 50, 100, 150, 200, 250, or 300 nm) constitutes at least or more than about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% of the total range of primary particle sizes. In other embodiments, a particular range of primary particle sizes (e.g., less than about 1, 2, 3, 5, or 10 nm, or greater than about 15, 20, 25, 30, 35, 40, 45, or 50 nm) or a particular range of aggregate particle sizes (e.g., less than about 20, 10, or 5 nm, or greater than about 25, 50, 100, 150, 200, 250, or 300 nm) constitutes no more than or less than about 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% of the total range of primary particle sizes.

[0404] Aggregates of magnetic nanoparticles (i.e., "aggregates") or their BNCs may have any degree of porosity, including substantial lack of porosity depending on the number of individual primary crystals that constitute them. In a particular embodiment, the aggregates are mesoporous because they contain interstitial mesopores (i.e., mesopores between primary magnetic nanoparticles, formed by packing arrangements). The size of the mesopores is typically at least 2 nm and up to 50 nm. In various embodiments, the mesopores may have an aperture of precisely or approximately, for example, 2, 3, 4, 5, 10, 12, 15, 20, 25, 30, 35, 40, 45, or 50 nm, or an aperture within the range defined by any two of the exemplary apertures described above. Similar to the case of particle size, mesopores typically have a size distribution, i.e., their sizes are typically dispersed, narrowly dispersed, or broadly dispersed. In various embodiments, any range of mesopore sizes may constitute a majority or a minority of the total range of mesopore sizes or total pore volume. For example, in some embodiments, a specific range of mesopore sizes (e.g., at least about 2, 3 or 5, and up to 8, 10, 15, 20, 25 or 30 nm) constitutes at least or more than about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% of the total range of mesopore sizes or total pore volume. In other embodiments, a specific range of mesopore sizes (e.g., less than about 2, 3, 4 or 5 nm, or greater than about 10, 15, 20, 25, 30, 35, 40, 45 or 50 nm) constitutes no more than or less than about 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1%, 0.5% or 0.1% of the total range of mesopore sizes or total pore volume.

[0405] The magnetic nanoparticles may have any composition known in the art. In some embodiments, the magnetic nanoparticles are or include a zero-valent metal portion having magnetism. Some examples of such zero-valent metals include cobalt, nickel and iron, and mixtures and alloys thereof. In other embodiments, the magnetic nanoparticles are or include oxides of magnetic metals (e.g., oxides of cobalt, nickel or iron, or mixtures thereof). In some embodiments, the magnetic nanoparticles have different core portions and surface portions. For example, the magnetic nanoparticles may have a core portion consisting of elemental iron, cobalt or nickel and a surface portion consisting of a passivation layer (e.g., a metal oxide or a noble metal coating, such as a gold, platinum, palladium or silver layer). In other embodiments, the metal oxide magnetic nanoparticles or their aggregates are coated with a layer of noble metal coating. The noble metal coating may, for example, reduce the number of charges on the surface of the magnetic nanoparticles, which may be beneficial in increasing dispersibility in the solution and better controlling the size of the BNC. The noble metal coating protects the magnetic nanoparticles from oxidation, leaching dissolution or chelating dissolution (when a chelating organic acid (e.g., citrate, malonate or tartrate) is used in a biochemical reaction or process). The passivation layer can have any suitable thickness, and specifically, at least, at most, or less than about, for example, 0.1 nm, 0.2 nm, 0.3 nm, 0.4 nm, 0.5 nm, 0.6 nm, 0.7 nm, 0.8 nm, 0.9 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, or 10 nm, or a thickness within a range bounded by any two of these values.

[0406] Magnetic materials useful in the present invention are well known in the art. Non-limiting examples include ferromagnetic and ferromagnetic materials, including ores such as iron ore (magnetite or lodestone), cobalt and nickel. In other embodiments, rare earth magnets are used. Non-limiting examples include neodymium, gadolinium, samarium, samarium cobalt, neodymium iron boron, etc. In further embodiments, the magnet comprises a composite material. Non-limiting examples include ceramics, ferrites and aluminum nickel cobalt magnets. In a preferred embodiment, the magnetic nanoparticles have an iron oxide composition. The iron oxide composition can be any magnetic or superparamagnetic iron oxide composition known in the art, such as magnetite (Fe3O4), hematite (α-Fe2O3), magnetic hematite (γ-Fe2O3) or spinel ferrite according to the formula AB2O4, where A is a divalent metal (e.g., Xn 2+ 、Ni 2+ , Mn 2+ 、Co 2+ , Ba 2+ , Sr 2+ or a combination thereof), B is a trivalent metal (e.g. Fe 3+ Cr 3+ or a combination thereof).

[0407] Individual magnetic nanoparticles or their aggregates or their BNCs have any appropriate degree of magnetism. For example, magnetic nanoparticles, BNCs or BNC scaffold components may have a saturation magnetization (Ms) of at least or up to about 5, 10, 15, 20, 25, 30, 40, 45, 50, 60, 70, 80, 90 or 100 emu / g. Magnetic nanoparticles, BNCs or BNC scaffold components preferably have a permanent magnetization (Mr) of no more than (i.e., at most) or less than 5 emu / g, and more preferably, at most or less than 4 emu / g, 3 emu / g, 2 emu / g, 1 emu / g, 0.5 emu / g or 0.1 emu / g. The surface magnetic field of the magnetic nanoparticles, BNCs or BNC-scaffold components can be about or at least, for example, about 0.5, 1, 5, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 Gauss (G), or a magnetic field within the range defined by any two of the above values. If microparticles are included, the microparticles can also have any of the above magnetic strengths.

[0408] Can make magnetic nanoparticles or its aggregates adsorb the appropriate amount of enzyme until saturation level or below saturation level, to produce final BNC, depending on application.In different embodiments, magnetic nanoparticles or its aggregates can adsorb about, at least, at most or less than, for example, 1, 5, 10, 15, 20, 25 or 30pmol / m2 of enzyme.Or, magnetic nanoparticles or its aggregates can adsorb about, at least, at most or less than, for example, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the amount of enzyme at saturation level.

[0409] The magnetic nanoparticles or their aggregates or their BNCs have any suitable pore volume. For example, the magnetic nanoparticles or their aggregates can have a pore volume of about, at least, at most, or less than, for example, about 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1 cm3 / g, or a pore volume within the range defined by any two of the foregoing values.

[0410] The magnetic nanoparticles or their aggregates or their BNCs have any suitable specific surface area. For example, the specific surface area of ​​the magnetic nanoparticles or their aggregates can be about, at least, at most, or less than, for example, about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 m2 / g.

[0411] The size of the magnetic macroporous matrix material used according to the present invention is precisely, approximately, at most or less than, for example, 100-1000, 50-100, 10-50 μm, or 5-10, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, less than 5, greater than 100, 150 average size, 75 average size, 40 average size, 20 average size or about 15 average size. In certain embodiments, the average particle size of the material is precisely, approximately, at most or less than 20-40 μm, 20 μm or 40 μm. In certain embodiments, the material has a tight size distribution with an average particle size of 20 μm or 40 μm.

[0412] In some embodiments, the methods described herein use recombinant cells expressing enzymes used in the present invention. Recombinant DNA technology is known in the art. In some embodiments, cells are transformed with expression vectors (e.g., plasmids) expressing enzymes. In other embodiments, the vector has one or more genetic signals, such as for transcription initiation, transcription termination, translation initiation, and translation termination. Here, nucleic acids encoding enzymes can be cloned in vectors so that they are expressed when appropriately transformed into suitable host organisms. Suitable host cells can be derived from bacteria, fungi, plants, or animals well known in the art.

[0413] Although BNCs (level 1) provide most of the enzyme immobilization capacity, they are sometimes too small to be easily captured by standard strength magnets. Therefore, submicron magnetic materials (level 2) are used to provide bulk magnetization and add stability to level 1. Commercially available free magnetite powders with particle sizes ranging from 50-500 nanometers are highly hydrophilic and tend to adhere to plastic and metal surfaces, which over time can reduce the amount of effective enzyme in a given reactor system. In addition, powdered magnetite is extremely dense, which increases shipping costs. It is also quite expensive, especially for particle sizes less than 100 nanometers. To overcome these limitations, low-density hybrid materials composed of magnetite, non-water-soluble cross-linked polymers such as polyvinyl alcohol (PVA) and carboxymethyl cellulose (CMC) have been developed. These materials are formed by freeze casting and freeze drying water-soluble polymers followed by cross-linking. These materials have reduced adhesion to external surfaces, require less magnetite, and achieve level 1 capture at least comparable to pure magnetite powder.

[0414] In one embodiment, the continuous macroporous scaffold has a cross-linked polymer composition. The polymer composition can be any solid organic, inorganic or mixed organic-inorganic polymer composition known in the art, and can be a biopolymer that is synthesized or acts as an adhesive. Preferably, the polymer scaffold will not dissolve or degrade in water or other media that the layered catalyst is intended to use. Some examples of synthetic organic polymers include vinyl addition polymers (e.g., polyethylene, polypropylene, polystyrene, polyacrylic acid or polyacrylic acid salts, polymethacrylic acid or polymethacrylates, polymethyl methacrylate, polyvinyl acetate, polyvinyl alcohol, etc.), fluoropolymers (e.g., polyvinyl fluoride, polyvinylidene fluoride, polytetrafluoroethylene, etc.), epoxides (e.g., phenolic resins, resorcinol-formaldehyde resins), polyamides, polyurethanes, polyesters, polyimides, polybenzimidazoles and copolymers thereof. Some examples of biopolymers include polysaccharides (e.g., cellulose, hemicellulose, xylan, chitosan, inulin, dextran, agarose and alginic acid), polylactic acid and polyglycolic acid. In the specific case of cellulose, cellulose can be microorganism- or algae-derived cellulose. Some examples of inorganic or hybrid organic-inorganic polymers include polysiloxanes (e.g., prepared by sol-gel synthesis, such as polydimethylsiloxane) and polyphosphazenes. In some embodiments, any one or more of the classes or specific types of polymer compositions provided above are extruded as macroporous scaffolds. In some embodiments, the 3D model is an electronic file.

[0415] Any of these compositions and methods can be used to immobilize enzymes in embodiments of the present invention.

[0416] It should be understood that "comprising" should be interpreted non-exhaustively where the context permits. Each comprising may be alternatively "consisting essentially of" or "consisting of" where the context permits.

[0417] Implementation

[0418] 1. A biocatalytic method for preparing a sugar receptor, comprising the following steps:

[0419] a. contacting a sugar with a nucleotide to form a sugar-nucleotide, wherein the nucleotide is present in a catalytic amount; and

[0420] b. contacting the sugar-nucleotide and the acceptor in the presence of a transferase to form a sugar-acceptor.

[0421] 2. The method according to embodiment 1, wherein the sugar is galactose, sialic acid or l-fucose.

[0422] 3. A method for preparing a sugar compound, comprising the following steps:

[0423] a. contacting a monosaccharide selected from the group consisting of galactose, sialic acid and l-fucose with a catalytic amount of a nucleotide selected from the group consisting of UDP, ADP, CMP and GDP to form a sugar-nucleotide compound.

[0424] b. contacting the sugar-nucleotide compound with an acceptor compound in the presence of a transferase to obtain the sugar.

[0425] 4. A method for preparing a sugar compound, comprising the following steps:

[0426] a. contacting a galactose sugar unit with a catalytic amount of a nucleotide selected from UDP and ADP or contacting a sialic acid or l-fucose sugar unit with a catalytic amount of a nucleotide selected from CMP and GDP to form a sugar-nucleotide compound.

[0427] b. contacting the sugar-nucleotide compound with an acceptor compound in the presence of a transferase to obtain the sugar.

[0428] 5. A method for galactosylating a receptor compound, comprising the following steps:

[0429] a. contacting galactose with a nucleotide to form a galactose-nucleotide compound, wherein the nucleotide is present in a catalytic amount; and

[0430] b. contacting the galactose-nucleotide compound with the acceptor compound in the presence of a transferase to galactosylate the acceptor compound.

[0431] 6. The method according to embodiment 5, wherein galactose is obtained in situ from sucrose.

[0432] 7. The method according to embodiment 5 or embodiment 6, wherein the method achieves a high conversion rate by converting sucrose into Glc-UDP and fructose.

[0433] 8. A method for sialylation of a receptor compound, comprising the following steps:

[0434] a. contacting sialic acid with a nucleotide to form a sialic acid-nucleotide compound, wherein the nucleotide is present in a catalytic amount; and

[0435] b. contacting the sialic acid-nucleotide compound and the compound with an acceptor compound in the presence of a transferase to sialylate the acceptor compound.

[0436] 9. The method according to embodiment 8, wherein the sialic acid is obtained in situ from 3'-SL or 6'-SL.

[0437] 10. The method of embodiment 8 or embodiment 9, wherein the method achieves a high conversion rate by converting lactose into galactose and glucose.

[0438] 11. A method for fucosylation of a receptor compound, comprising the following steps:

[0439] a. contacting fucose with a nucleotide to form a fucose-nucleotide compound, wherein the nucleotide is present in a catalytic amount; and

[0440] b. contacting the fucose-nucleotide compound with the acceptor compound in the presence of a transferase to fucosylate the acceptor compound.

[0441] 12. The method according to embodiment 11, wherein the fucose is l-fucose and is obtained in situ from 2'-FL.

[0442] 13. The method of embodiment 11 or embodiment 12, wherein the method achieves a high conversion rate by converting sucrose into Glc-UDP and fructose.

[0443] 14. The method according to any one of embodiments 1-13, wherein the acceptor compound is a monosaccharide, a disaccharide, an oligosaccharide or a polysaccharide.

[0444] 15. The method of any one of embodiments 1-14, wherein substantially no ATP is added to or produced by the method.

[0445] 16. The method of any one of embodiments 1-15, wherein the catalytic amount of nucleotide is about 0.01 to about 10 mole percent relative to the amount of sugar.

[0446] 17. A method of producing LNT and fructose comprising the steps of contacting LNTII, sucrose, a transferase, a sucrose synthase, an epimerase, and UDP to produce LNT and fructose, wherein galactose-UDP is produced, and wherein UDP is present in an amount of about 0.01 mole percent to about 10 mole percent of the galactose or sucrose.

[0447] 18. The method of embodiment 17, wherein the transferase is Cvb3GalT.

[0448] 19. The method according to embodiment 18, wherein the transferase is B3GALT5

[0449] 20. The method of any one of embodiments 17-19, wherein the epimerase is GalE.

[0450] 21. A method of producing LNnT and fructose comprising contacting LNTII, sucrose, a glycosyltransferase, a sucrose synthase, an epimerase, and UDP to produce LNnT and fructose, wherein UDP is present at about 0.01 mole percent to about 10 mole percent of galactose or sucrose.

[0451] 22. The method of embodiment 21, wherein the glycosyltransferase is LgtB.

[0452] 23. The method of embodiment 22, wherein LgtB is NmLgtB.

[0453] 24. The method of embodiment 23, wherein the glycosyltransferase is B4GALT1.

[0454] 25. The method of any one of embodiments 21-24, wherein the epimerase is GalE.

[0455] 26. A method for preparing LNT and fructose, comprising the steps of: contacting GlcNAc, 2-chloro-1,3-dimethylimidazolinium chloride, and a base to obtain a reaction mixture comprising GlcNAc-Oxa; and contacting a reaction mixture comprising GlcNAc-Oxa, lactose, sucrose, an amidase, a transferase, a sucrose synthase, and an epimerase to provide LNT and fructose, wherein UDP is present at about 0.01 mole percent to about 10 mole percent of the lactose or sucrose.

[0456] 27. The method of embodiment 26, wherein the amidase is Bbh1.

[0457] 28. The method of embodiment 26 or embodiment 27, wherein the transferase is Cvb3GalT.

[0458] 29. The method of any one of embodiments 26-28, wherein the epimerase is GalE.

[0459] 30. The method of any one of embodiments 26-29, wherein the base is triethylamine.

[0460] 31. A method for preparing lactose and fructose comprising contacting glucose, sucrose, lactose synthase, sucrose synthase, an epimerase, and UDP to provide lactose and fructose, wherein UDP is present at about 0.01 mole percent to about 10 mole percent of the glucose and / or sucrose.

[0461] 32. A method according to embodiment 31, wherein the isomerase is GalE.

[0462] 33. A method of preparing LSTa, galactose and glucose comprising contacting 3'-SL, LNT, CMP, sialyltransferase and lactase to provide LSTa, galactose and glucose, wherein CMP is present at about 0.01 mole percent to about 10 mole percent of 3'-SL.

[0463] 34. The method of embodiment 33, wherein the sialyltransferase is ST3GAL3.

[0464] 35. A method of preparing LSTc, galactose, and glucose comprising contacting 3'-SL, LNnT, CMP, a sialyltransferase, and lactase to provide LSTc, galactose, and glucose, wherein CMP is present at about 0.01 mole percent to about 10 mole percent of 3'-SL.

[0465] 36. The method of embodiment 35, wherein the sialyltransferase is ST3GAL3.

[0466] 37. A method of preparing LSTd, galactose, and glucose comprising contacting 3'-SL, LNnT, CMP, a sialyltransferase, and lactase to provide LSTd, galactose, and glucose, wherein CMP is present at about 0.01 mole percent to about 10 mole percent of 3'-SL.

[0467] 38. The method of embodiment 37, wherein the sialyltransferase is ST3GAL3.

[0468] 39. A method of preparing DSLNT, galactose and glucose comprising contacting 3'-SL, LNT, CMP, sialyltransferase and lactase to provide DSLNT, galactose and glucose, wherein CMP is present at about 0.01 mole percent to about 10 mole percent of 3'-SL.

[0469] 40. The method of embodiment 39, wherein the sialyltransferases are ST3GAL3 and ST6GALNAC5.

[0470] 41. A method of preparing LNFPI, galactose, and glucose comprising contacting 2'-FL, LNT, GDP, Te2FT, HmFucT, and lactase to provide LNFPI, galactose, and glucose, wherein GDP is present at about 0.01 mole percent to about 10 mole percent of 2'-FL.

[0471] 42. A method according to embodiment 41, wherein the transferases are Te2FT and HmFucT.

[0472] 43. A method of preparing LNFPII, galactose, and glucose comprising contacting 2'-FL, LNT, GDP, Hp34FT, HmFucT, and lactase, wherein GDP is present at about 0.01 mole percent to about 10 mole percent of 2'-FL.

[0473] 44. A method according to embodiment 43, wherein the transferases are Hp34FT and HmFucT.

[0474] 45. A method of preparing LNFPIII, galactose, and glucose comprising contacting 2'-FL, LNnT, GDP, FUT9, HmFucT, and lactase to provide LNFPIII, wherein GDP is present at about 0.01 mole percent to about 10 mole percent of 2'-FL.

[0475] 46. ​​The method of embodiment 45, wherein the transferases are FUT and HmFucT.

[0476] 47. The method of any one of embodiments 1-46, wherein one or more enzymes are immobilized.

[0477] 48. The method of any one of embodiment 47, wherein each enzyme is immobilized.

[0478] 49. The method of any one of embodiments 1-48, wherein the method occurs in a single reaction vessel.

[0479] 50. A machine configured for the method of any one of embodiments 1-49.

[0480] 51. The machine of embodiment 50, wherein the method is carried out in a single reaction vessel.

[0481] 52. LNT prepared by the method according to any one of embodiments 1-7, 14-20 and 47-49.

[0482] 53. LNnT prepared by the method according to any one of embodiments 1-7, 14-16, 21-30 and 47-49.

[0483] 54. Lactose prepared by the method according to any one of embodiments 1-7, 14-16, 31-32 and 47-49.

[0484] 55. LSTa prepared by the method according to any one of embodiments 1-4, 7-9, 14-16, 33-34 and 47-49.

[0485] 56. LSTc prepared by the method according to any one of embodiments 1-4, 7-9, 14-16, 35-36 and 47-49.

[0486] 57. LSTd prepared by the method according to any one of embodiments 1-4, 7-9, 14-16, 37-38 and 47-49.

[0487] 58. DSLNT prepared by the method according to any one of embodiments 1-4, 7-9, 14-16, 39-40 and 47-49.

[0488] 59. LNFPI prepared by the method according to any one of embodiments 1-4, 10-15, 41-42 and 47-49.

[0489] 60. LNFPII prepared by the method according to any one of embodiments 1-4, 10-16, 43-44 and 47-49.

[0490] 61. LNFPIII prepared by the method according to any one of embodiments 1-7, 10-16 and 45-49.

[0491] 62. A lactose derivative prepared by the method according to any one of embodiments 1-7, 14-16 and 47-49.

[0492] 63. The lactose derivative according to embodiment 62, which is selected from lactobionic acid, lactitol, lactulooligosaccharides, galactoligosaccharides, lactulose and HMO.

[0493] 64. A compound prepared by the method of any one of embodiments 1-49, wherein the compound is obtained from a non-animal based plant material.

[0494] Other implementations are as follows.

[0495] 1. A method for producing a glycosylation main product, comprising the following steps:

[0496] a. in the presence of a transferase, adding a catalytic amount of a sugar-nucleotide donor to a stoichiometric acceptor to obtain a glycosylation primary product and a catalytic amount of a nucleotide; and

[0497] The nucleotide is regenerated into a regenerated sugar-nucleotide donor by contacting a catalytic amount of the nucleotide with a stoichiometric amount of the sugar donor precursor in the presence of a transferase to obtain a regenerated sugar-nucleotide donor and a secondary product.

[0498] 2. The method of embodiment 1, further comprising:

[0499] a. in the presence of a transferase, adding a catalytic amount of a regenerated sugar-nucleotide donor to a stoichiometric acceptor to obtain a glycosylation primary product and a catalytic amount of nucleotide; and

[0500] b. regenerating the nucleotide into a regenerated sugar-nucleotide donor by contacting a catalytic amount of the nucleotide with a stoichiometric amount of a sugar donor precursor in the presence of a transferase to obtain a regenerated sugar-nucleotide donor and a secondary product.

[0501] 3. A method for producing a glycosylation main product, comprising the steps of adding a sugar donor precursor, an acceptor, a transferase and a catalytic amount of nucleotides to obtain a glycosylation main product and a secondary product.

[0502] 4. A method according to embodiment 3, wherein the sugar donor precursor and the nucleotide provide the sugar-nucleotide donor.

[0503] 5. The method of any one of embodiments 1-4, wherein the sugar donor precursor and the nucleotide provide the sugar-nucleotide donor precursor, and wherein the auxiliary enzyme and the sugar-nucleotide donor precursor provide the sugar-nucleotide donor.

[0504] 6. The method according to any one of embodiments 1-5, wherein the sugar donor precursor is a sugar comprising 2 or more sugar units.

[0505] 7. The method according to embodiment 6, wherein the sugar donor precursor is a sugar comprising 2, 3 or 4 sugar units.

[0506] 8. The method of embodiment 7, wherein the sugar donor precursor is a disaccharide.

[0507] 9. The method according to embodiment 7, wherein the sugar donor precursor is a trisaccharide.

[0508] 10. The method of any one of embodiments 1-9, wherein the sugar donor precursor comprises galactose, sialic acid, fucose, or N-acetylglucosamine.

[0509] 11. The method according to embodiment 10, wherein galactose, sialic acid, fucose or N-acetylglucosamine is located at the terminal position of the sugar donor precursor.

[0510] 12. The method of any one of embodiments 1-9, wherein the sugar-nucleotide donor comprises galactose, sialic acid, fucose, or N-acetylglucosamine.

[0511] 13. The method according to any one of embodiments 1-9, wherein the sugar-nucleotide donor is galactose-nucleotide, sialic acid-nucleotide, fucose-nucleotide or N-acetylglucosamine-nucleotide.

[0512] 14. The method according to any one of embodiments 1-13, wherein the secondary product is a monosaccharide, a disaccharide or a trisaccharide.

[0513] 15. The method according to any one of embodiments 1-14, wherein the secondary product is converted into a secondary product derivative in the presence of a processing enzyme.

[0514] 16. The method of any one of embodiments 1-15, wherein the secondary product derivative is converted to a modified secondary product derivative in the presence of a second processing enzyme.

[0515] 17. The method according to any one of embodiments 1-16, wherein the acceptor is an organic compound containing a hydroxyl group.

[0516] 18. The method according to embodiment 17, wherein the organic compound containing a hydroxyl group is a sugar.

[0517] 19. The method of any one of embodiments 1-18, wherein the acceptor is obtained by converting a secondary product into the acceptor by a processing enzyme.

[0518] 20. The method of any one of embodiments 1-19, wherein the nucleotide is uridine diphosphate, cytidine monophosphate, or guanosine diphosphate.

[0519] 21. The method according to any one of embodiments 1-20, wherein the amount of nucleotides is 0.001 mol% to 10 mol% relative to the sugar donor precursor or acceptor.

[0520] 22. The method according to any one of embodiments 1-21, wherein the amount of nucleotides is 0.01 mole percent to 1 mole percent relative to the sugar donor precursor or acceptor.

[0521] 23. The method of any one of embodiments 1-22, wherein the transferase is β-1,3-galactosyltransferase, sucrase synthase, β-1,4-galactosyltransferase, sialyltransferase, fucosyltransferase, or glucosaminyl (N-acetyl) transferase 2.

[0522] 24. The method of any one of embodiments 1-23, wherein the transferase comprises a first transferase and a second transferase.

[0523] 25. A method according to any one of embodiments 1-24, wherein the first transferase catalyzes the transfer of a sugar from a sugar-nucleotide donor to an acceptor to obtain a glycosylation primary product, and the second transferase catalyzes the reaction of a nucleotide and a sugar donor precursor to obtain a sugar-nucleotide donor and a secondary product.

[0524] 26. A method for producing a galactosylation main product, comprising the following steps:

[0525] a. adding a catalytic amount of a sugar-nucleotide donor to a stoichiometric acceptor to obtain a galactosylation primary product and a catalytic amount of nucleotide; and

[0526] b. regenerating the nucleotide into a regenerated sugar-nucleotide donor by contacting a catalytic amount of the nucleotide with a stoichiometric amount of a sugar donor precursor to obtain a regenerated sugar-nucleotide donor and a secondary product.

[0527] 27. The method of embodiment 26, further comprising:

[0528] a. adding a catalytic amount of a regenerated sugar-nucleotide donor to a stoichiometric acceptor to obtain a glycosylation primary product and a catalytic amount of nucleotide; and

[0529] b. regenerating the nucleotide into a regenerated sugar-nucleotide donor by contacting a catalytic amount of the nucleotide with a stoichiometric amount of a sugar donor precursor to obtain a regenerated sugar-nucleotide donor and a secondary product.

[0530] 28. A method for producing a galactosylation major product, comprising the steps of adding a sugar donor precursor, an acceptor, a transferase and a catalytic amount of nucleotides to obtain a glycosylation major product and a minor product.

[0531] 29. The method of any one of embodiments 26-28, wherein the acceptor is lactose-N-trisaccharide II (LNTII), glucose, or galacto-oligosaccharide.

[0532] 30. The method of any one of embodiments 26-29, wherein the secondary product is fructose.

[0533] 31. The method according to any one of embodiments 26-30, comprising the step of contacting fructose in the presence of glucose isomerase to obtain glucose.

[0534] 32. The method according to any one of embodiments 26-31, comprising the step of contacting lactase and N-acetylglucosamine in the presence of β-N-acetyhexosaminidase (Bbh1) to obtain LNTII.

[0535] 33. The method of any one of embodiments 26-32, wherein the sugar donor precursor is sucrose.

[0536] 34. A method according to any one of embodiments 26-33, wherein the sugar-nucleotide donor precursor is a glucose nucleotide.

[0537] 35. The method of any one of embodiments 26-34, wherein the secondary product is fructose.

[0538] 36. The method of any one of embodiments 26-35, wherein the auxiliary enzyme is galactose epimerase.

[0539] 37. The method of any one of embodiments 26-36, wherein the nucleotide is uridine diphosphate.

[0540] 38. The method of any one of embodiments 26-36, wherein the transferase comprises a first transferase and a second transferase.

[0541] 39. The method of embodiment 38, wherein the first transferase is β-1,3-galactosyltransferase from Chromobacterium violaceum (Cvb3GalT), β-1,4-galactosyltransferase from Neisseria meningitidis (NmLgtB), and the second transferase is sucrose synthase from Arabidopsis thaliana (AtSuSy1).

[0542] 40. A method for producing a sialylated primary product, comprising the steps of:

[0543] a. adding a catalytic amount of a sugar-nucleotide donor to a stoichiometric amount of an acceptor to obtain a sialylated primary product and a catalytic amount of a nucleotide; and

[0544] b. regenerating the nucleotide into a regenerated sugar-nucleotide donor by contacting a catalytic amount of the nucleotide with a stoichiometric amount of a sugar donor precursor to obtain a regenerated sugar-nucleotide donor and a secondary product.

[0545] 41. The method of embodiment 40, further comprising:

[0546] a. adding a catalytic amount of a regenerated sugar-nucleotide donor to a stoichiometric acceptor to obtain a glycosylation primary product and a catalytic amount of nucleotide; and

[0547] b. regenerating the nucleotide into a regenerated sugar-nucleotide donor by contacting a catalytic amount of the nucleotide with a stoichiometric amount of a sugar donor precursor to obtain a regenerated sugar-nucleotide donor and a secondary product.

[0548] 42. A method for producing a sialylated major product, comprising the steps of adding a sugar donor precursor, an acceptor, a transferase and a catalytic amount of nucleotides to obtain a sialylated major product and a minor product.

[0549] 43. The method of any one of embodiments 40-42, wherein the sugar donor precursor is 3'-sialyllactose.

[0550] 44. The method of any one of embodiments 40-43, wherein the sugar-nucleotide donor is N-acetylneuraminic acid nucleotide.

[0551] 45. The method of any one of embodiments 40-44, wherein the secondary product is lactose.

[0552] 46. ​​The method of any one of embodiments 40-45, wherein the secondary product is converted to a secondary product derivative in the presence of a processing enzyme.

[0553] 47. The method of embodiment 46, wherein the processing enzyme is lactase.

[0554] 48. A method according to embodiment 47, wherein the secondary product derivatives are glucose and galactose.

[0555] 49. The method of any one of embodiments 40-48, wherein the nucleotide is cytidine monophosphate.

[0556] 50. The method of any one of embodiments 40-49, wherein the transferase comprises a first transferase and a second transferase.

[0557] 51. The method according to embodiment 50, wherein the first transferase is β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1), CMP-N-acetylneuraminic acid-β-1,4-galactoside α-2,3-sialyltransferase (ST3GAL3) or α-N-acetylgalactosamine α-2,6-sialyltransferase 5 (ST6GALNAC5), and the second transferase is CMP-N-acetylneuraminic acid-β-galactosamide-α-2,3-sialyltransferase 4 (ST3GAL4).

[0558] 52. The method of embodiment 50, wherein the first transferase is ST3GAL3 and ST6GALNAC5.

[0559] 53. A method for producing a fucosylated main product, comprising the following steps:

[0560] a. adding a catalytic amount of a sugar-nucleotide donor to a stoichiometric amount of an acceptor to obtain a fucosylated primary product and a catalytic amount of a nucleotide; and

[0561] b. regenerating the nucleotide into a regenerated sugar-nucleotide donor by contacting a catalytic amount of the nucleotide with a stoichiometric amount of a sugar donor precursor to obtain a regenerated sugar-nucleotide donor and a secondary product.

[0562] 54. The method of embodiment 53, further comprising:

[0563] a. adding a catalytic amount of a regenerated sugar-nucleotide donor to a stoichiometric acceptor to obtain a glycosylation primary product and a catalytic amount of nucleotide; and

[0564] b. contacting a catalytic amount of a nucleotide with a stoichiometric amount of a sugar donor precursor to regenerate the nucleotide into a regenerated sugar-nucleotide donor to obtain a regenerated sugar-nucleotide donor and a secondary product.

[0565] 55. A method for producing a fucosylated major product, comprising adding a sugar donor precursor, an acceptor, a transferase, and a catalytic amount of nucleotides to obtain a fucosylated major product and a minor product.

[0566] 56. The method of any one of embodiments 53-55, wherein the sugar donor precursor is 2'-fucosyllactose.

[0567] 57. The method of any one of embodiments 53-56, wherein the sugar-nucleotide donor is a fucose nucleotide.

[0568] 58. The method of any one of embodiments 53-57, wherein the secondary product is lactose.

[0569] 59. The method of any one of embodiments 53-58, wherein the secondary product is converted to a secondary product derivative in the presence of a processing enzyme.

[0570] 60. The method of embodiment 59, wherein the processing enzyme is lactase.

[0571] 61. A method according to embodiment 59 or embodiment 60, wherein the secondary product derivatives are glucose and galactose.

[0572] 62. The method of any one of embodiments 59-61, wherein the secondary product derivative is converted to a modified secondary product derivative in the presence of a second processing enzyme.

[0573] 63. The method of claim 62, wherein the second processing enzyme is a source of D-galactose isomerase from Geobacillus stearothermophilus (GsAI) and the modified secondary product derivative is tagatose.

[0574] 64. The method of any one of embodiments 53-63, wherein the nucleotide is guanosine diphosphate.

[0575] 65. The method of any one of embodiments 53-64, wherein the transferase comprises a first transferase and a second transferase.

[0576] 66. The method of embodiment 65, wherein the first transferase is alpha-1,2-fucosyltransferase (Te2FT), fucosyltransferase 9 (FUT9), or fucosyltransferase 3 (FUT3) from Thermosynechococcus tricholoma, and the second transferase is alpha-1,2-fucosyltransferase (HmFucT) or fucosyltransferase (FUT1) from Helicobacter huitonii.

[0577] 67. A method for producing an N-acetylglucosaminylated primary product, comprising the steps of:

[0578] a. adding a catalytic amount of a sugar-nucleotide donor to a stoichiometric acceptor to obtain a major N-acetylglucosaminylation product and a catalytic amount of a nucleotide; and

[0579] b. Regenerating the nucleotide into a regenerated sugar-nucleotide donor by contacting a catalytic amount of the nucleotide with a stoichiometric amount of a sugar donor precursor to obtain a regenerated sugar-nucleotide donor and a secondary product.

[0580] The method of embodiment 1 further comprises:

[0581] a. adding a catalytic amount of a regenerated sugar-nucleotide donor to a stoichiometric acceptor to obtain a glycosylation primary product and a catalytic amount of nucleotide; and

[0582] b. regenerating the nucleotide into a regenerated sugar-nucleotide donor by contacting a catalytic amount of the nucleotide with a stoichiometric amount of a sugar donor precursor to obtain a regenerated sugar-nucleotide donor and a secondary product.

[0583] The method of embodiment 1 further comprises:

[0584] a. adding a catalytic amount of a regenerated sugar-nucleotide donor to a stoichiometric acceptor to obtain a glycosylation primary product and a catalytic amount of nucleotide; and

[0585] b. regenerating the nucleotide into a regenerated sugar-nucleotide donor by contacting a catalytic amount of the nucleotide with a stoichiometric amount of a sugar donor precursor to obtain a regenerated sugar-nucleotide donor and a secondary product.

[0586] 69. A method for producing an N-acetylglucosaminylation major product comprising adding a sugar donor precursor, an acceptor, a transferase, and a catalytic amount of a nucleotide to obtain an N-acetylglucosaminylation major product and a minor product.

[0587] 70. The method of any one of embodiments 67-69, wherein the sugar donor precursor is lactose-N-biose.

[0588] 71. The method of any one of embodiments 67-70, wherein the sugar-nucleotide donor is an N-acetylglucosamine nucleotide.

[0589] 72. The method of any one of embodiments 67-71, wherein the secondary product is galactose.

[0590] 73. The method of any one of embodiments 67-72, wherein the secondary product derivative is tagatose.

[0591] 74. The method according to any one of embodiments 61-73, wherein the processing enzyme D-galactose isomerase is derived from Geobacillus stearothermophilus (GsAI).

[0592] 75. The method of any one of embodiments 61-74, wherein the nucleotide is uridine diphosphate.

[0593] 76. The method of any one of embodiments 61-75, wherein the transferase is β-1,3-N-acetyl-hexosaminyltransferase (NmLgtA) from Neisseria meningitidis.

[0594] 77. The method of any one of embodiments 61-76, wherein the transferase comprises a first transferase and a second transferase.

[0595] 78. The method of embodiment 77, wherein the first transferase is glucosaminyl (N-acetyl) transferase 2 (GCNT2) and the second transferase is β-1,3-N-acetyl-hexosaminyl transferase (NmLgtA) from Neisseria meningitidis.

[0596] 79. The method of any one of embodiments 1-78, wherein the glucose is D-glucose.

[0597] 80. The method of any one of embodiments 1-79, wherein the galactose is D-galactose.

[0598] 81. The method of any one of embodiments 1-80, wherein the fucose is L-fucose.

[0599] 82. A glycosylated compound prepared according to any one of embodiments 1-81.

[0600] 83. A galactosylated compound prepared according to any one of embodiments 1-39.

[0601] 84. A sialylated compound prepared according to any one of embodiments 1-25, 40-52 or 79-81.

[0602] 85. A fucosylated compound prepared according to any one of embodiments 1-25, 53-66 or 79-81.

[0603] 86. An N-acetylglucosaminylated compound prepared according to any one of embodiments 1-25, 67-78 or 79-81.

[0604] 87. A lactose derivative prepared by the method according to any one of embodiments 1-81.

[0605] 88. The lactose derivative according to claim 87, which is selected from lactobionic acid, lactitol, lactulooligosaccharides, galactoligosaccharides, lactulose and HMO.

[0606] 89. A compound prepared by the method of any one of embodiments 1-81, wherein the compound is obtained from a non-animal based plant material.

[0607] 90. A machine configured for the method of any one of embodiments 1-81.

[0608] 91. A machine according to embodiment 90, wherein the process is carried out in a single reaction vessel.

[0609] In order to more fully understand the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and should not be construed as limiting the present invention in any way. Example

[0610] abbreviation

[0611] 3'-SL: 3' sialyllactose

[0612] 2'-FL: 2'-fucosyllactose

[0613] LNFPI: Lactose-N-fucopentaose I

[0614] LNFPII: Lactose-N-fucopentaose II

[0615] LNFPIII: Lactose-N-fucopentaose III

[0616] LNT: lactose-N-tetraose

[0617] LNnT: lactose-N-neotetraose

[0618] LNTII: Lactose-N-triose II

[0619] DSLNT: disialyl lactose-N-tetraose

[0620] UDP: Uridine 5'-diphosphate

[0621] UTP: Uridine 5'-triphosphate

[0622] ADP: adenosine 5'-diphosphate

[0623] ATP: Adenosine 5'-triphosphate

[0624] CMP: Cytidine 5'-monophosphate

[0625] CDP: Cytidine 5'-diphosphate

[0626] CTP: Cytidine 5'-triphosphate

[0627] GDP: Guanosine 5'-diphosphate

[0628] GTP: Guanosine 5'-triphosphate

[0629] TDP: thymidine 5'-diphosphate

[0630] Tris: tris(hydroxymethyl)aminomethane

[0631] DMC: 2-chloro-1,3-dimethylimidazolinium chloride

[0632] Bbh1: β-N-acetylglucosaminidase

[0633] GOS: Galacto-oligosaccharide

[0634] DFL: difucosyllactose

[0635] 3-FL: 3-fucosyllactose

[0636] LSTa: Sialyl lactose-N-tetrasaccharide a / LS-tetrasaccharide a

[0637] LSTb: Sialyl lactose-N-tetrasaccharide b / LS-tetrasaccharide b

[0638] LSTc: Sialyl lactose-N-neotetrasaccharide c / LS-tetrasaccharide c

[0639] LSTd: Sialyl lactose-N-neotetrasaccharide d / LS-tetrasaccharide d

[0640] Cvb3GalT: β-1,3-galactosyltransferase from Chromobacterium violaceum

[0641] AtSuSy1: sucrose synthase from Arabidopsis thaliana

[0642] β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1)

[0643] ST3GAL3: CMP-N-acetylneuraminic acid-β-1,4-galactoside α-2,3-sialyltransferase 3

[0644] ST3GAL4: CMP-N-acetylneuraminic acid-β-galactosylamide-α-2,3-sialyltransferase 4

[0645] ST6GALNAC5: α-N-acetylgalactosamine α-2,6-sialyltransferase 5

[0646] ST3GAL4: CMP-N-acetylneuraminic acid-β-galactosylamide-α-2,3-sialyltransferase 4

[0647] Te2FT: α-1,2-fucosyltransferase from Thermosynechococcus tricholoma

[0648] Hp34FT: α1-3 / 4-fucosyltransferase from Helicobacter pylori

[0649] FUT9: Fucosyltransferase 9

[0650] FUT3: Fucosyltransferase 3

[0651] HmFucT: α-1,2-fucosyltransferase from Helicobacter pylori (HmFucT)

[0652] FUT1: fucosyltransferase

[0653] Pyr: pyruvic acid

[0654] AcPi: acetyl phosphate

[0655] PPi: Inorganic pyrophosphate

[0656] Pi: inorganic phosphate

[0657] NADP+: oxidized form of nicotinamide adenine dinucleotide phosphate

[0658] NADPH: Nicotinamide adenine dinucleotide phosphate reduced form

[0659] PolyP n and PolyP n-1 : polyphosphate; n represents the number of phosphate units

[0660] HK: Hexokinase

[0661] FDH: Formate dehydrogenase

[0662] PtxD: phosphite oxidoreductase

[0663] GOS: Galacto-oligosaccharide

[0664] Lactose synthase EC 2.4.1.22: Produces lactose from glucose and UDP-galactose. It consists of a 1:1 complex of galactosyltransferase (B4GALT1 or B4GALT2) and α-lactalbumin.

[0665] UDP-sugar pyrophosphorylase (USP) EC 2.7.7.64: catalyzes the reversible transfer of a uridine group from UTP to sugar-1-phosphate, producing UDP-sugar and pyrophosphate (PPi)

[0666] UTP-glucose-1-phosphate uridyltransferase (GalU) EC 2.7.7.9: catalyzes the conversion of glucose-1-phosphate or galactose-1-phosphate into UDP-glucose or UDP-galactose, respectively.

[0667] Galactokinase (GalK) EC 2.7.1.6: catalyzes the phosphorylation of galactose to galactose-1-phosphate (Gal-1P) by consuming one ATP unit.

[0668] Inorganic pyrophosphatase (Ppa) EC 3.6.1.1: catalyzes the hydrolysis of pyrophosphate (PPi) into two monophosphate ions (Pi).

[0669] Alkaline phosphatase EC3.1.3.1: catalyzes the hydrolysis of nucleotides (NMP, NDP, NTP) into the corresponding nucleosides (N) and phosphate.

[0670] CIAP: calf intestinal alkaline phosphatase

[0671] Example 1: Cell-free production of simple glycans

[0672] 1a. Reagents and Materials:

[0673] The following chemical reagents were used to synthesize carbohydrates, glycans, or human milk oligosaccharides: sucrose (Carbosynth: OS02339), glucose (α-D(+)-glucose, 99+%, anhydrous, Thermo Scientific: AC170080010), CMP (cytidine 5′-monophosphate disodium salt; Carbosynth: NC05637), UDP (uridine 5′-diphosphate disodium salt; Carbosynth: NU03399), GDP (guanosine 5′-diphosphate disodium salt; Carbosynth: NG09782), 2′-fucosyllactose (2′-FL; Carbosynth: OF06739), 3′-sialyllactose (3′-SL; Carbosynth: OS04397), lactose (D-lactose monohydrate; Fisher Scientific: L5-500), GlcNAc (N-acetyl-D-glucosamine; Sigma-Aldrich: Sigma-Aldrich: Sigma-Aldrich: Sigma-Aldrich: Sigma-Aldrich: Sigma-Aldrich: Sigma-Aldrich: Sigma-Aldrich: Sigma-Aldrich: Sigma-Aldrich: Aldrich: A8625), Tris (TRIS, 1.0 M buffer solution, pH 7.5; Alfa Aesar: J62993AP), HEPES (Thermo Scientific: AAA1477730), PIPES (Thermo Scientific: AC172615000) and MgCl2 (magnesium chloride; Macron Fine Chemicals: 595804). All water was obtained from BarnStead Nanopure water purifier (Thermo Scientific, 18.5 MOhm-cm). LNTII, LNT and LNnT were all produced in-house from lactose.

[0674] Mammalian enzymes ST3GAL3, ST3GAL4, ST6GAL1, B4GALT1, B3GALT5, B3GNT2, FUT9, and ST6GalNAc5 were purchased from Glyco Expression Technologies Inc. (Athens, Georgia). Lactase was purchased from Sunson Enzymes, and human α-lactalbumin was purchased from Athens Research & Technology. SuSy (sucrose synthase; source: Glycine Max), GalE (UDP-glucose 4-epimerase; source: Bifidobacterium longum), NmLgtB (β-1,4-galactosyltransferase; source: Neisseria meningitidis), Cvβ3GalT (β-1,3-galactosyltransferase; source: Chromobacterium violaceum), Te2FT (α-1,2-fucosyltransferase; source: Thermosynechococcus tricholoma), HmFucT (α-1,2-fucosyltransferase; source: Helicobacter pylori) and Hp34FT (α1-3 / 4-fucosyltransferase; source: Helicobacter pylori) were all recombinantly produced in-house in Escherichia coli (BL21 / DE3). The enzymes were purified from soluble lysates by affinity chromatography (NiNTA) and buffer exchanged by dialysis against 50 mM Tris pH 7.5. The enzymes were supplemented with 10% (w / w) glycerol and stored frozen at -80° C. Plasmids for protein expression were generated by Genewiz or Genscript by custom synthesis of inserts and splicing into a commercial pET28a vector (Novagen).

[0675] Example 1b: LSTa and LNT

[0676] 1b. Reagents and Materials: The following chemical reagents were used to synthesize lactose, LSTa, and LNT: sucrose (Carbosynth: OS02339), glucose (α-D(+)-glucose, 99+%, anhydrous, Thermo Scientific: AC170080010), CMP (cytidine 5'-monophosphate disodium salt; Carbosynth: NC05637), UDP (uridine 5'-diphosphate disodium salt; Carbosynth: NU03399), 3'-sialyllactose (3'-SL; Carbosynth: OS04397), lactose (D-lactose monohydrate; Fisher Scientific: L5-500), Tris (TRIS, 1.0 M buffer, pH 7.5; Alfa Aesar: J62993AP), HEPES (Thermo Scientific: AAA1477730), PIPES (Thermo Scientific: AC172615000) and MgCl2 (magnesium chloride; Macron Fine Chemicals: 595804). All water was obtained from BarnStead Nanopure water purifier (Thermo Scientific, 18.5 MOhm-cm). Both LNTII and LNT were produced in-house from lactose.

[0677] Mammalian enzymes ST3GAL3, ST3GAL4, ST6GAL1, B4GALT1, B3GALT5, B3GNT2, FUT9, and ST6GalNAc5 were purchased from Glyco Expression Technologies Inc. (Athens, Georgia). Lactase was purchased from Sunson Enzymes, and human α-lactalbumin was purchased from Athens Research & Technology. SuSy (sucrose synthase; source: soybean), GalE (UDP-glucose 4-epimerase; source: Bifidobacterium longum), NmLgtB (β-1,4-galactosyltransferase; source: Neisseria meningitidis), Cvβ3GalT (β-1,3-galactosyltransferase; source: Chromobacterium violaceum), Te2FT (α-1,2-fucosyltransferase; source: Thermosynechococcus fasciatus), HmFucT (α-1,2-fucosyltransferase; source: Helicobacter pylori) and Hp34FT (α1-3 / 4-fucosyltransferase; source: Helicobacter pylori) were all recombinantly produced in-house in Escherichia coli (BL21 / DE3). The enzymes were purified from soluble lysates by affinity chromatography (NiNTA) and buffer exchanged by dialysis against 50 mM Tris pH 7.5. The enzymes were supplemented with 10% (w / w) glycerol and stored frozen at -80° C. Plasmids for protein expression were generated by Genewiz or Genscript by custom synthesis of inserts and splicing into a commercial pET28a vector (Novagen).

[0678] Example 2a: Production of LNT, LNnT and lactose by galactosylation

[0679] The following examples illustrate the stepwise synthesis of LNT, LNnT, lactose, GOS, LNFPI, LNFPII, LNFPIII, LSTa, LSTc, LSTd, and DSLNT, as shown in Figures 1-3. The target glycan can be synthesized stepwise by sequentially combining the following examples. Alternatively, the target glycan can be synthesized from any higher glycan building blocks.

[0680] A. Synthesis of LNT from LNTII

[0681] LNT( Figure 1B The reaction mixture consisted of 25 mM LNTII, 50 mM sucrose, 5 mM UDP, 400 μg / ml SuSy, 800 μg / ml Cvb3GalT, 400 μg / ml GalE and 10 mM MgCl2 in 50 mM Tris pH 7.0 at 37°C.

[0682] B. Synthesis of LNnT from LNTII

[0683] LNnT ( Figure 1C The reaction mixture consisted of 25 mM LNTII, 50 mM sucrose, 0.1-10 mM UDP, 400 μg / ml SuSy, 800 μg / ml Cvb3GalT, 400 μg / ml GalE and 10 mM MgCl2 in 20-100 mM buffer with a pH range of 6.5 to 7.5 at 37°C.

[0684] C. Synthesis of lactose from glucose

[0685] Four enzymes, B4GALT1, α-lactalbumin, SuSy, and GalE, are used to produce lactose from glucose, sucrose, and a catalytic amount of UDP ( Figure 1E The reaction mixture consisted of 25 mM glucose, 100 mM sucrose, 0.1-10 mM UDP, 100 μg / ml B4GALT1, 100 μg / ml α-lactalbumin, 400 μg / ml SuSy, 400 μg / ml GalE, and 10 mM MgCl2 in 20-100 mM buffer with a pH range of 6.5 to 7.5 at 37°C.

[0686] D. Synthesis of galacto-oligosaccharides (GOS) from sucrose

[0687] GOS ( Figure 1F The reaction mixture consisted of 2 M sucrose, 0.1-10 mM UDP, 400 μg / ml SuSy, 800 μg / ml β-1,4-galactosyltransferase, 400 μg / ml GalE, and 10 mM MgCl2 in 20-100 mM buffer with a pH range of 6.5 to 7.5 at 37°C.

[0688] Example 2b: Production of LSTa, LSTc, LSTd and DSLNT. Sialylation.

[0689] A. Synthesis of LSTa from LNT

[0690] Three enzymes, ST3GAL4, ST3GAL3 and lactase, were used to generate LSTa from LNT, 3'-SL and a catalytic amount of CMP. Figure 2AThe reaction mixture consisted of 25 mM LNT, 30 mM 3'-SL, 0.1-10 mM CMP, 100 μg / ml ST3GAL4, 150 μg / ml ST3GAL3, 400 μg / ml lactase and 10 mM MgCl2 in 20-100 mM buffer with a pH range of 6.5 to 7.5 at 37°C.

[0691] B. Synthesis of LSTc from LNnT

[0692] LSTc ( Figure 2B The reaction mixture consisted of 25 mM LNnT, 30 mM 3'-SL, 0.1-10 mM CMP, 400 μg / ml ST3GAL4, 400 μg / ml ST6GAL1, 400 μg / ml lactase and 10 mM MgCl2 in 20-100 mM buffer with a pH range of 6.5 to 7.5 at 37°C.

[0693] C. Synthesis of LSTd from LNnT

[0694] LSTd ( Figure 2C The reaction mixture consisted of 25 mM LNnT, 30 mM 3'-SL, 0.1-10 mM CMP, 400 μg / ml ST3GAL4, 400 μg / ml ST3GAL3, 400 μg / ml lactase and 10 mM MgCl2 in 20-100 mM buffer with a pH range of 6.5 to 7.5 at 37°C.

[0695] D. Synthesis of DSLNT from LNT

[0696] Four enzymes, ST3GAL4, ST3GAL3, ST6GALNAC5 and lactase, were used to generate DSLNT from LNT, 3'-SL and a catalytic amount of CMP ( Figure 2D The reaction mixture consisted of 25 mM LNT, 60 mM 3'-SL, 0.1-10 mM CMP, 400 μg / ml ST3GAL4, 400 μg / ml ST3GAL3, 400 μg / ml ST6GALNAC5, 400 μg / ml lactase and 10 mM MgCl2 in 20-100 mM buffer with a pH range of 6.5 to 7.5 at 37°C.

[0697] Example 2c. Production of LNFPI, II, III, etc. by fucosylation.

[0698] A. Synthesis of LNFPI from LNT

[0699] LNFPI was produced from LNT, 2'-FL and a catalytic amount of CMP using three enzymes: HmFucT, Te2FT and lactase. Figure 3A The reaction mixture consisted of 25 mM LNT, 30 mM 2'-FL, 0.1-10 mM GDP, 400 μg / ml HmFucT, 400 μg / ml Te2FT, 400 μg / ml lactase and 10 mM MgCl2 in 20-100 mM buffer with a pH range of 6.5 to 7.5 at 37°C.

[0700] B. Synthesis of LNFPII from LNT

[0701] LNFPII was generated from LNT, 2'-FL and a catalytic amount of CMP using three enzymes HmFucT, Hp34FT or FUT5 and lactase ( Figure 3B The reaction mixture consisted of 25 mM LNT, 30 mM 2'-FL, 0.1-10 mM GDP, 400 μg / ml HmFucT, 400 μg / ml Hp34FT or FUT5, 400 μg / ml lactase and 10 mM MgCl2 in 20-100 mM buffer with a pH range of 6.5 to 7.5 at 37°C.

[0702] C. Synthesis of LNFPIII from LNnT

[0703] LNFPIII was produced from LNnT, 2'-FL and a catalytic amount of CMP using three enzymes HmFucT, FUT9 and lactase. Figure 3C The reaction mixture consisted of 25 mM LNnT, 30 mM 2'-FL, 0.1-10 mM GDP, 400 μg / ml HmFucT, 400 μg / ml FUT9, 400 μg / ml lactase and 10 mM MgCl2 in 20-100 mM buffer with a pH range of 6.5 to 7.5 at 37°C.

[0704] Example 3a. Production of LNT and lactose by galactosylation.

[0705] A. Synthesis of LNT from LNTII

[0706] LNT( Figure 1B). The reaction mixture consisted of 25mM LNTII, 50mM sucrose, 5mM UDP, 400μg / ml SuSy, 800μg / ml Cvb3GalT, 400μg / ml GalE and 10mM MgCl2 in 50mM Tris pH7.0 at 37°C. The conversion from LNTII to LNT was monitored by HPLC (ThermoVanquish, Trinity P1 column, electrospray detector). Conversions of 10.6%, 37.4% and 77.7% were observed at 0.5 hours, 1 hour and 17 hours, respectively.

[0707] B. Synthesis of lactose from glucose

[0708] Lactose was produced from glucose, sucrose and a catalytic amount of UDP in a four-step process using four enzymes, B4GALT1, α-lactalbumin, SuSy and GalE. Figure 1E ). In the first step, the lactose synthase complex was assembled in a mixture consisting of 25mM glucose, 200μg / ml B4GALT1, 80μg / ml α-lactalbumin, 10mM MnCl2, 1mM CaCl2, and 50mM HEPES pH6.8, and incubated at 4°C for 1 hour. In the second step, UDP-galactose was synthesized in a reaction mixture consisting of 100μg / ml SuSy, 100μg / ml GalE, 6mM UDP, 50mM sucrose, and 10mM MnCl2 in 50mM HEPES pH6.8, and incubated at 37°C for 1 hour. In the third step, equal volumes of the two mixtures from the first and second steps were combined and incubated at 4°C for 30 minutes. During this process, UDP-galactose produced in the second step was used to stabilize the lactose synthase complex. In the fourth step, the reaction mixture was transferred to 37°C to promote the conversion of glucose to lactose. The conversion from glucose to lactose was monitored by HPLC (Thermo Vanquish, Trinity P1 column, electrospray detector). Conversions of 8.9%, 14.3%, 20.8% and 25.4% were observed at 0.25 hours, 0.5 hours, 1 hour and 17 hours, respectively.

[0709] Example 3b. Production of LSTa by sialylation of LNT.

[0710] Synthesis of LSTa from LNT

[0711] Three enzymes, ST3GAL4, ST3GAL3 and lactase, were used to generate LSTa from LNT, 3'-SL and a catalytic amount of CMP. Figure 2A). The reaction mixture consisted of 25 mM LNT, 30 mM 3'-SL, 5 mM CMP, 100 μg / ml ST3GAL4, 150 μg / ml ST3GAL3, 400 μg / ml lactase and 10 mM MgCl2 in 50 mM Tris pH 7.0 at 37°C. The conversion from LNT to LSTa was monitored by HPLC (ThermoVanquish, Trinity P1 column, electrospray detector). About 5% conversion was observed after 21 hours.

[0712] Example 4. Cell-free production of simple glycans

[0713] 4a. Reagents and Materials

[0714] The following chemical reagents were used to synthesize carbohydrates, glycans, or human milk oligosaccharides: sucrose (Carbosynth: OS02339), glucose (α-D(+)-glucose, 99+%, anhydrous, Thermo Scientific: AC170080010), CMP (cytidine 5'-monophosphate disodium salt; Carbosynth: NC05637), UDP (uridine 5'-diphosphate disodium salt; Carbosynth: NU03399), GDP (guanosine 5'-diphosphate disodium salt; Carbosynth: NG09782), 2'-fucosyllactose (2'-FL; Carbosynth: OF06739), 3'-sialyllactose (3'-SL; Carbosynth: OS04397), lactose (D-lactose monohydrate; Fisher Scientific: L5-500), lactose-N-disaccharide (LNB; Chemily Scientific: L5-500), and lactose-N-disaccharide (LNB; Chemily Scientific: L5-500). GlycoScience: OS03002); GlcNAc (N-acetyl-D-glucosamine; Sigma Aldrich: A8625), Tris (TRIS, 1.0 M buffer solution, pH 7.5; AlfaAesar: J62993AP), HEPES (Thermo Scientific: AAA1477730), PIPES (Thermo Scientific: AC172615000) and MgCl2 (magnesium chloride; Macron Fine Chemicals: 595804). All water was obtained from BarnStead Nanopure water purifier (Thermo Scientific, 18.5 MOhm-cm). LNTII, LNT and LNnT were all produced in-house from lactose.

[0715] Mammalian enzymes ST3GAL3, ST3GAL4, ST6GAL1, B4GALT1, B3GALT5, GCNT2, B3GNT2, FUT1, FUT3, FUT9, and ST6GalNAc5 were purchased from Glyco Expression Technologies Inc. (Athens, Georgia). Lactase was purchased from Sunson Enzymes, and human α-lactalbumin was purchased from Athens Research & Technology. SuSy (sucrose synthase; source: soybean), GalE (UDP-glucose 4-epimerase; source: Bifidobacterium longum), NmLgtB (β-1,4-galactosyltransferase; source: Neisseria meningitidis), NmLgtA (β1,3-N-acetyl-hexosaminyltransferase; source: Neisseria meningitidis), Cvβ3GalT (β-1,3-galactosyltransferase; source: Chromobacterium violaceum), Te2FT (α-1,2-fucosyltransferase; source: Thermosynechococcus tricholoma), HmFucT (α-1,2-fucosyltransferase; source: Helicobacter pylori), GsAI (D-galactose isomerase; source: Geobacillus stearothermophilus), and Hp34FT (α1-3 / 4-fucosyltransferase; source: Helicobacter pylori) were recombinantly produced in-house in Escherichia coli (BL21 / DE3). The enzyme was purified from the soluble lysate by affinity chromatography (NiNTA) and the buffer was exchanged by dialysis against 50 mM Tris pH 7.5. The enzyme was supplemented with 10% (w / w) glycerol and stored frozen at -80°C. Plasmids for protein expression were generated by Genewiz or Genscript by custom synthesizing inserts and splicing into commercial pET28a vectors (Novagen).

[0716] 4b. Reagents and Materials:

[0717] Lactose, LNT and LNnT were synthesized using the following chemical reagents: sucrose (Carbosynth: OS02339), glucose (α-D(+)-glucose, 99+%, anhydrous, Thermo Scientific: AC170080010), UDP (uridine 5'-diphosphate disodium salt; Carbosynth: NU03399), lactose (D-lactose monohydrate; Fisher Scientific: L5-500), disodium phosphate heptahydrate (Fisher Chemical: S373-500) and MgCl2 (magnesium chloride; Macron Fine Chemicals: 595804). All water was obtained from BarnStead Nanopure water purifier (Thermo Scientific, 18.5 MOhm-cm). LNTII was produced in-house from lactose.

[0718] AtSuSy1 (sucrose synthase; source: Arabidopsis thaliana), GalE (UDP-glucose 4-epimerase; source: Bifidobacterium longum), NmLgtB (β-1,4-galactosyltransferase; source: Neisseria meningitidis) and Cvβ3GalT (β-1,3-galactosyltransferase; source: Chromobacterium violaceum) were recombinantly produced in-house in Escherichia coli (BL21 / DE3). The enzymes were purified from soluble lysates by affinity chromatography (NiNTA) and the buffer was exchanged by dialysis against 50 mM Tris pH 7.5. The enzymes were supplemented with 10% (w / w) glycerol and stored frozen at -80°C. Plasmids for protein expression were generated by Genewiz or Genscript by custom synthesizing inserts and splicing into commercial pET28a vectors (Novagen).

[0719] Example 5a. Fucosylation to produce LNFPI, LNFPII, LNFPIII, DFL and 3-FL.

[0720] A. Synthesis of LNFPI from LNT

[0721] LNFPI was generated from LNT, 2'-FL and a catalytic amount of GDP using four enzymes: HmFucT, Te2FT, lactase and GsAI. Fig.11A The reaction mixture consisted of 25 mM LNT, 30 mM 2'-FL, 0.1-10 mM GDP, 400 μg / ml HmFucT, 400 μg / ml Te2FT, 400 μg / ml lactase and 10 mM MgCl2 in 20-100 mM buffer with a pH range of 4.5 to 7.5 at 37°C.

[0722] B. Synthesis of LNFPII from LNT

[0723] LNFPII was generated from LNT, 2'-FL and a catalytic amount of GDP using four enzymes: HmFucT, Hp34FT, lactase and GsAI. Fig. 11B The reaction mixture consisted of 25 mM LNT, 30 mM 2'-FL, 0.1-10 mM GDP, 400 μg / ml HmFucT, 400 μg / ml Hp34FT or FUT5, 400 μg / ml lactase and 10 mM MgCl2 in 20-100 mM buffer with a pH range of 4.5 to 7.5 at 37°C.

[0724] C. Synthesis of LNFPIII from LNnT

[0725] LNFPIII ( Fig. 11C The reaction mixture consisted of 25 mM LNnT, 30 mM 2'-FL, 0.1-10 mM GDP, 400 μg / ml HmFucT, 400 μg / ml FUT9, 400 μg / ml lactase and 10 mM MgCl2 in 20-100 mM buffer with a pH range of 4.5 to 7.5 at 37°C.

[0726] D. Synthesis of DFL from 2'-FL

[0727] HP34FT, lactase and GsAI ( Fig.11D The reaction mixture consisted of 50 mM 2'-FL, 0.1-10 mM GDP, 400 μg / ml HmFucT, 400 μg / ml Hp34FT, 400 μg / ml lactase, 400 μg / ml GsAI and 10 mM MgCl2 in 20-100 mM buffer with a pH range of 4.5 to 7.5 at 37°C.

[0728] E. Synthesis of 3-FL from 2'-FL

[0729] Five enzymes, HmFucT, HP34FT, lactase, GsAI, and fucosidase, were used to generate 3-FL from 2'-FL and a catalytic amount of GDP. Fig.11E The reaction mixture consisted of 50 mM 2'-FL, 0.1-10 mM GDP, 400 μg / ml HmFucT, 400 μg / ml Hp34FT, 400 μg / ml lactase, 400 μg / ml GsAI, 400 μg / ml fucosidase and 10 mM MgCl2 in 20-100 mM buffer with a pH range of 4.5 to 7.5 at 37°C.

[0730] Example 5b. N-acetylglucosamination with lactose-N-disaccharide.

[0731] A. Synthesis of LNTII from lactose-N-biose (LNB).

[0732] LNTII was produced from LNB, lactose and a catalytic amount of UDP using two enzymes, NmLgtA and GsAI ( FIG. 12A ). The reaction mixture consisted of 25 mM LNB, 30 mM lactose, 0.1-10 mM UDP, 400 μg / ml NmLgtA and 400 μg / ml GsAI and 10 mM MgCl in a 20-100 mM buffer with a pH range of 4.5 to 7.5 at 37°C.

[0733] B. Synthesis of β-1,6-GlcNAc-LNnT from LNnT.

[0734] The three enzymes NmLgtA (or B3GNT2), GCNT2, and GsAI were used to generate β-1,6-GlcNAc-LNnT ( Fig. 12B The reaction mixture consisted of 25 mM LNnT, 30 mM LNB, 0.1-10 mM UDP, 400 μg / ml NmLgtA, 400 μg / ml GCNT2 and 400 μg / ml GsAI and 10 mM MgCl2 in 20-100 mM buffer with a pH range of 4.5 to 7.5 at 37°C.

[0735] Example 6

[0736] A. Synthesis of LNT from LNTII

[0737] LNT( Fig. 9B ). The reaction mixture consisted of 250 mM LNTII, 1.0 M sucrose, 1 mM UDP, 250 μg / ml AtSuSy1, 500 μg / ml Cvb3GalT, 500 μg / ml GalE and 10 mM MgCl2 in 50 mM phosphate buffer pH 6.6 at 37°C. The conversion from LNTII to LNT was monitored by HPLC (Agilent 1100, amino column, evaporative light scattering detector). A conversion of 86% was observed after 16 hours, 1 hour and 17 hours.

[0738] B. Synthesis of LNnT from LNTII

[0739] LNnT ( Fig. 9C ). The reaction mixture consisted of 500mM LNTII, 1.0M sucrose, 5mM UDP, 400μg / ml AtSuSy1, 200μg / ml NmLgtB, 400μg / ml GalE and 10mM MgCl2 in 250mM phosphate pH 6.0 at 37°C. The conversion from LNTII to LNT was monitored by HPLC (Agilent 1100, amino column, evaporative light scattering detector). 36%, 64%, 77% and 96% conversion were observed at 2 hours, 4 hours, 6 hours and 24 hours, respectively, to produce 500mg LNnT product.

[0740] C. Synthesis of lactose from glucose

[0741] Three enzymes, NmLgtB, AtSuSy1, and GalE, are used to produce lactose from glucose, sucrose, and a catalytic amount of UDP ( Fig.9E ). The reaction mixture consisted of 500 mM glucose, 1.0 M sucrose, 5 mM UDP, 400 μg / ml NmLgtB, 400 μg / ml AtSuSy1, 400 μg / ml GalE and 10 mM MgCl2 in 250 mM phosphate pH 6.0 at 37°C. The conversion from glucose to lactose was monitored by TLC using p-anisaldehyde stain. Approximately 50% conversion was observed after 24 hours. This yielded 2.5 grams of lactose product.

[0742] Example 7: Synthesis of lactose from glucose

[0743] Lactose ( Fig.9F ). The reaction mixture consisted of 500 mM glucose, 1.0 M sucrose, 5 mM UDP, 400 μg / ml NmLgtB, 400 μg / ml AtSuSy1, 400 μg / ml GalE, 400 μg / ml glucose isomerase and 10 mM MgCl2 in 250 mM phosphate pH 6.0 at 37°C. The conversion from glucose to lactose was monitored by TLC using p-anisaldehyde stain. About 70% conversion was observed after 24 hours. Thus yielding (predicted) 3.0 grams of lactose product.

[0744] Example 8: Synthesis of lactose from glucose

[0745] Lactose ( Figure 9G ). The reaction mixture consisted of 500 mM glucose, 500 mM fructose, 500 mM sucrose, 5 mM UDP, 400 μg / ml NmLgtB, 400 μg / ml AtSuSy1, 400 μg / ml GalE, 400 μg / ml glucose isomerase and 10 mM MgCl2 in 250 mM phosphate pH 6.0 at 37°C. The conversion from glucose to lactose was monitored by TLC using p-anisaldehyde stain. About 70% conversion was observed after 24 hours. Thus yielding (predicted) 3.0 grams of lactose product.

[0746] Example 9: Synthesis of LSTa from LNT

[0747] LSTa was produced from LNT, 3'-SL and a catalytic amount of CMP using three enzymes, ST3GAL4, ST3GAL3 and lactase. The reaction mixture consisted of 5 mM LNT, 25 mM 3'-SL, 5 mM CMP, 300 μg / ml ST3GAL4, 300 μg / ml ST3GAL3, 300 μg / ml lactase and 20 mM MgCl2 in 25 mM phosphate pH 6.0 at 37°C. The formation of LSTa was confirmed by comparison with commercial LSTa (purchased from Biosynth) by silica gel TLC using a butanol / water / acetic acid mobile phase system (4:2.5:2), where Rf (LSTa) 0.16, Rf (LNT) It is 0.33Sss.

[0748] LSTa synthesis was further optimized by varying the equivalents of buffer (50 mM NaOAc; pH 5.5 and 50 mM Na3PO4; pH 6.0), 3'-SL (500 mM and 1 M), CMP (5 mM and 25 mM) at 250 mM LNT with and without lactase in the reaction mixture. It was observed that the presence of lactase and the use of a higher concentration of 3'-SL (1 M compared to 500 mM) resulted in higher LSTa conversion (about 20-25% based on TLC). Both 5 mM and 25 mM CMP reactions produced similar results. NaOAc buffer (pH 5.5) and Na3PO4 (pH 6.0) buffer also produced similar results.

[0749] Example 10: Synthesis of LSTb from LNT

[0750] LSTb was produced from LNT, 3'-SL and a catalytic amount of CMP using five enzymes, ST3GAL4, ST3GAL3, ST6GALNAC5, lactase and α2-3 neuraminidase S. The reaction mixture consisted of 25 mM LNT, 60 mM 3'-SL, 0.1-10 mM CMP, 400 μg / ml ST3GAL4, 400 μg / ml ST3GAL3, 400 μg / ml ST6GALNAC5, 400 μg / ml lactase, 100 μg / ml α2-3 neuraminidase S and 10 mM MgCl2 in 20-100 mM buffer with a pH range of 6.5 to 7.5 at 37°C.

[0751] Example 11: Glycosylase

[0752] The enzymes in Table 1 are used in the methods of the present invention. Other enzymes are disclosed herein. Table 1 provides E numbers, enzyme names, EC numbers, and examples of enzymes that can be used. Table 1 may refer to enzymes or genes encoding enzymes.

[0753] Table 1: Selected enzymes

[0754]

[0755]

[0756] Example 12: In situ GDP-L-fucose production using pyruvate-AcK / PyrOx+FDH regenerated 2'-FL synthesis

[0757] The enzymes E1, E1', E2, E3, E4, E5, E6, E7, E8, E9, E10, E11 and E12 used in Example 1 are immobilized. All reagents and cofactors, buffers, salts are added at the beginning. The enzymes are immobilized and, in the case of a flow reactor (e.g., a packed bed reactor), the above mixture of reagents flows through the flow reactor. Glucose, fructose or a mixture of glucose and fructose as well as the feedstock and the immobilized enzyme are introduced into the reactor. Fig.15 The conversions shown provide 2'FL or another fucosylated oligosaccharide or a fucosylated antibody-glycan conjugate.

[0758] D-mannose isomerase converts glucose or fructose into mannose. Glk (HK) converts mannose into mannose-6-phosphate and simultaneously converts ATP into ADP. RfbK (ManB) converts mannose-6-phosphate into mannose-1-phosphate. RfbM (ManC) converts mannose-1-phosphate into GDP-D-Man and simultaneously converts GTP into PPi. Gmd converts GDP-D-Man into GDP-4-keto-6-deoxymannose. GFS (WcaG) converts GDP-4-keto-6-deoxymannose into GDP-L-fucose and simultaneously converts NADPH into NADP+. Fucosyltransferase converts lactose and GDP-L-fucose into 2'FL. In the same reactor, the following reactions occur. PmPpa converts inorganic diphosphate into inorganic phosphate. AcK and pyruvate oxidase convert pyruvate and inorganic phosphate (generated by PmPpa) into CO2 and peroxide (catalase converts CO2 and peroxide into oxygen and water). FDH converts formate into CO2 while converting NADP+ into NADPH. Also in the same reactor, GDP is converted into GTP. 2'FL is obtained. See Fig.15 .

[0759] Example 13: In situ GDP-L-fucose production for 2'-FL synthesis using pyruvate-AcK / PyrOx+PtxD regeneration

[0760] The enzymes (immobilized form) E1, E1', E2, E3, E4, E5, E6, E7, E8', E9, E10, E11 and E12 are combined with the feedstock to carry out Fig.16 The reactions shown are performed to provide 2'FL or another fucosylated oligosaccharide or a fucosylated antibody-glycan conjugate.

[0761] Example 14: In situ GDP-L-fucose production for 2'-FL synthesis using PolyP-PPK+FDH regeneration

[0762] The enzymes (immobilized form) E1, E1', E2, E3, E4, E5, E6, E7, E8, E9' and E12 are combined with the feedstock to carry out Fig.17 The reactions shown are performed to provide 2'FL or another fucosylated oligosaccharide or a fucosylated antibody-glycan conjugate.

[0763] Example 15: In situ GDP-L-fucose production for 2'-FL synthesis using PolyP-PPK+PtxD regeneration

[0764] Enzymes (immobilized form) E1, E1', E2, E3, E4, E5, E6, E7, E8', E9' and E12 are combined with the raw material to carry out Fig.18 The reactions shown are performed to provide 2'FL or another fucosylated oligosaccharide or a fucosylated antibody-glycan conjugate.

[0765] Example 16: Enzymes

[0766] The enzymes in Table 2 are used in the methods of the present invention. Table 2 provides Figure 15-18 The E numbers used in the present invention, enzyme names, EC numbers and examples of enzymes that can be used are shown in Table 2. Table 2 may refer to enzymes or genes encoding enzymes.

[0767] Table 2: Integrated fucosylation pathway enzymes

[0768]

[0769] https: / / www.researchgate.net / figure / Production-of-D-mannose-from-D-fructose-and-D-glucose-using-different-enzymes-MIase_fig1_336675669. Lui et al., Foods, 9(12), 1809.

[0770] All publications and patent documents disclosed or cited herein are incorporated by reference in their entirety.

[0771] The above description is for illustration and description purposes only. The description is not intended to limit the invention to the precise form disclosed. The scope of the invention is intended to be defined by the appended claims.

Claims

1. A method for producing a glycosylation main product, comprising the following steps: a. contacting a catalytic amount of a sugar-nucleotide donor with a stoichiometric amount of an acceptor in the presence of a transferase to obtain a glycosylation main product and a catalytic amount of a nucleotide; as well as b. regenerating said nucleotide into a regenerated sugar-nucleotide donor by contacting said catalytic amount of said nucleotide with a stoichiometric amount of a sugar donor precursor in the presence of a transferase to obtain said regenerated sugar-nucleotide donor and a secondary product.

2. The method according to claim 1, further comprising: a. contacting a catalytic amount of the regenerated sugar-nucleotide donor with the stoichiometric acceptor in the presence of a transferase to obtain the glycosylation main product and the catalytic amount of nucleotide; as well as b. regenerating said nucleotide into said regenerated sugar-nucleotide donor by contacting said catalytic amount of said nucleotide with said stoichiometric amount of said sugar donor precursor in the presence of a transferase to obtain said regenerated sugar-nucleotide donor and said secondary product.

3. A method for producing a glycosylation main product, comprising the steps of adding a sugar donor precursor, an acceptor, a transferase and a catalytic amount of nucleotides to obtain a glycosylation main product and a secondary product.

4. The method of claim 3, wherein the sugar donor precursor and the nucleotide provide a sugar-nucleotide donor.

5. The method according to any one of claims 1 to 4, wherein the sugar donor precursor and the nucleotide provide a sugar-nucleotide donor precursor, wherein an auxiliary enzyme and the sugar-nucleotide donor precursor provide the sugar-nucleotide donor.

6. The method according to any one of claims 1 to 5, wherein the sugar donor precursor is a sugar comprising 2 or more sugar units.

7. The method of claim 6, wherein the sugar donor precursor is a sugar comprising 2, 3 or 4 sugar units.

8. The method of claim 7, wherein the sugar donor precursor is a disaccharide.

9. The method of claim 7, wherein the sugar donor precursor is a trisaccharide.

10. The method according to any one of claims 1 to 9, wherein the sugar donor precursor comprises galactose, sialic acid, fucose or N-acetylglucosamine.

11. The method according to claim 10, wherein the galactose, sialic acid, fucose or N-acetylglucosamine is located at the terminal position of the sugar donor precursor.

12. The method of any one of claims 1-11, wherein the sugar-nucleotide donor comprises galactose, sialic acid, fucose or N-acetylglucosamine.

13. The method of claim 13, wherein the sugar-nucleotide donor is galactose-nucleotide, sialic acid-nucleotide, fucose-nucleotide or N-acetylglucosamine-nucleotide.

14. The process according to any one of claims 1 to 13, wherein the secondary product is a monosaccharide, a disaccharide or a trisaccharide.

15. The method according to any one of claims 1 to 14, further comprising the step of contacting the secondary product with a processing enzyme to convert the secondary product into a secondary product derivative.

16. The method of any one of claims 1-15, wherein the secondary product derivative is converted to a modified secondary product derivative in the presence of a second processing enzyme.

17. The method according to any one of claims 1 to 16, further comprising the step of contacting the main product with a processing enzyme to convert the main product into a main product derivative.

18. The method according to any one of claims 15-17, wherein the processing enzyme is an oxidase, an isomerase or a hydrolase.

19. The method according to any one of claims 1 to 18, wherein the acceptor is an organic compound containing a hydroxyl group.

20. The method according to claim 19, wherein the organic compound containing a hydroxyl group is a sugar.

21. The method according to any one of claims 1 to 20, wherein the acceptor is obtained by converting the secondary product into the acceptor by the processing enzyme.

22. The method of any one of claims 1-21, wherein the nucleotide is uridine diphosphate, cytidine monophosphate or guanosine diphosphate.

23. The method according to any one of claims 1 to 22, wherein the amount of the nucleotide relative to the sugar donor precursor or the acceptor is 0.001 mol% to 10 mol%.

24. The method according to any one of claims 1 to 23, wherein the amount of the nucleotide relative to the sugar donor precursor or the acceptor is 0.01 mol% to 1 mol%.

25. The method of any one of claims 1-24, wherein the transferase is β-1,3-galactosyltransferase, sucrase synthase, β-1,4-galactosyltransferase, sialyltransferase, fucosyltransferase, or glucosaminyl (N-acetyl) transferase 2.

26. The method of any one of claims 1-25, wherein the transferase comprises a first transferase and a second transferase.

27. The method of any one of claims 1-26, wherein the first transferase catalyzes the transfer of a sugar from the sugar-nucleotide donor to the acceptor to obtain the glycosylation primary product, and the second transferase catalyzes the reaction of the nucleotide and the sugar donor precursor to obtain the sugar-nucleotide donor and the secondary product.

28. A method for producing a galactosylation main product, comprising the following steps: a. contacting a catalytic amount of a sugar-nucleotide donor with a stoichiometric amount of an acceptor to obtain a galactosylation main product and a catalytic amount of a nucleotide; as well as b. regenerating said nucleotide into a regenerated sugar-nucleotide donor by contacting said catalytic amount of said nucleotide with a stoichiometric amount of a sugar donor precursor to obtain said regenerated sugar-nucleotide donor and a secondary product.

29. The method of claim 1, further comprising: a. contacting a catalytic amount of the regenerated sugar-nucleotide donor with the stoichiometric acceptor to obtain the glycosylation main product and the catalytic amount of nucleotides; as well as b. regenerating said nucleotide into said regenerated sugar-nucleotide donor by contacting said catalytic amount of nucleotide with said stoichiometric sugar donor precursor to obtain said regenerated sugar-nucleotide donor and said secondary product.

30. A method for producing a galactosylation major product, comprising the steps of adding a sugar donor precursor, an acceptor, a transferase and a catalytic amount of nucleotides to obtain a glycosylation major product and a minor product.

31. The method of any one of claims 28-30, wherein the acceptor is lactose-N-trisaccharide II (LNTII), glucose, or galacto-oligosaccharide (GOS).

32. The method of any one of claims 28-31, wherein the secondary product is fructose.

33. The method according to any one of claims 28-32, comprising the step of obtaining said glucose by contacting said fructose in the presence of glucose isomerase, to obtain said glucose.

34. The method according to any one of claims 28 to 33, comprising the step of obtaining said LNTII by contacting lactose and N-acetylglucosamine in the presence of β-N-acetyhexosaminidase (Bbhl) to obtain said LNTII.

35. The method of any one of claims 28-34, wherein the sugar donor precursor is sucrose.

36. The method of any one of claims 28-35, wherein the sugar donor precursor and the nucleotide provide a sugar-nucleotide donor precursor, and wherein an auxiliary enzyme and the sugar-nucleotide donor precursor provide the sugar-nucleotide donor.

37. The method of any one of claims 28-36, wherein the sugar-nucleotide donor precursor is a glucose nucleotide.

38. The method according to any one of claims 28 to 37, wherein the auxiliary enzyme is galactose epimerase.

39. The method of any one of claims 28-38, wherein the nucleotide is uridine diphosphate.

40. The method of any one of claims 28-39, wherein the transferase comprises a first transferase and a second transferase.

41. The method of claim 40, wherein the first transferase is a β-1,3-galactosyltransferase from Chromobacterium violaceum (Cvb3GalT), a β-1,4-galactosyltransferase from Neisseria meningitidis (NmLgtB), and the second transferase is a sucrose synthase from Arabidopsis thaliana (AtSuSy1).

42. The method of any one of claims 28-41, wherein the main galactosylation product is lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lactose or GOS.

43. The method of any one of claims 28-42, further comprising the step of contacting the secondary product or the primary product with a processing enzyme to convert the secondary product into a secondary product derivative or to convert the primary product into a primary product derivative.

44. The method of claim 43, wherein the processing enzyme is an oxidase, an isomerase or a hydrolase.

45. The method of claim 44, wherein the processing enzyme is an oxidase and the galactosylation main product derivative is oxidized fructose or oxidized glucose.

46. ​​A method for producing a sialylated main product, comprising the steps of: a. contacting a catalytic amount of a sugar-nucleotide donor with a stoichiometric amount of an acceptor to obtain a sialylated main product and a catalytic amount of a nucleotide; as well as b. regenerating said nucleotide into a regenerated sugar-nucleotide donor by contacting a catalytic amount of said nucleotide with a stoichiometric amount of a sugar donor precursor to obtain said regenerated sugar-nucleotide donor and a secondary product.

47. The method of claim 40, further comprising: a. contacting a catalytic amount of the regenerated sugar-nucleotide donor with the stoichiometric acceptor to obtain the glycosylation main product and the catalytic amount of nucleotides; as well as b. regenerating said nucleotide into said regenerated sugar-nucleotide donor by contacting said catalytic amount of nucleotide with said stoichiometric sugar donor precursor to obtain said regenerated sugar-nucleotide donor and said secondary product.

48. A method for producing a sialylated major product, comprising the steps of adding a sugar donor precursor, an acceptor, a transferase and a catalytic amount of nucleotides to obtain a sialylated major product and a minor product.

49. The method of any one of claims 46-48, wherein the sugar donor precursor is 3'-sialyllactose.

50. The method of any one of claims 46-49, wherein the sugar-nucleotide donor is N-acetylneuraminic acid nucleotide.

51. The method of any one of claims 46-50, wherein the secondary product is lactose.

52. The method of any one of claims 46-51, wherein the main sialylation product is sialyllactose-N-tetraose a (LSTa), sialyllactose-N-tetraose b (LSTb), sialyllactose-N-neotetraose c (LSTc), sialyllactose-N-neotetraose d (LSTd) or disialyllactose-N-tetraose (DSLNT).

53. The method of any one of claims 46-52, further comprising the step of contacting the secondary product or the primary product with a processing enzyme to convert the secondary product into a secondary product derivative or to convert the primary product into a primary product derivative.

54. The method of claim 53, wherein the processing enzyme is an oxidase, an isomerase, or a hydrolase.

55. A method according to claim 53 or claim 54, wherein the secondary product is converted to a secondary product derivative in the presence of a processing enzyme.

56. The method of claim 55, wherein the processing enzyme is lactase.

57. The method of claim 55, wherein the secondary product derivatives are glucose and galactose.

58. The method of claim 53, wherein the processing enzyme is an enzyme that oxidizes the lactose to lactobionic acid.

59. The method of claim 58, wherein the processing enzyme is lactose oxidase.

60. The method of claim 57, wherein the glucose is converted to a modified secondary product derivative in the presence of a second processing enzyme.

61. The method of claim 60, wherein the second processing enzyme is D-galactose isomerase and the modified secondary product derivative is tagatose.

62. The method of claim 61, wherein the galactose isomerase is derived from Geobacillus stearothermophilus (GsAI).

63. The method of claim 60, wherein the second processing enzyme is glucose oxidase and the modified secondary product derivative is gluconolactone.

64. The method of any one of claims 46-63, wherein the processing enzyme is an oxidase, an isomerase, or a hydrolase, and the main product derivative is a main product of oxidation, a main product of isomerization, or a main product of hydrolysis.

65. The method of claim 64, wherein the main product derivative is oxidized, isomerized or hydrolyzed LSTa, LSTb, LSTc, LSTd or DSLNT.

66. The method of any one of claims 46-65, wherein the nucleotide is cytidine monophosphate.

67. The method of any one of claims 46-66, wherein the transferase comprises a first transferase and a second transferase.

68. The method of claim 67, wherein the first transferase is β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1), CMP-N-acetylneuraminic acid-β-1,4-galactoside α-2,3-sialyltransferase 3 (ST3GAL3), or α-N-acetylgalactosamine α-2,6-sialyltransferase 5 (ST6GALNAC5), and the second transferase is CMP-N-acetylneuraminic acid-β-galactosamide-α-2,3-sialyltransferase 4 (ST3GAL4).

69. The method of claim 68, wherein the first transferase is ST3GAL3 and ST6GALNAC5.

70. A method for producing a fucosylated main product, comprising the steps of: a. contacting a catalytic amount of a sugar-nucleotide donor with a stoichiometric amount of an acceptor to obtain a fucosylated primary product and a catalytic amount of a nucleotide; as well as b. regenerating said nucleotide into a regenerated sugar-nucleotide donor by contacting a catalytic amount of said nucleotide with a stoichiometric amount of a sugar donor precursor to obtain said regenerated sugar-nucleotide donor and a secondary product.

71. The method of claim 70, further comprising: a. contacting a catalytic amount of the regenerated sugar-nucleotide donor with the stoichiometric acceptor to obtain the glycosylation main product and the catalytic amount of nucleotides; as well as b. regenerating said nucleotide into said regenerated sugar-nucleotide donor by contacting said catalytic amount of nucleotide with said stoichiometric sugar donor precursor to obtain said regenerated sugar-nucleotide donor and said secondary product.

72. A method for producing a fucosylated major product, comprising the steps of adding a sugar donor precursor, an acceptor, a transferase and a catalytic amount of nucleotides to obtain a fucosylated major product and a minor product.

73. The method of any one of claims 70-72, wherein the sugar donor precursor is 2'-fucosyllactose.

74. The method of any one of claims 70-73, wherein the sugar-nucleotide donor is a fucose nucleotide.

75. The method of any one of claims 70-74, wherein the secondary product is lactose.

76. The method of any one of claims 70-75, wherein the major product is lactose-N-fucopentaose I (LNFPI), lactose-N-fucopentaose II (LNFPII), lactose-N-fucopentaose III (LNFPIII), difucosyllactose (DFL) or 3-fucosyllactose (3-FL).

77. The method of any one of claims 70-76, further comprising the step of contacting the secondary product or the primary product with a processing enzyme to convert the secondary product into a secondary product derivative or to convert the primary product into a primary product derivative.

78. The method according to claim 77, wherein the processing enzyme is an oxidase, an isomerase or a hydrolase.

79. The method of any one of claims 77 or claim 78, wherein the secondary product is converted to a secondary product derivative in the presence of a processing enzyme.

80. The method of claim 77, wherein the processing enzyme is an enzyme that oxidizes the lactose to lactobionic acid.

81. The method of claim 80, wherein the processing enzyme is lactose oxidase.

82. The method of claim 77, wherein the processing enzyme is lactase.

83. The method of claim 77, wherein the secondary product derivatives are glucose and galactose.

84. The method of any one of claims 77-83, wherein the secondary product derivative is converted to a modified secondary product derivative in the presence of a second processing enzyme.

85. The method of claim 84, wherein the second processing enzyme is D-galactose isomerase and the modified secondary product derivative is tagatose.

86. The method of claim 85, wherein the D-galactose isomerase is derived from Geobacillus stearothermophilus (GsAI).

87. The method of claim 84, wherein the second processing enzyme is glucose oxidase and the modified secondary product derivative is gluconolactone.

88. The method of any one of claims 70-87, wherein the principal product is converted to a principal product derivative in the presence of a processing enzyme.

89. The method of any one of claims 70-88, wherein the processing enzyme is an oxidase, an isomerase, or a hydrolase, and the main product derivative is a main product of oxidation, a main product of isomerization, or a main product of hydrolysis.

90. The method of claim 89, wherein the main product derivative is oxidized, isomerized or hydrolyzed LNFPI, LNFPII, LNFPIII, DFL or 3-FL.

91. The method of any one of claims 70-88, comprising the further step of oxidizing the fucosylated main product in the presence of an oxidase to provide a main product derivative.

92. The method of claim 91, wherein the major product derivative is oxidized LNFPI, LNFPII, LNFPIII, DFL or 3-FL.

93. The method of any one of claims 70-92, wherein the nucleotide is guanosine diphosphate.

94. The method of any one of claims 70-93, wherein the transferase comprises a first transferase and a second transferase.

95. The method of claim 94, wherein the first transferase is alpha-1,2-fucosyltransferase (Te2FT) from Thermosynechococcus vestitus, alpha 1-3 / 4-fucosyltransferase (Hp34FT), fucosyltransferase 9 (FUT9), or fucosyltransferase 3 (FUT3) from Helicobacter pylori, and the second transferase is alpha-1,2-fucosyltransferase (HmFucT) or fucosyltransferase (FUT1) from Helicobacter mustelae.

96. A method for producing an N-acetylglucosaminylated main product, comprising the steps of: a. contacting a catalytic amount of a sugar-nucleotide donor with a stoichiometric amount of an acceptor to obtain a major N-acetylglucosamine product and a catalytic amount of a nucleotide; as well as b. regenerating said nucleotide into a regenerated sugar-nucleotide donor by contacting a catalytic amount of said nucleotide with a stoichiometric amount of a sugar donor precursor to obtain said regenerated sugar-nucleotide donor and a secondary product.

97. The method of claim 1, further comprising: a. contacting a catalytic amount of the regenerated sugar-nucleotide donor with the stoichiometric acceptor to obtain the glycosylation main product and the catalytic amount of nucleotides; as well as b. regenerating said nucleotide into said regenerated sugar-nucleotide donor by contacting said catalytic amount of nucleotide with said stoichiometric sugar donor precursor to obtain said regenerated sugar-nucleotide donor and said secondary product.

98. A method for producing an N-acetylglucosaminylation major product, comprising the steps of adding a sugar donor precursor, an acceptor, a transferase, and a catalytic amount of nucleotides to obtain an N-acetylglucosaminylation major product and a minor product.

99. The method of any one of claims 96-98, wherein the sugar donor precursor is lactose-N-biose.

100. The method of any one of claims 96-99, wherein the sugar-nucleotide donor is an N-acetylglucosamine nucleotide.

101. The method of any one of claims 96-100, wherein the secondary product is galactose.

102. The method of any one of claims 96-101, wherein the major product is LNTII or β-1,6-GlcNAc-LNnT.

103. The method of any one of claims 96-102, further comprising the step of contacting the secondary product or the primary product with a processing enzyme to convert the secondary product into a secondary product derivative or to convert the primary product into a primary product derivative.

104. The method of claim 103, wherein the processing enzyme is an oxidase, an isomerase, or a hydrolase.

105. The method of any one of claims 103 or claim 104, wherein the secondary product is converted to a secondary product derivative in the presence of a processing enzyme.

106. The method of claim 105, wherein the processing enzyme is D-galactose isomerase and the secondary product derivative is tagatose.

107. The method of claim 106, wherein the D-galactose isomerase is derived from Geobacillus stearothermophilus (GsAI).

108. The method of any one of claims 96-107, comprising the further step of oxidizing the N-acetylglucosaminylation main product in the presence of an oxidase to provide a main product derivative.

109. The method of claim 107, wherein the major product derivative is oxidized and the major product is LNTII or β-1,6-GlcNAc-LNnT.

110. The method of any one of claims 96-109, wherein the nucleotide is uridine diphosphate.

111. The method of any one of claims 96-110, wherein the transferase is β-1,3-N-acetyl-hexosaminyltransferase (NmLgtA) from Neisseria meningitidis.

112. The method of any one of claims 96-110, wherein the transferase comprises a first transferase and a second transferase.

113. The method of claim 112, wherein the first transferase is glucosaminyl (N-acetyl) transferase 2 (GCNT2) and the second transferase is β-1,3-N-acetyl-hexosaminyl transferase (NmLgtA) from Neisseria meningitidis.

114. The method of any one of claims 96-113, comprising the further step of oxidizing the N-acetylglucosaminylated main product in the presence of an oxidase to provide a main product derivative.

115. The method of claim 114, wherein the main product derivative is oxidized LNTII or oxidized β-1,6-GlcNAc-LNnT.

116. The method of any one of claims 1-115, wherein the sugar is in a naturally occurring isomeric form.

117. The method of claim 116, wherein the glucose is D-glucose.

118. The method of claim 116, wherein the galactose is D-galactose.

119. The method of claim 116, wherein the fucose is L-fucose.

120. A glycosylated compound prepared according to any one of claims 1-119.

121. A galactosylated compound prepared according to any one of claims 1-27, 28-45 and 116-119.

122. A sialylated compound prepared according to any one of claims 1-27, 46-69 or 116-119.

123. A fucosylated compound prepared according to any one of claims 1-27, 70-95 or 116-119.

124. An N-acetylglucosaminated compound prepared according to any one of claims 1-27, 96-119.

125. A lactose derivative prepared by the method according to any one of claims 1-119.

126. The lactose derivative according to claim 125, which is selected from lactobionic acid, lactitol, lactulooligosaccharides, galactoligosaccharides, lactulose and HMO.

127. A compound prepared according to the method of any one of claims 1-119.

128. The compound of claim 127, wherein the compound is lactose, DSLNT, LNT, LNnT, LSTa, LSTb, LSTc, or LSTd.

129. A compound prepared by the method of claim 127 or claim 128, wherein the compound is obtained from a non-animal based plant material.

130. A machine configured for the method of any one of claims 1-119.

131. A machine according to claim 130, wherein the method process is carried out in a single reaction vessel.

132. A method for producing fucosylated oligosaccharides or fucosylated antibody-glycan conjugates, comprising the following steps: a. contacting the glucose or fructose in the presence of an enzyme that converts fructose or glucose into mannose; b. contacting the mannose with an enzyme that converts mannose into mannose-6-phosphate; c. contacting the mannose-6-phosphate with an enzyme that converts mannose-6-phosphate into mannose-1-phosphate; d. contacting the mannose-1-phosphate with an enzyme that converts the mannose-1-phosphate into GDP-D-mannose; e. contacting the GDP-D-mannose with an enzyme that converts GDP-D-mannose into GDP-4-keto-6-deoxymannose; f. contacting the GDP-4-keto-6-deoxymannose with an enzyme that converts the GDP-4-keto-6-deoxymannose into GDP-L-fucose; g. contacting the GDP-L-fucose with a disaccharide, an oligosaccharide or an antibody-glycan conjugate and an enzyme that fucosylates the disaccharide, the oligosaccharide or the antibody-glycan conjugate; as well as h. obtaining a fucosylated disaccharide, oligosaccharide or fucosylated antibody-glycan conjugate, wherein each of said enzymes is immobilized, and wherein said method comprises a set of regenerating enzyme systems to convert ADP to ATP, PPi to Pi, GDP to GTP and NADP+ to NADPH.

133. The method of claim 132, comprising immobilized regeneration enzymes formate dehydrogenase, pyruvate oxidase, acetate kinase, catalase and inorganic pyrophosphatase.

134. The method of claim 132, comprising immobilized regeneration enzymes phosphite dehydrogenase, pyruvate oxidase, acetate kinase, catalase, and inorganic pyrophosphatase.

135. The method of claim 132 comprising immobilized regeneration enzymes formate dehydrogenase, polyphosphate kinase, and inorganic pyrophosphatase.

136. The method of claim 132 comprising immobilized regeneration enzymes phosphite dehydrogenase, polyphosphate kinase and inorganic pyrophosphatase.

137. The method of any one of claims 132-136, wherein the enzyme that converts the glucose to mannose is immobilized D-mannose isomerase.

138. The method of any one of claims 132-136, wherein the enzyme that converts the fructose to mannose is immobilized N-acetyl-d-glucosamine-2-epimerase.

139. The method of any one of claims 132-136, wherein the enzyme that converts mannose to mannose-6-phosphate is immobilized hexokinase.

140. The method of any one of claims 132-136, wherein the enzyme that converts mannose-6-phosphate to mannose-1-phosphate is immobilized phosphomannose mutase.

141. The method of any one of claims 132-136, wherein the enzyme that converts mannose-1-phosphate to GDP-D-mannose is immobilized GDP-mannose pyrophosphorylase.

142. The method of any one of claims 132-136, wherein the enzyme that converts GDP-D-Man to GDP-4-keto-6-deoxymannose is immobilized GDP-mannose-4,6-dehydratase.

143. The method of any one of claims 132-136, wherein the enzyme that converts GDP-4-keto-6-deoxymannose to GDP-L-fucose is immobilized GDP-fucose synthase.

144. The method of any one of claims 132-136, wherein the enzyme that fucosylates the disaccharide, oligosaccharide or antibody-glycan conjugate is an immobilized fucosyltransferase.

145. The method of claim 132, wherein the GDP-mannose is polymerized to mannan by a transferase.

146. The method of any one of claims 132-144, wherein the oligosaccharide is 3'SL, LNTII, LNT, LNnT, LNFPI, LNFPII, LNFPIII, LSTa, LSTb, LSTc, LSTd and DSLNT, LNnH, 3"'3,3"'6-bis-O-α-Sia-LNnH3"'3,3"'6-bis-O-α-Sia-(3"3,3"6-bis-O-α-Fuc)-LNnH, dilinear sialylated or fucosylated lactose-N-neohexaose and neoheptasaccharides, α-2,3-sialyllactose-N-neopentose, linear fucosyl- and sialyllactose-N-neopentose, linear lactose-N-neopentose or dilinear lactose-N-neohexaose and heptasaccharides, and in certain embodiments, the polysaccharide is 3'SL In some embodiments, the polysaccharide is selected from the group consisting of LNTII, LNT, LNnT, LNFPI, LNFPII, LNFPIII, LSTa, LSTb, LSTc, LSTd, DSLNT, 2'-FL, LNnH, DSLNnH, or DSDFLNNH. In other embodiments, the polysaccharide is 3'SL, LNTII, 2'-FL, LNFPI, LSTa.

147. The method of any one of claims 132-144, wherein the oligosaccharide is lactose, LNT, LNnT, LNnH or LNH.

148. The method of any one of claims 132-147, wherein the fucosylated oligosaccharide is 2'FL or 3-FL.

149. The method of any one of claims 132-147, wherein the fucosylated oligosaccharide is a fucosylated HMO.

150. The method of claims 132-147, wherein the fucosylated oligosaccharide is fucosylated LDFT, fucosylated LNFP I, fucosylated LNFP II, fucosylated LNFP V, fucosylated LNDFHI, fucosylated LNDFHII, fucosylated DFLNHa, or fucosylated DFLNHc.

151. The method of any one of claims 132-150, wherein the method occurs in a single reaction vessel.

152. A machine comprising the immobilized enzyme of any one of claims 132-150, wherein the machine produces fucosylated disaccharides, fucosylated oligosaccharides, or fucosylated antibody-glycan conjugates.

153. The machine of claim 152, wherein the immobilized enzyme is contained within a single reaction vessel.

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