Biocatalytic production of sugar nucleotides

Abstract

The present invention provides production and isolation of sugar nucleotides. In some embodiments, the production or separation is accomplished under acidic conditions. Production is cell-free synthesis using enzymes, including immobilized enzymes. They may be accomplished using a one-pot reaction protocol. The synthesis can be used as a highly customizable and efficient cell-free manufacturing process. In some embodiments, sugar nucleotides are used to prepare UDP-Gal, lactose derivatives, and human milk oligosaccharides (HMOs).

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 / 355,450, filed on June 24, 2022, and is incorporated herein by reference in its entirety. Field of the Invention

[0003] The present invention provides compositions and methods for producing sugar nucleotide compounds and purification thereof. The method is a highly customizable, scalable and efficient manufacturing process. Background of the Invention

[0005] Cell-free systems have been of interest in biomanufacturing, but challenges remain (MP Cordoso Marques et al., Adv. Biochem. Eng. Biotechnol., https: / / doi.org / 10.1007 / 10_2020_160; C. You, Adv. Biochem. Engl, DOI: 10.1007 / 10_2012_159 (2012)); CH Hokke, et al., Glycoconjugate Journal 13.4:687-692 (1996); A. Zervosen and L. 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" J. Am. Chem. Soc. 118.8: 1836-1840 (1996). The aforementioned document is incorporated herein by reference in its entirety.

[0006] Therefore, the art seeks an economical and efficient method to produce polysaccharides, including human milk oligosaccharides (HMOs). These include, but are not limited to, polysaccharides of two sugar units or greater or four sugar units or greater. This would overcome the major obstacles in advancing the use of these polysaccharides and HMOs for probiotic, preventive and therapeutic purposes. Economical and efficient HMO production would be an important advance in global infant nutrition and disease prevention. Importantly, the present invention provides improved production efficiency of sugars and complex carbohydrates while reducing costs. This is a huge improvement over the prior art as it increases the accessibility and affordability of these molecules.

[0007] Glycans are complex carbohydrate structures that are major molecules on the cell surface and serve as the first contact point between cells and the extracellular matrix and with pathogens. Glycans are involved in a variety of 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.

[0008] Activated sugars are often used as research reagents to achieve the use of glycans or the discovery of new glycans. The use of glycans and activated sugars is a new technology that can be widely applied. Galactose-UDP is an activated sugar and a precursor building block of glycans in organisms. T. Jaroentomeechai et al., Frontiers in Chemistry, 8:645, (2020); M. Lemmerer et al., Advanced Synthesis & Catalysis 358, 3113-3122 (2016); Y. Zou et al., Carbohydrate Res. 373: 76-81 (2013); EP3819381A1; CN113265434A; G. Zhao et al., Nat. Protoc. 5: 636-646 (2010); A. Zervosen et al., J. Mol. Catal. B: Enzym. 5: 25-28 (1998); H. Tanaka et al., Angew. Chem. 124: 11699-11702 ( 2012), Angew.Chem.Int.Ed.51:11531-11534 (2012); S.Amann et al., Carbohydr.Res.,335:23-32 (2001); Y.Zhai et al., Biotechnol.Lett.,34:1321-1326 (2012); R.Marquardt et al., USPat.No.5,866,378; E.Baroja-Fernandez et al., Proc.Natl.Acad.Sci.USA 109:321-326 (2012). The foregoing documents are incorporated herein by reference in their entirety.

[0009] Therefore, the art seeks an economical and efficient method to produce nucleotide activated sugars, including but not limited to nucleotide-galactose compounds and UDP-galactose (https: / / pubchem.ncbi.nlm.nih.gov / compound / UDP-alpha-D-galactose, https: / / www.sigmaaldrich.com / US / en / product / sigma / u4500). This will overcome the major obstacles to advancing the production of nucleotide sugars (such as UDP-galactose, galactosylated polysaccharides and HMOs) for probiotic, preventive and therapeutic purposes. The economical and efficient production of nucleotide sugars and HMOs will be an important advance in global infant nutrition, drug development, disease treatment, preventive treatment and prevention. SUMMARY OF THE INVENTION

[0011] The invention provides methods for preparing sugar nucleotide compounds and methods for purifying them. These methods use phosphatase, which hydrolyzes nucleotides under the condition of not hydrolyzing sugar nucleotides in the presence of sugar nucleotides. In some embodiments, the invention provides methods for preparing nucleotide-galactose compounds starting from galactose. These methods are cost-effective and can be expanded industrially to produce UDP-galactose and other galactose compounds by increasing feed concentration, minimizing feed cost, reducing operating cost and adopting cGMP production process in some embodiments. In one embodiment, continuous manufacturing using immobilized enzymes provides a significant reduction in operating costs and allows easier adoption of cGMP processes.

[0012] The present invention provides a simple, cost-effective purification method that does not rely on chromatography. Sugar nucleotides can be used to synthesize sugar-containing compounds, and sugar nucleotides can be used in therapeutic, preventive and prophylactic treatment methods. This includes, but is not limited to, cancer treatment and triggering immune responses, including but not limited to immune responses in humans.

[0013] In one embodiment, the present invention provides a method for converting adenosine diphosphate into adenosine, comprising the step of contacting a first composition comprising adenosine diphosphate and sugar nucleotides with a phosphatase at an acidic pH to convert the first composition into a second composition comprising adenosine, phosphate and sugar nucleotides. Another embodiment further comprises an initial step of generating the first composition by contacting sugars, nucleotide triphosphates and adenosine triphosphate with a kinase, a nucleotide sugar pyrophosphorylase and an inorganic pyrophosphorylase.

[0014] In certain embodiments, the phosphatase is an alkaline phosphatase or an acid phosphatase. In certain embodiments, the alkaline phosphatase is an Antarctic phosphatase. In certain embodiments, the Antarctic phosphatase is a bacterial Antarctic phosphatase. In certain embodiments, the bacterial Antarctic phosphatase has Tab5 activity and has at least 90% sequence identity to SEQ ID NO: 1. In a preferred embodiment, Tab5 has the sequence of SEQ ID NO: 1.

[0015] Enzymes with sequence variations compared to SEQ ID NO: 1 but functioning as phosphatases are included in the present invention. In some embodiments, the enzyme used in the present invention has Tab5 activity and has at least 90% sequence identity to SEQ ID NO: 1. Enzymes contemplated by the present invention include, but are not limited to, enzymes with phosphatase activity similar to Tab5 but with sequences different from SEQ ID NO: 1. In some embodiments, Tab5-related phosphatases can be obtained, for example, by protein engineering.

[0016] In certain embodiments, the acidic pH is 6.9 or below. In other embodiments, the acidic pH is 5.0 to pH 6.9.

[0017] Some embodiments also include a processing step of separating sugar nucleotides and adenosine. In certain embodiments, the processing step is to selectively precipitate sugar nucleotides in the presence of an antisolvent or a water-soluble solvent. Some embodiments also include the following steps: concentrating the second composition to obtain a concentrate, adding salt to the concentrate to obtain a salted concentrate, adding an antisolvent or a water-soluble solvent to the salted concentrate to provide a precipitation solution, separating the precipitation solution to obtain a filtrate and a precipitate, and drying the precipitate to obtain sugar nucleotides. Some embodiments also include removing kinases, nucleotide sugar pyrophosphorylases, and inorganic pyrophosphorylases to provide an initial step of a filtered output composition. In some embodiments, filtering removes phosphatases, kinases, nucleotide sugar pyrophosphorylases, and inorganic pyrophosphorylases by filtering or adsorption methods. In certain embodiments, filtering is ultrafiltration, tangential flow filtration, or diafiltration. In some embodiments, concentration is about 2 to about 100 times to obtain a concentrate. In some embodiments, concentration is carried out by tangential flow nanofiltration.

[0018] In some embodiments, the salt is an ammonium salt, a lithium salt, a sodium salt, or a potassium salt. In certain embodiments, the sodium salt is sodium acetate or the potassium salt is potassium acetate. In some embodiments, sodium acetate is present at about 10 mM to about 200 mM. In other embodiments, potassium acetate is present at about 10 mM to about 200 mM.

[0019] In some embodiments, the water-soluble solvent is an alcohol. In certain embodiments, the alcohol is ethanol, methanol or isopropanol. In a preferred embodiment, the alcohol is ethanol. In some embodiments, the solvent volume of the water-soluble solvent is 2, 3, 4, 5, 6, 7, 8, 9 or 10 volumes of solvent relative to the volume of the salified concentrate. Certain embodiments of the present invention also include an incubation step, wherein the water-soluble solvent and the salified concentrate are incubated. In some embodiments, the temperature of the incubation step is about -20°C to about 4°C. In some embodiments, the incubation time of the incubation step is about 2 hours to about 24 hours.

[0020] In some embodiments, the separation is performed by a physical separation method. In certain embodiments, the physical separation method is filtration, centrifugation or decantation.

[0021] In some embodiments, a drying step is employed, and the drying is performed by spray drying, freeze drying, vacuum drying, or evaporative drying. In more specific embodiments, the drying is performed by spray drying.

[0022] In one embodiment, the present invention provides a method for preparing UDP-galactose, comprising the steps of contacting galactose, UTP, ATP, galactokinase, UDP-sugar pyrophosphorylase and inorganic pyrophosphatase to prepare UDP-galactose.

[0023] In one embodiment, the present invention provides a method for preparing UDP-galactose, comprising the step of contacting galactose, UTP, ATP, BiGalK, BlUSP, and PmPpa to prepare UDP-galactose.

[0024] In one embodiment, the present invention provides a method for preparing UDP-galactose, comprising the following steps:

[0025] a. contacting galactose, UTP, ATP, BiGalK, BlUSP, PmPpa to produce UDP-galactose in the reaction mixture and alkaline phosphatase; and

[0026] b. Purification of UDP-galactose by filtration.

[0027] In certain embodiments, the sugar is glucose, galactose, fructose, rhamnose, mannose, ribose, xylose, arabinose, fucose, apiose, glucuronic acid, galacturonic acid, N-acetylglucosamine, N-acetylgalactosamine, or N-acetylneuraminic acid.

[0028] In certain embodiments, the sugar is D-glucose, D-galactose, D-fructose, L-rhamnose, D-mannose, D-ribose, D-xylose, L-arabinose, D-xylose, L-fucose, D-apiose, D-glucuronic acid, D-galacturonic acid, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, or N-acetylneuraminic acid.

[0029] In other embodiments, the nucleotide is uridine 5'-diphosphate (UDP) and the sugar is D-glucose, D-galactose, D-fructose, L-rhamnose, D-mannose, D-ribose, D-xylose, L-arabinose, D-xylose, L-fucose, D-apiose, D-glucuronic acid, D-galacturonic acid, N-acetyl-D-glucosamine, or N-acetyl-D-galactosamine.

[0030] In other embodiments, the nucleoside is adenosine 5'-diphosphate (ADP) and the sugar is D-glucose, D-galactose, D-fructose, D-mannose, D-ribose, L-arabinose, D-xylose, or D-glucuronic acid.

[0031] In other embodiments, the nucleotide is guanosine 5'-diphosphate (GDP) and the sugar is D-glucose, D-galactose, D-mannose, L-rhamnose, D-ribose, D-xylose, L-arabinose, or L-fucose.

[0032] In other embodiments, the nucleotide is cytidine 5'-monophosphate (CMP) and the sugar is N-acetylneuraminic acid.

[0033] In other embodiments, the nucleotide is thymidine 5'-diphosphate (TDP) and the sugar is D-glucose or D-galacturonic acid.

[0034] In certain embodiments, phosphatase, kinase, nucleotide sugar pyrophosphorylase or inorganic pyrophosphorylase is a free enzyme. In other embodiments, phosphatase, kinase, nucleotide sugar pyrophosphorylase or inorganic pyrophosphorylase is an immobilized enzyme. In the embodiment using the immobilized enzyme, fixation is carried out by N-terminal tag or C-terminal tag. In such an embodiment, N-terminal tag or C-terminal tag is poly-arginine, poly-lysine or histidine-arginine repeat sequence.

[0035] In some embodiments, the sugar nucleotide pyrophosphorylase is uridine 5'-diphosphate-sugar-pyrophosphorylase (UDP-sugar pyrophosphorylase). In other embodiments, the nucleotide sugar pyrophosphorylase is UDP-sugar pyrophosphorylase (USP) or UTP-glucose 1-phosphate uridyl transferase (GalU). In other embodiments, the UDP-sugar pyrophosphorylase is BlUSP, EcUSP or SpUSP. In specific embodiments, the UDP-sugar pyrophosphorylase is BlUSP. In other embodiments, the UTP-glucose 1-phosphate uridyl transferase is SpGalU.

[0036] In some embodiments, the kinase is a galactokinase. In certain embodiments, the galactokinase is a galactokinase (BiGalK) from Bifidobacterium infantis, a galactokinase (SpGalK) from Streptococcus pneumoniae, a galactokinase (EcGalK) from Escherichia coli, or a galactokinase (LgGalK) from Leminorella grimontii. In a specific embodiment, the galactokinase is from Bifidobacterium infantis (BiGalK). L. Li et al., Carbohydrate Research 355: 35-39 (2012).

[0037] In certain embodiments, the inorganic pyrophosphatase is an inorganic pyrophosphatase from Pasteurella multocida (PmPpa) or an inorganic pyrophosphatase from Escherichia coli (EcPpa). In a specific embodiment, the inorganic pyrophosphatase is from Pasteurella multocida (PmPpa).

[0038] In some embodiments, the immobilized enzyme is immobilized by an iron oxide material. In certain embodiments, the iron oxide material is hematite, magnetite, or strontium ferrite. In certain embodiments, the immobilized enzyme is an A-type scaffold BNC. In other embodiments, the immobilized enzyme is a B-type scaffold BNC.

[0039] In some embodiments, one or more enzymes are immobilized in a bionanocatalyst (BNC), which in turn is embedded in a scaffold. The bionanocatalyst (BNC) according to the present invention comprises an enzyme self-assembled with magnetic nanoparticles (MNPs). The BNC self-assembles with the scaffold. In some embodiments, each enzyme is immobilized in a BNC.

[0040] 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, wherein each step of the synthesis is carried out in a module. In production mode, these modules contain a complete system for enzymes (sugar activation and sugar transfer) for the specialized construction of polysaccharides. In some embodiments, polysaccharides are oligosaccharides.

[0041] In some embodiments, the scaffold comprises a magnetic metal oxide. In some embodiments, the scaffold is a highly magnetic and high porosity composite blend of thermoplastics, which includes magnetic particles forming a powder. They can be a single layer or multilayer material that accommodates BNCs. Objects of this design can be produced by sintering composite magnetic powders 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. In a preferred embodiment, human milk oligosaccharides (HMOs) are produced.

[0042] Thus, the present invention provides cell-free production of sugar nucleotides using combinatorial bionanocatalysts (BNCs) organized in serial modules.

[0043] In preferred embodiments, the sugar nucleotide is UDP-galactose.In certain embodiments, the UDP-galactose has a purity of about or at least 97%, about or at least 98%, or about or at least 99%.

[0044] In certain embodiments, the method of the present invention occurs in a single reaction vessel. In some embodiments, the method is in batch, flow, semi-continuous or continuous flow conditions. In specific embodiments, the method does not include chromatography or chromatographic purification methods. In certain embodiments, the method is a cGMP production method.

[0045] In some embodiments, the invention provides sugar nucleotides prepared by a method according to any embodiment of the invention. In a preferred embodiment, UDP-galactose is prepared. In certain embodiments, the compounds of the invention are obtained from non-animal based plant materials.

[0046] Certain embodiments provide a machine configured for use in a method according to any embodiment of the invention.In some embodiments, the method is performed in one or two reaction vessels.

[0047] BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1A and Figure 1B A flow chart of the galactose-UDP synthesis is depicted.

[0049] Figure 2 Immobilized enzymes and methods for their preparation are described.

[0050] Figure 3A and Figure 3B Depicted is a flow chart of galactose-UDP synthesis using Tab5.

[0051] Figure 4 : Depicted is an SDS PAGE gel of Tab5 and the sequence of Tab5 SEQ ID NO: 1.

[0052] Figure 5 Depicted are HPLC traces showing the conversion of ADP to AMP and adenosine catalyzed by Tab in two different buffers at the 4 hour and 20 hour time points.

[0053] Detailed description of the invention

[0054] The present invention provides enhanced production efficiency of sugar nucleotides and related compounds (including but not limited to galactose-containing nucleotides, galactose-containing sugars and galactose-containing complex carbohydrates) while reducing costs. This improves the accessibility and affordability of these molecules. https: / / www.xenimmune.com / index.html (see the third picture of UDP-Gal); https: / / www.dsm.com / human-nutrition / en / products / hmos.html; M. Wiciński et al., Nutrients 12(1):266(2020); CA Autran et al., British Journal of Nutrition 116(2):294-299(2016); A. Rousseaux et al., Frontiers in Immunology 12:680911(2021); DR Hill et al., Nutrients 13(10):3364(2021); M. Zuurveld et al., Frontiers in Immunology 11:801(2020). The foregoing is incorporated herein by reference in its entirety.

[0055] The present invention provides methods for obtaining sugar nucleotide compounds and methods for purifying the obtained sugar nucleotide compounds. In this article, the terms sugar nucleotide and nucleotide sugar are used interchangeably. In one embodiment, the present invention provides a method for obtaining sugar nucleotides from a reaction mixture containing sugar nucleotides and adenosine diphosphate. In one embodiment, the present invention provides a method for preparing sugar nucleotides and then separating and purifying the sugar nucleotides from the reaction mixture. In certain embodiments, the method for preparing sugar nucleotides such as Figure 1A and Figure 3A As shown, the method for separating sugar nucleotides is as follows Figure 1B and Figure 3B shown.

[0056] In some embodiments, the present invention provides a method for obtaining sugar nucleotides from a reaction mixture comprising sugar nucleotides and adenosine diphosphate. In other embodiments, the present invention provides a method for preparing sugar nucleotides and then separating and purifying the sugar nucleotides from the reaction mixture. In certain embodiments, the method for preparing sugar nucleotides is as follows: Figure 1A and Figure 3A As shown, the method for separating sugar nucleotides is as follows Figure 1B and Figure 3B shown.

[0057] In some embodiments, the invention provides a method for reducing the amount of adenosine diphosphate in a sugar nucleotide composition by hydrolyzing adenosine diphosphate to adenosine and phosphate under neutral or acidic reaction conditions using phosphatase. Under these conditions, adenosine diphosphate is selectively hydrolyzed relative to sugar nucleotides. This allows the preparation of sugar nucleotides that are substantially free of nucleotide starting materials and nucleotide by-products, including but not limited to adenosine triphosphate, adenosine diphosphate and adenosine. Enzymatic hydrolysis under alkaline conditions at higher pH causes sugar nucleotides to be hydrolyzed to sugar and nucleotides.

[0058] In certain embodiments, a method for separating sugar nucleotides and nucleotide triphosphates is provided, comprising providing a composition comprising sugar nucleotides and nucleotide triphosphates, contacting the composition with a phosphatase at a pH of 6.9 or less. In some embodiments, a method for converting adenosine diphosphate to adenosine in the presence of sugar nucleotides is provided, comprising providing a composition comprising adenosine diphosphate and sugar nucleotides, contacting the composition with a phosphatase at a pH of 6.9 or less to convert adenosine diphosphate to adenosine and phosphate.

[0059] In certain embodiments, the present invention provides a method for converting adenosine diphosphate into adenosine in the presence of sugar nucleotides, comprising contacting a phosphatase with a composition comprising adenosine diphosphate and sugar nucleotides at an acidic pH to convert adenosine diphosphate into adenosine and phosphate. In other embodiments, a method for preparing a composition comprising adenosine and sugar nucleotides is provided, comprising providing a first composition comprising adenosine diphosphate, sugar nucleotides and a phosphatase at a pH of 6.9 or less to convert adenosine diphosphate into adenosine and phosphate, thereby providing a second composition comprising adenosine and sugar nucleotides.

[0060] Table 1 summarizes the nucleotide and sugar unit combinations that can be prepared according to the present invention.

[0061] Table 1: Nucleotide and sugar combinations

[0062]

[0063]

[0064] In certain embodiments, the sugar nucleotide is UDP-glucose, UDP-galactose, UDP-fructose, UDP-rhamnose, UDP-mannose, UDP-ribose, UDP-xylose, UDP-arabinose, UDP-xylose, UDP-fucose, UDP-apiose, UDP-glucuronic acid, UDP-galacturonic acid, UDP-N-acetyl-D-glucosamine, or UDP-N-acetyl-D-galactosamine.

[0065] 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.

[0066] In certain embodiments, the sugar nucleotide is UDP-D-glucose, UDP-D-galactose, UDP-D-fructose, UDP-L-rhamnose, UDP-D-mannose, UDP-D-ribose, UDP-D-xylose, UDP-L-arabinose, UDP-D-xylose, UDP-L-fucose, UDP-D-apiose, UDP-D-glucuronic acid, UDP-D-galacturonic acid, UDP-N-acetyl-D-glucosamine, UDP-N-acetyl-D-galactosamine.

[0067] In certain embodiments, the sugar nucleotide is ADP-glucose, ADP-galactose, ADP-fructose, ADP-mannose, ADP-ribose, ADP-arabinose, ADP-xylose or ADP-glucuronic acid. In certain embodiments, the sugar nucleotide is ADP-D-glucose, ADP-D-galactose, ADP-D-fructose, ADP-D-mannose, ADP-D-ribose, ADP-L-arabinose, ADP-D-xylose or ADP-D-glucuronic acid.

[0068] In certain embodiments, the sugar nucleotide is GDP-glucose, GDP-galactose, GDP-mannose, GDP-rhamnose, GDP-ribose, GDP-xylose, GDP-arabinose or GDP-fucose. In certain embodiments, the sugar nucleotide is GDP-D-glucose, GDP-D-galactose, GDP-D-mannose, GDP-L-rhamnose, GDP-D-ribose, GDP-D-xylose, GDP-L-arabinose or GDP-L-fucose.

[0069] In certain embodiments, the sugar nucleotide is CMP-N-acetylneuraminic acid. In certain embodiments, the sugar nucleotide is TDP-glucose or TDP-galacturonic acid. In certain embodiments, the sugar nucleotide is TDP-D-glucose or TDP-D-galacturonic acid.

[0070] Figure 1A and Figure 1B The synthesis route of Gal-UDP according to the present invention is depicted. In the presence of BiGalK, BlUSP and PmPpa, galactose, UTP, ATP, phosphate buffer and MgCl2 are mixed in a flow reactor. In the presence of ATP, the enzyme BiGalK converts galactose into galactose monophosphate and generates ADP. In the presence of UTP, the enzyme BlUSP converts galactose monophosphate into galactose diphosphate and generates pyrophosphate. In the presence of PmPpa, the generated pyrophosphate is converted into inorganic phosphate. The reaction mixture output from flow reactor 1 is galactose-UDP, ADP, ATP and phosphate. The output of flow reactor 1 is added to flow reactor 2. In the presence of bovine intestinal alkaline phosphatase, ADP and water are converted into adenosine and inorganic phosphate. The conversion of ADP into adenosine helps the purification process of the present invention. Nucleosides (here adenosine) rather than nucleotides (here ADP) will remain in the solution instead of being precipitated with sugar nucleotides in the precipitation step. Therefore, unreacted nucleotides are removed before precipitation. Therefore, in Figure 1A In the embodiment of the present invention, the output of flow reactor 2 is galactose-UDP, adenosine and phosphate. Then, the output mixture of flow reactor 2 is purified.

[0071] First, if free enzymes are used in the embodiments, they are removed prior to the concentration step. If immobilized enzymes are used, they will remain in their respective reaction vessels and will not be present in the reaction output (or will be present in small amounts). If free enzymes are used, they will be present in the output of each reaction and are preferably removed prior to product purification. Removal can be achieved by methods including, but not limited to, ultrafiltration. For clarity, if immobilized enzymes are used, an ultrafiltration step is not required, but it can still be used to ensure removal of any free enzyme. Therefore, if immobilized enzymes are used, the ultrafiltration step is not required, but it can still be used to ensure removal of any free enzyme. Figure 1B As shown, UDP-galactose is purified by concentration, addition of salt, addition of ethanol in the precipitation step, filtration and spray drying. In certain embodiments, the UDP-galactose method of the present invention uses free enzymes and performs a removal step, such as ultrafiltration, before concentration. Proteins are retained and removed after reactor 1 or reactor 2.

[0072] In certain embodiments, the methods of the invention involve selective precipitation of the obtained sugar nucleotides. Figure 1BDepicted is ethanol precipitation for selective precipitation in certain embodiments.

[0073] The aqueous product is added to ethanol (EtOH: 90-100% v / v) to selectively precipitate galactose-UDP while the remaining reactants are kept in solution. In certain embodiments, phosphate, MgCl2, adenosine and galactose remain in solution, and any remaining AMP, ADP and ATP and unreacted UTP are precipitated from the solution. The mixture is filtered and the retentate (galactose-UDP) is spray dried to obtain a dry product. The ethanol precipitation step can be repeated to further improve purity.

[0074] In certain embodiments of the invention, Tab5 is used to hydrolyze adenosine diphosphate into adenosine and phosphate. Figure 3A and Figure 3B Embodiments of the invention using Tab5 phosphatase are depicted. Figure 4 Involves Tab5 cloning, expression and characterization. Figure 4 Depicted are Tab5 sequences used in embodiments of the invention and an SDS PAGE gel of Tab5 shown to be expressed at >95% purity. Figure 5 Depicted is Tab5 activity assessment, which shows ADP hydrolysis under acidic pH conditions. Depicted is the percentage conversion of ADP to AMP and the qualitative conversion value to adenosine. Tab5 is a psychrophilic alkaline phosphatase from Antarctic bacteria Tab5 (Uniprot main accession number Q9KWY4: https: / / www.uniprot.org / uniprotkb / Q9KWY4 / entry); D. Koutsioulis, et al., Protein Engineering, Design & Selection 21, 5: 319-327 (2008); M. Rina, et al., Eur. J. Biochem. 267, 1230-1238 (2000); D.-H. Lee, et al., Biotechnology 15, 1 (2015), DOI 10.1186 / s12896-015-0115-2. The foregoing is incorporated herein by reference in its entirety. Without being bound by theory, the addition of MgCl2 and / or ZnCl2 can accelerate hydrolysis (buffer 2>buffer 1), while the addition of inorganic phosphate can inhibit hydrolysis. See Example 5.

[0075] Certain embodiments of the present invention provide a method for preparing a composition comprising adenosine and sugar nucleotides, comprising providing a first composition comprising adenosine diphosphate, sugar nucleotides, and Antarctic phosphatase (including but not limited to Tab5) to convert adenosine diphosphate into adenosine and phosphate, thereby providing a second composition comprising adenosine and sugar nucleotides.

[0076] The present invention provides methods for treating, separating, purifying, isolating, and concentrating to separate sugar nucleotides from a reaction mixture and separate them from adenosine.

[0077] In some embodiments, the present invention provides a method for preparing a sugar nucleotide compound, comprising contacting a kinase, an enzyme that converts galactose-1-phosphate and UTP into galactose-UDP, an inorganic pyrophosphatase, a sugar, a nucleotide and ATP to prepare a sugar nucleotide. In certain embodiments, the method comprises a step of further purifying the sugar nucleotide.

[0078] In some embodiments, the present invention provides a method for preparing a sugar nucleotide compound, comprising the steps of: contacting a kinase, an enzyme that converts galactose-1-phosphate and UTP to galactose-UDP, an inorganic pyrophosphatase, a sugar, a nucleotide, and ATP to obtain a first output; contacting the first output in the presence of alkaline phosphatase to obtain a second output; and purifying the second output to obtain a sugar nucleotide.

[0079] In certain embodiments, the method of the present invention purifies sugar nucleotides by a multi-step process, comprising the steps of filtering the second output to remove the enzyme, concentrating the second output to obtain a concentrate, adding salt to the concentrate to obtain a salted concentrate, adding an anti-solvent or a water-soluble solvent to the salted concentrate to provide a precipitation solution, separating the precipitation solution to obtain a filtrate and a precipitate, and drying the precipitate to obtain nucleotide sugars. If free (non-immobilized) enzymes are used, this process is useful. Certain embodiments include filtering the first output to remove the enzyme. Other embodiments include filtering the first output and the second output to remove the enzyme.

[0080] In certain embodiments, the filtering step removes the enzyme by filtration or adsorption. In certain embodiments, the filtration is ultrafiltration, tangential flow filtration or diafiltration. In specific embodiments, the filtering step is ultrafiltration.

[0081] Some embodiments of the present invention employ nucleotide sugar pyrophosphorylase to transfer nucleotide monophosphate groups from triphosphate nucleotides to phosphorylated sugars to provide sugar dinucleotides. In certain embodiments, the nucleotide sugar pyrophosphorylase is a UDP-sugar pyrophosphorylase. In certain embodiments, the enzyme that converts galactose-1-phosphate and UTP to galactose-UDP is a UDP-sugar pyrophosphorylase (USP) or a UTP-glucose 1-phosphate uridyltransferase. In other embodiments, the UDP-sugar pyrophosphorylase is BlUSP, EcUSP, or SpUSP. In certain embodiments, the UTP-glucose 1-phosphate uridyltransferase is SpGalU. Figure 1A and Figure 3AIn the embodiment shown, the nucleotide sugar pyrophosphorylase is BlUSP. MM Muthana et al., Chemical Communications 48.21:2728-2730 (2012) (incorporated herein by reference in its entirety).

[0082] Some embodiments of the invention employ a kinase to transfer a phosphate group from ATP to a sugar. In certain embodiments, the kinase is a galactokinase. In some embodiments, the galactokinase is BiGalK, SpGalK, EcGalK, or LgGalK. Figure 1A and Figure 3A In the embodiment shown, the kinase is BiGal K. L. Li et al., "A highly efficient galactokinase from Bifidobacterium infantis with broad substrate specificity" Carbohydrate Research 355:35-39 (2012) (incorporated herein by reference in its entirety).

[0083] Some embodiments of the invention employ an inorganic pyrophosphatase to convert an inorganic diphosphate into a monophosphate. In certain embodiments, the inorganic pyrophosphatase is PmPpa or EcPpa. Figure 1A and Figure 3A In the embodiment shown, the inorganic pyrophosphatase is PmPpa. J. Kapyla et al., "Effect of D97E substitution on the kinetic and thermodynamic properties of Escherichia coliinorganic pyrophosphatase" Biochemistry 34.3:792-800 (1995). The foregoing is incorporated herein by reference in its entirety. Figure 1B and Figure 3B In the embodiment shown, the monophosphate produced is removed from the reaction mixture in a filtration step.

[0084] Some embodiments of the present invention use phosphatase to convert adenosine diphosphate into adenosine and phosphate. In certain embodiments, the phosphatase is alkaline phosphatase (EC3.1.3.1) or acid phosphatase (EC3.1.3.2). In certain embodiments, the reaction mixture containing the phosphatase is in neutral or acidic pH. Not bound by theory, having non-alkaline reaction conditions allows ADP to be hydrolyzed into adenosine without a large amount of sugar nucleotides being hydrolyzed into sugar and nucleotides. This helps to separate and purify sugar nucleotides by allowing precipitation and filtration methods from sugar nucleotides rather than separation of adenosine by chromatography.

[0085] In certain embodiments, the alkaline phosphatase is calf intestinal alkaline phosphatase. In other embodiments, the phosphatase is Antarctic phosphatase. In certain embodiments, the Antarctic phosphatase is Tab5.

[0086] In embodiments of the invention employing phosphatases, the pH is maintained in the acidic range to allow ADP hydrolysis while minimizing chemical hydrolysis of sugar nucleotides, a deleterious non-enzymatic reaction catalyzed by magnesium chloride (MgCl2) and particularly evident at pH>6.9. In some embodiments, the phosphatase reaction (e.g. Figure 1A and Figure 3A Reactor 2 (two) shown) is at an acidic pH. In some embodiments, the pH of the phosphatase reaction is precisely, about, at least, up to or below, for example, pH 5.0 to pH 6.9. In some embodiments, the pH of the phosphate reaction mixture is precisely, about, at least, up to or below, for example, pH 5, 6, or 7. In some embodiments, the pH of the phosphatase reaction mixture is precisely, about, at least, up to or below, for example, pH 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, or pH 6.9.

[0087] In some embodiments, the reaction to obtain a sugar nucleotide ( Figure 3A Reactor 1 (a) and Figure 3A ) The pH of reactor 1 is acidic, neutral or alkaline. In certain embodiments, the pH in reactor 1 is precisely or approximately, for example, 5.0 to 8.5. The buffer in the input can be a phosphate buffer or other buffer that maintains the pH range of the reaction, including but not limited to PIPES, MOPS, BisTris, Tris, imidazole, sodium phosphate, potassium phosphate, PBS, imidazole, glycine, acetate, citrate or Good buffer (including but not limited to MES, Bis-tris, PIPES, MOPs, TES, HEPES, TEA, Tris). In some embodiments, acetate is sodium acetate and citrate is sodium citrate.

[0088] In certain embodiments, a buffer is not used, but the pH is continuously adjusted by adding concentrated acid, base, or both. In such embodiments, Figure 1A and Figure 3A No buffer was used in Reactor 1 or Reactor 2 as shown.

[0089] It is understood that the pH may fluctuate during the reaction. Such fluctuations are within the scope of the present invention.

[0090] Certain embodiments of the present invention include the steps of filtering the second output to remove the enzyme, thereby providing a filtered output composition; concentrating the filtered output composition to obtain a concentrate; adding salt to the concentrate to obtain a salted concentrate; adding an anti-solvent or a water-soluble solvent to the salted concentrate to provide a precipitation composition, wherein the precipitation composition includes a solid phase and a solution phase; separating the precipitation composition to obtain a filtrate and a precipitate; drying the precipitate to obtain sugar nucleotides. Certain embodiments include the step of filtering the first output composition. Other embodiments include the steps of filtering the first output composition and the second output composition.

[0091] In certain embodiments, the present invention adopts relatively high reaction concentration.Use higher reaction concentration to avoid or minimize the need to remove water from the reaction mixture.It also allows the use of smaller reaction vessels, thereby reducing capital expenditure and production costs.In certain embodiments, the method of the present invention is carried out under 5mM to 500mM galactose.In certain embodiments, the method of the present invention is carried out under 250mM to 500mM galactose.

[0092] In some embodiments, the concentration of adenosine diphosphate in the second composition is 5-100% of the concentration of adenosine diphosphate in the first composition.

[0093] In some embodiments, the conversion rate of adenosine diphosphate to adenosine and phosphate is about 80-100%. In some embodiments, such as reactor 2 ( Figure 1A and Figure 3A ) shows that the conversion rate of adenosine diphosphate to adenosine and phosphate is about 80-100%.

[0094] In certain embodiments, based on molar percentage, sugar nucleotide is not hydrolyzed to corresponding sugar and nucleotide basically.In certain embodiments, accurately, approximately, up to or less than for example 20%, 15%, 10% or 5% is hydrolyzed.In other embodiments, accurately, approximately, up to or less than for example 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% sugar nucleotide essence is hydrolyzed.In other embodiments, accurately, approximately, up to or less than for example 0.5% sugar nucleotide is hydrolyzed.In certain embodiments, greater than 95% or greater than 99% sugar nucleotide is not hydrolyzed.In some embodiments, the conversion rate that sugar nucleotide is converted into sugar and nucleotide is accurately or less than about 1%.

[0095] In some embodiments, the concentration of sugar nucleotides in the second composition is exactly or at least about 90-100% of the concentration of sugar nucleotides in the first composition. In certain embodiments, the concentration of sugar nucleotides in the second composition is exactly, at least, or greater than about 95% of the concentration of sugar nucleotides in the first composition or is exactly, at least, or greater than 99% of the concentration of sugar nucleotides in the first composition. In certain embodiments, the concentration of sugar nucleotides in the second composition is exactly, at least, or greater than about 0.5% of the concentration of sugar nucleotides in the first composition.

[0096] exist Figure 1A , Figure 1B , Figure 3A and Figure 3B In the embodiment shown in , the first composition is the output of flow reactor 2 and the second composition is the output of flow reactor 1 .

[0097] The present invention provides a robust purification method that does not require chromatography or chromatographic purification methods.

[0098] In one embodiment, the invention provides a method for purifying sugar nucleotides, comprising the steps of: concentrating the second output to obtain a concentrate, adding salt to the concentrate to obtain a salted concentrate, adding an antisolvent or a water-soluble solvent to the salted concentrate to provide a precipitation solution, separating the precipitation solution to obtain a filtrate and a precipitate, and drying the precipitate to obtain nucleotide sugars. The precipitation solution comprises a solid phase comprising sugar nucleotides and a solution phase comprising soluble reaction components. When using an immobilized enzyme, the method is useful. In certain embodiments, the concentrate is concentrated by about or at least 2 to about or at least 100 times. In other embodiments, the concentrate is concentrated by about or at least 2 to about or at least 100 times. In other embodiments, the concentrate is concentrated precisely, about, at least, higher than, up to or less than, for example, 2, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 times. In certain embodiments, the concentration is carried out by tangential flow nanofiltration (TFNF).

[0099] In certain embodiments, the salt added to the concentrate to obtain a salted concentrate is an ammonium salt, a lithium salt, a sodium salt or a potassium salt. In some embodiments, the sodium salt is sodium acetate. In other embodiments, the potassium salt is potassium acetate. In other embodiments, the salt is present in an amount of exactly, about, at least, above, up to or less than, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mM. In certain embodiments, sodium acetate is present in about 10 mM to about 200 mM. In other embodiments, potassium acetate is present in about 10 mM to about 200 mM.

[0100] In certain embodiments, the anti-solvent causes precipitation of certain reaction mixture components, while other components remain in solution. Anti-solvents include but are not limited to ethyl acetate, acetonitrile, tetrahydrofuran, toluene, acetone, amyl alcohol and octanol. In certain embodiments, the anti-solvent includes but is not limited to water-soluble solvents. In certain embodiments, the water-soluble solvent is an alcohol, including but not limited to an alcohol with 1-8 carbon atoms or an alcohol with 1-6 carbon atoms. In certain embodiments, the alcohol is ethanol, methanol or isopropanol. In other embodiments, the alcohol is ethanol.

[0101] In embodiments involving anti-solvents or water-soluble solvents, the solvent volume of such solvents is an amount that is exactly, about, at least, more than, up to, or less than, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times the solvent volume relative to the volume of the salified concentrate.

[0102] In certain embodiments, an antisolvent or water-soluble solvent is incubated with the salt concentrate to provide a precipitation solution. In such embodiments, the incubation temperature is precisely, about, at least, above, up to or below, for example, -20°C, -15°C, -10°C, -5°C, 0°C or 4°C. In certain embodiments, the incubation temperature is about -20°C to about 4°C.

[0103] In certain embodiments, the antisolvent or water-soluble solvent and the salted concentrate are incubated for exactly, about, at least, more than, up to, or less than, for example, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours. In other embodiments, the antisolvent or water-soluble solvent and the salted concentrate are incubated for about 2 hours to about 24 hours.

[0104] In embodiments of the present invention, separation is performed by a physical separation method. In certain embodiments, the physical separation method is filtration, centrifugation or decantation.

[0105] In embodiments of the present invention, drying is carried out by spray drying, freeze drying, vacuum drying or evaporative drying. In certain embodiments, drying is carried out by spray drying. Spray-dried sugar nucleotides are in the form of salts based on the salts used herein. In certain embodiments, sugar nucleotides are ammonium salts, lithium salts, sodium salts or potassium salts.

[0106] In certain embodiments, sugar nucleotides are purified to a level of, for example, 80%, 85%, 90%, 95%, or 100% exactly, about, at least, above, up to, or below. In certain embodiments, sugar nucleotides are free of, for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% from the reagents, reactants, or contaminants used in the methods of the invention.

[0107] In certain embodiments, UDP-galactose of the present invention (including but not limited to spray-dried UDP-galactose) is purified to a level of exactly, about, at least, above, up to or below, for example, 80%, 85%, 90%, 95% or 100%. In certain embodiments, sugar nucleotides are exactly, about, at least, above, up to or below, for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% free of reagents, reactants or contaminants used in the methods of the present invention.

[0108] In certain embodiments, the present invention provides methods of preparing UDP-galactose.

[0109] Therefore, the present invention provides a method for preparing UDP-galactose, comprising the step of contacting galactokinase, UDP-sugar pyrophosphorylase, inorganic pyrophosphatase, galactose, UTP and ATP to prepare UDP-galactose. In certain embodiments, the method further comprises the step of purifying UDP-galactose.

[0110] Therefore, the present invention provides a method for preparing UDP-galactose, comprising the following steps:

[0111] a. contacting galactokinase, UDP-sugar pyrophosphorylase, inorganic pyrophosphatase, galactose, UTP and ATP to form a reaction mixture,

[0112] b. Purify the reaction mixture to obtain UDP-galactose.

[0113] The method of the present invention provides sugar nucleotides of high purity of precisely, about, at least, above or up to, for example, 97%, 98% or 99%. In some embodiments, the UDP-galactose obtained has a purity of about or at least 97%, about or at least 98% or about or at least 99%.

[0114] The invention provides a process carried out in a single reaction vessel to carry out a one-pot reaction or more than one reaction vessel. One-pot synthesis can be carried out in batches or flow, including but not limited to repeated batches or continuous flow. In a flow reactor (e.g., a packed bed reactor), a reagent mixture is passed through by a flow mode. Therefore, in one embodiment, the invention provides a commercially applicable flow mode biocatalysis. In a flow operation, the flow rate is set to control the residence time to achieve optimal conversion while maximizing the productivity of product production. Embodiments of the invention using the CIAP process are preferably carried out in a flow reactor.

[0115] The present invention also provides products, including but not limited to sugar nucleotides prepared by any of the methods described herein. In one embodiment, UDP-galactose is prepared by the method of the present invention.

[0116] The present invention also provides a machine configured to perform the method of the present invention. In certain embodiments, the method is carried out in a single reaction vessel. The enzyme and reactant used in the method of the present invention are contacted with each other by combining in a reaction vessel.

[0117] In certain embodiments, sugar nucleotides of the present invention are used to add sugars to other compounds, including but not limited to polysaccharides, carbohydrates and oligosaccharides, including but not limited to two sugar units or more. Thus, sugar-containing compounds are synthesized. The synthesis is a biocatalytic synthesis performed using a transferase that transfers an activated sugar (sugar nucleotide) to an acceptor sugar or other compound.

[0118] Sugar nucleotides (e.g., Gal-UDP) can be used to add galactose to the sugar. Other sugar nucleotides can be used to make HMOs, including but not limited to LNT, LNnT, and lactose.

[0119] 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.

[0120] The nucleotides can also be recovered. In the embodiments exemplified herein, UDP, GDP or ADP can be recovered in the galactosylation reaction. In certain embodiments, the nucleotides are converted into the corresponding nucleosides which are easily purified.

[0121] Glycans are carbohydrate-based compounds characterized by one or more monosaccharides connected by glycosidic bonds (including N-linked and O-linked bonds). Activated monosaccharides, oligosaccharides, polysaccharides, plant glycans, animal glycans and microbial glycans are all within the scope of the present invention, as are glycoconjugates such as glycolipids, glycopeptides, glycoproteins and proteoglycans. Glycans also include humanized glycoproteins, humanized antibodies and glycoconjugate vaccines. C. Reiley et al., Nature Reviews Nephrology 15(6): 346-366 (2019); R. Rappuoli, "Glycoconjugate vaccines: Principles and mechanisms" Science Translational Medicine, 20(456) (2018). The foregoing is incorporated herein by reference in its entirety.

[0122] Sugar nucleotides according to the present invention can be used to add sugars to receptor compounds. For example, compounds can be galactosylated using UDP-galactose prepared according to the methods 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, flavoring agents, and spices.

[0123] The polysaccharides synthesized according to the present invention can be simple or complex polysaccharides, which can be linear or branched. Include polysaccharides with one or more sugar units. In certain embodiments, polysaccharides have two, three, four or five sugar units or more units. In some embodiments, polysaccharides have 1-18 units. In some embodiments, polysaccharides have 1-10 units. In some embodiments, polysaccharides have 1-5 units. In certain embodiments, polysaccharides have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 units. In certain embodiments, polysaccharides have 1 unit, 2 units, 3 units, 4 units, 5 units or 6 units. In some embodiments, polysaccharides are oligosaccharides. In some embodiments, polysaccharides are linear or branched. In certain embodiments, polysaccharides have 1-6 units and are linear. In other embodiments, polysaccharides have 1-6 units and are branched. In certain embodiments, n is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0124] The compounds obtained according to the present invention (including but not limited to galactosylated compounds) can be used as components in the synthesis of any polysaccharide-containing compound. Materials functionalized with enzymes or enzyme systems can be used to produce pharmaceuticals, biological products, nutritional products, cosmeceuticals and food ingredients. In certain embodiments, sugars and oligosaccharides are of non-animal origin.

[0125] In certain embodiments, the methods provide galactosylated oligosaccharides or galactosylated antibody-glycan conjugates.In preferred embodiments, galactosylated human milk oligosaccharides (HMOs) are produced.

[0126] 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-di-O-α-Sia-LNnH3"'3,3"'6-di-O-α-Sia-(3"3,3"6-di-O-α-Fuc)-LNnH, biantenary 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 biantenary lactose-N-neohexaose and neoheptasaccharides. In certain embodiments, sialic acid is obtained from 3'-SL or 6'-SL, including but not limited to in situ.

[0127] 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).

[0128] In embodiments of the invention, the enzyme is a free enzyme or an immobilized enzyme. Without being bound by theory, it is assumed that substantially all enzymes in certain embodiments are free or immobilized.

[0129] In certain embodiments, the enzyme is immobilized. Enzyme immobilization can be performed by covalent immobilization, embedding, adsorption, molecular labeling with affinity tags, protein affinity tags, non-covalent adsorption, non-covalent deposition, embedding, physical embedding, bioconjugation, chelation, cross-linking, disulfide bonds. See, for example, A. Basso & S. Serban, Mol. Catal. 479: 110607 (2019); Xu et al., Frontiers in Bioeng & Biotech., published on June 30, 2020, publdoi: 10.3389 / fbioe.2020.00660; and R. A. Sheldon & S. van Pelt Chem. Soc. Rev., 42: 6223 (2013). The foregoing is incorporated herein by reference in its entirety.

[0130] In certain embodiments, the present invention employs the use of N-terminal or C-terminal tags (including but not limited to: a. polyarginine (R6-R15 ); b. Polylysine (K6-K 15 ); or c. Histidine-arginine repeat sequence (HR) 4-12 ) immobilized enzyme.

[0131] 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, WO2022 / 119982 and U.S. Application Nos. 63 / 285,082, 63 / 430,271 and 63 / 448,218. The foregoing is incorporated herein by reference in its entirety.

[0132] 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.

[0133] In some embodiments, the immobilized enzyme is non-magnetic.In certain embodiments, such as for oligosaccharides or sugar nucleotides for use in food ingredients, the immobilized enzyme does not comprise nanoparticles.

[0134] 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.

[0135] 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.

[0136] The bionanocatalyst (BNC) includes an enzyme self-assembled with magnetic nanoparticles (MNPs). The self-assembled mesoporous aggregates of magnetic nanoparticles include a polysaccharide synthase, wherein the mesoporous aggregates are immobilized on a magnetic macroporous support. In one embodiment, the immobilized enzyme includes (i.) a polysaccharide synthase self-assembled in magnetic nanoparticles, and (ii.) a magnetic support. The polysaccharide enzyme 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. In one embodiment, the nanoparticles include magnetite (Fe3O4) or hematite (Fe2O3). In another embodiment, the nanoparticles include a product synthesized from FeCl2 and FeCl3, in particular a product 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 contain magnetite (Fe3O4).

[0137] Glycan synthase is any enzyme that can be used for glycan synthesis. Steps in glycan synthesis may include activation of sugars, transfer of sugar units to extend sugars, cofactor recycling, and equilibrium shifts. Glycan synthases include, but are not limited to, sugar activating enzymes, sugar elongating enzymes, reagent regenerating enzymes, sugar functionalizing enzymes, sugar supporting enzymes, and sugar removing enzymes.

[0138] 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.

[0139] 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.

[0140] 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 SC Corgie et al., Catalysis & Biocatalysis, Chemistry 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.

[0141] For example, you can Figure 2 The immobilized enzyme is prepared as shown and used in the method of the present invention (WO2022 / 119982). In the first step, the 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, the polysaccharide synthase is added to the product of the first step to support the BNC. The B-type scaffold BNC composition is made by this method.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] One embodiment provides a modular method for producing glycans, comprising a module that can be a flow cell, wherein: the module comprises a magnetic macroporous powder, the magnetic macroporous powder comprising magnetic microparticles, wherein the powder has fixed thereto a preparation of self-assembled mesoporous aggregates of magnetic nanoparticles containing glycan synthase; wherein a substrate is introduced into the module (or passed through the flow cell) and the substrate is modified to provide glycans.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] The method for preparing the magnetic scaffold is flexible because the method used in the present invention is a convenient and flexible reaction.

[0156] 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.

[0157] 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.

[0158] 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).

[0159] 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.

[0160] In one embodiment, the polypropylene-magnetite material can be 3D printed via SLS in any shape and form.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 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 (JP Kruth et al., Assembly Automation 23(4):357-371 (2003), which is incorporated herein by reference in its entirety.

[0170] 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.

[0171] 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).

[0172] 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.

[0173] 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.

[0174] 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).

[0175] 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.

[0176] 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.

[0177] 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 complex features and 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.

[0178] 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.

[0179] 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.

[0180] 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).

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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) (R. 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.

[0185] 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.

[0186] The scaffold may include a cross-linked water-insoluble polymer and a substantially uniform distribution of embedded magnetic microparticles (MMP). The scaffold may include a thermosetting resin, including epoxy resins, polyesters, polyurethanes, melamine resins, vinyl esters, silicones (polysiloxanes), furan resins, polyureas, phenolic resins, phenol formaldehyde, urea formaldehyde, diallyl phthalate (DAP), benzophenone, Oxazines, polyimides and bismaleimides, cyanate esters can be used. By combining natural or engineered enzymes, and in some embodiments, cofactors and cofactor recovery systems, the scaffold technology disclosed herein allows innovations in biocatalysis to be quickly translated into production innovations in batch and flow processes. Magnetic powders are suitable for flow chemistry applications, such as packed bed reactors.

[0187] Self-assembled mesoporous nanoclusters containing embedded enzymes are highly active and stable. 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 as "bionanocatalysts" (BNCs) in this article. Clusters can be magnetically templated onto formable magnetic supports.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 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 includes 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).

[0197] 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.

[0198] 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.

[0199] 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.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] 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.

[0204] 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.

[0205] Any of these compositions and methods can be used to immobilize enzymes in embodiments of the invention.In some embodiments, the glycan synthase is a phosphatase, a kinase, a nucleotide sugar pyrophosphorylase, or an inorganic pyrophosphorylase.

[0206] It should be understood that "comprising" should be interpreted non-exhaustively where the context permits. Where the context permits, each comprising may be alternatively "consisting essentially of" or "consisting of".

[0207] Some implementations are as follows.

[0208] A method for preparing a sugar nucleotide compound comprises the step of contacting a kinase, an enzyme that converts galactose-1-phosphate and UTP into galactose-UDP, an inorganic pyrophosphatase, a sugar, a nucleotide and ATP to prepare a sugar nucleotide.

[0209] A method according to embodiment 1, wherein the method comprises a further step of purifying the sugar nucleotides.

[0210] A method for preparing a sugar nucleotide compound comprises the steps of: contacting a kinase, an enzyme that converts galactose-1-phosphate and UTP to galactose-UDP, an inorganic pyrophosphatase, a sugar, a nucleotide, and ATP to obtain a first output; contacting the first output in the presence of alkaline phosphatase to obtain a second output; and purifying the second output to obtain a sugar nucleotide.

[0211] According to the method of any one of embodiments 1-3, the nucleotide is UDP, and the sugar is D-glucose, D-galactose, D-fructose, L-rhamnose, D-mannose, D-ribose, D-xylose, L-arabinose, D-xylose, L-fucose, D-apiose, D-glucuronic acid, D-galacturonic acid, N-acetyl-D-glucosamine or N-acetyl-D-galactosamine. According to the method of any one of embodiments 1-3, the nucleoside is ADP, and the sugar is D-glucose, D-galactose, D-fructose, D-mannose, D-ribose, L-arabinose, D-xylose or D-glucuronic acid. According to the method of any one of embodiments 1-3, the nucleotide is GDP, and the sugar is D-glucose, D-galactose, D-mannose, L-rhamnose, D-ribose, D-xylose, L-arabinose or L-fucose. The method according to any one of embodiments 1-3, wherein the nucleotide is CMP and the sugar is N-acetylneuraminic acid. The method according to any one of embodiments 1-3, wherein the nucleotide is TDP and the sugar is D-glucose or D-galacturonic acid.

[0212] A method according to any one of embodiments 2-8, wherein purification is performed by selective precipitation.

[0213] The method according to any one of embodiments 2-8, wherein the purification comprises the steps of filtering the second output to remove the enzyme, concentrating the second output to obtain a concentrate, adding salt to the concentrate to obtain a salted concentrate, adding a water-soluble solvent to the salted concentrate to provide a precipitation solution, separating the precipitation solution to obtain a filtrate and a precipitate, and drying the precipitate to obtain sugar nucleotides. The method according to embodiment 10, wherein the separation step removes the enzyme by filtration or adsorption. The method according to embodiment 11, wherein the filtration is ultrafiltration, tangential flow filtration or diafiltration.

[0214] The method according to any one of embodiments 10-12, wherein the purification comprises the steps of concentrating the second output to obtain a concentrate, adding salt to the concentrate to obtain a salified concentrate, adding a water-soluble solvent to the salified concentrate to provide a precipitation solution, separating the precipitation solution to obtain a filtrate and a precipitate, and drying the precipitate to obtain sugar nucleotides. The method according to embodiment 13, wherein the concentration to obtain the concentrate is about 2 to about 100 times. The method according to embodiment 13, wherein the concentration is performed by tangential flow nanofiltration.

[0215] The method according to any one of embodiments 10-15, wherein the salt is an ammonium salt, a lithium salt, a sodium salt, or a potassium salt. The method according to embodiment 16, wherein the sodium salt is sodium acetate or the potassium salt is potassium acetate. The method according to embodiment 17, wherein the sodium acetate is present at about 10 mM to about 200 mM. The method according to embodiment 17, wherein the potassium acetate is present at about 10 mM to about 200 mM.

[0216] The method according to any one of embodiments 10-19, wherein the water-soluble solvent is an alcohol. The method according to embodiment 20, wherein the alcohol is ethanol, methanol or isopropanol. The method according to embodiment 21, wherein the alcohol is ethanol. The method according to any one of embodiments 10-22, wherein the solvent volume of the water-soluble solvent is 2, 3, 4, 5, 6, 7, 8, 9 or 10 volumes of solvent relative to the volume of the salified concentrate.

[0217] The method according to any one of embodiments 2-23, further comprising an incubation step, wherein the water-soluble solvent and the salted concentrate are incubated. The method according to embodiment 24, wherein the incubation temperature of the incubation step is about -20°C to about 4°C. The method according to embodiment 24, wherein the incubation time of the incubation step is about 2 hours to about 24 hours.

[0218] The method according to any one of embodiments 2-26, wherein the separation is performed by a physical separation method. The method according to embodiment 27, wherein the physical separation method is filtration, centrifugation or decantation.

[0219] The method according to any one of embodiments 10-28, wherein the drying is performed by spray drying, freeze drying, vacuum drying or evaporative drying. The method according to embodiment 29, wherein the drying is performed by spray drying.

[0220] The method according to any one of embodiments 1-30, wherein the enzyme is a free enzyme.

[0221] The method according to any one of embodiments 1-30, wherein the enzyme is an immobilized enzyme. The method according to embodiment 32, wherein the immobilized enzyme is fixed by an N-terminal tag or a C-terminal tag. The method according to embodiment 33, wherein the N-terminal tag or the C-terminal tag is a polyarginine, polylysine or a histidine-arginine repeat sequence. The method according to embodiment 32, wherein the immobilized enzyme is immobilized by an iron oxide material. The method according to embodiment 35, wherein the iron oxide material is hematite, magnetite or strontium ferrite. The method according to embodiment 36, wherein the immobilized enzyme is an A-type scaffold BNC. The method according to embodiment 36, wherein the immobilized enzyme is a B-type scaffold BNC.

[0222] According to the method described in any one of embodiments 1-4 and 9-38, the sugar nucleotide pyrophosphorylase is UDP-sugar pyrophosphorylase. According to the method described in embodiment 39, the enzyme that galactose-1-phosphate and UTP are converted into galactose-UDP is UDP-sugar pyrophosphorylase (USP) or UTP-glucose 1-phosphate uridyl transferase. According to the method described in embodiment 40, the UDP-sugar pyrophosphorylase is BlUSP, EcUSP or SpUSP. According to the method described in embodiment 40, the UTP-glucose 1-phosphate uridyl transferase is SpGalU.

[0223] The method according to any one of embodiments 1-6 or 9-42, wherein the kinase is galactokinase. The method according to embodiment 43, wherein the galactokinase is BiGalK, SpGalK, EcGalK or LgGalK.

[0224] The method of any one of embodiments 1-44, wherein the inorganic pyrophosphatase is PmPpa or EcPpa.

[0225] The method according to any one of embodiments 3-45, wherein the alkaline phosphatase is calf intestinal alkaline phosphatase.

[0226] The method according to any one of embodiments 1-4 or 9-42, wherein the sugar nucleotide is UDP-galactose. The method according to any one of embodiments 1-4 or 9-47, wherein the UDP-galactose has a purity of about or at least 97%, about or at least 98%, or about or at least 99%. Spec; further precipitate.

[0227] The method according to any one of embodiments 1-48, wherein the method occurs in a single reaction vessel. The method according to any one of embodiments 1-49, wherein the method is carried out under batch, flow, semi-continuous or continuous flow conditions. The method according to embodiment 50, wherein the method is carried out in a continuous flow reactor.

[0228] The method according to any one of embodiments 1-51, wherein the method does not include a chromatography method or a chromatographic purification method. The method according to any one of embodiments 1-52, wherein the method is a cGMP production method.

[0229] A sugar nucleotide prepared by the method according to any one of embodiments 1 to 53. A UDP-galactose prepared by the method according to any one of embodiments 1 to 4 or 9 to 54.

[0230] A compound prepared by the method according to any one of embodiments 1-55, wherein the compound is obtained from a non-animal based plant material.

[0231] A machine configured for the method of any one of embodiments 1 to 53. The machine of embodiment 57, wherein the method is performed in one or two reaction vessels.

[0232] 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 manner. Example

[0233] abbreviation

[0234] PBS: Phosphate buffered saline

[0235] MES: 2-(N-morpholino)ethanesulfonic acid

[0236] Bis-tris: 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol PIPES: piperazine-N,N'-bis(2-ethanesulfonic acid)

[0237] MOPS: 3-(N-morpholino)propanesulfonic acid

[0238] TES: 2-{[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}ethane-1-sulfonic acid HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

[0239] TEA: triethanolamine

[0240] Tris: 2-amino-2-(hydroxymethyl)propane-1,3-diol

[0241] 3'-SL: 3'-sialyllactose

[0242] 6'-SL: 6'-sialyllactose

[0243] 2'-FL: 2'-fucosyllactose

[0244] LNFPI: Lactose-N-fucosylopentaose I

[0245] LNT: lactose-N-tetraose

[0246] LNnT: lactose-N-neotetraose

[0247] LNTII: lactose-N-trisaccharide II DSLNT: disialyl lactose-N-tetrasaccharide

[0248] UDP: Uridine 5'-diphosphate

[0249] UTP: Uridine 5'-triphosphate

[0250] ADP: adenosine 5'-diphosphate

[0251] ATP: Adenosine 5'-triphosphate

[0252] CMP: Cytidine 5'-monophosphate

[0253] CDP: Cytidine 5'-diphosphate

[0254] CTP: Cytidine 5'-triphosphate

[0255] GDP: Guanosine 5'-diphosphate

[0256] GTP: Guanosine 5'-triphosphate

[0257] TDP: thymidine 5'-diphosphate

[0258] Tris: tris(hydroxymethyl)aminomethane

[0259] DMC: 2-chloro-1,3-dimethylimidazolinium chloride

[0260] Bbh1: β-N-acetylglucosaminidase

[0261] GOS: galacto-oligosaccharide

[0262] Lactose synthase EC2.4.1.22: Produces lactose from glucose and UDP-galactose. It consists of galactosyltransferase (B4GALT1 or B4GALT2) and α-lactalbumin

[0263] USP: UDP-sugar pyrophosphorylase EC 2.7.7.64, which catalyzes the reversible transfer of a uridine group from UTP to sugar-1-phosphate, generating UDP-sugar and pyrophosphate (PPi)

[0264] BlUSP: USP from Bifidobacterium longum

[0265] EcUSP: USP from Escherichia coli

[0266] SpUSP: USP from Streptococcus pneumoniae

[0267] 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.

[0268] Inorganic pyrophosphatase (Ppa) EC 3.6.1.1: catalyzes the hydrolysis of pyrophosphate (PPi) into two monophosphate ions (Pi).

[0269] Alkaline phosphatase EC3.1.3.1: catalyzes the hydrolysis of nucleotides (NMP, NDP, NTP) into the corresponding nucleosides (N) and phosphates

[0270] Acid phosphatase EC3.1.3.2:

[0271] GalK: Galactokinase EC2.7.1.6: catalyzes the phosphorylation of galactose to galactose-1-phosphate (Gal-1P) by consuming one ATP unit.

[0272] BiGalK: galactokinase from Bifidobacterium infantis

[0273] SpGalK: galactokinase from Streptococcus pneumoniae

[0274] EcGalK: galactokinase from Escherichia coli

[0275] LgGalK: galactokinase from Lerminolium griseum

[0276] BlUSP: UDP-sugar pyrophosphorylase from Bifidobacterium longum

[0277] Ppa: Inorganic pyrophosphatase

[0278] PmPpa: inorganic pyrophosphatase from Pasteurella multocida

[0279] EcPpa: Inorganic pyrophosphatase from Escherichia coli

[0280] GalU: UTP-glucose 1-phosphate uridyltransferase

[0281] CIAP: Calf Intestinal Alkaline Phosphatase

[0282] SpGalU: UTP-glucose-1-phosphate uridyltransferase

[0283] Embodiment 1: Reaction Description

[0284] In the preparation of sugar nucleotides, the following conditions may be used.

[0285] 1. pH range: 6.0-9.0 for flow reactor 1 (e.g., BiGalK, BlUSP, PmPpa)

[0286] 2. pH range: 6.0-9.0 for flow reactor 2 (e.g., CIAP)

[0287] 3. Buffer: sodium phosphate, potassium phosphate, PBS, imidazole, glycine, Good buffer (including MES, Bis-tris, PIPES, MOPs, TES, HEPES, TEA, Tris).

[0288] 3. Reagent concentration:

[0289] a. Galactose: 1-1000mM

[0290] b. UTP: 1-1.5 molar equivalent relative to galactose (1-1500mM)

[0291] c.ATP: 1-1.5 molar equivalent relative to galactose (1-1500mM)

[0292] d. Buffer: 5-250mM

[0293] e.MgCl2 or MnCl2: 0.2-100mM

[0294] f. USP or GalU enzyme: 0.1-15 mg / ml (in solution or in packing bed)

[0295] g. GalK enzyme: 0.1-15 mg / ml (in solution or in packing bed)

[0296] h. Ppa enzyme: 0.1-15 mg / ml (in solution or in packing bed)

[0297] i.CIAP: 0.1-15 mg / ml (in solution or in packed bed)

[0298] 4. Temperature range: 20℃-70℃

[0299] Embodiment 2: Downstream Processing Description

[0300] The following conditions can be used in the method of the present invention.

[0301] 1. Selective precipitation of UDP-galactose

[0302] a.Solvents: alcohols, including ethanol, methanol, isopropanol

[0303] b.Solvent volume: 2, 3, 4, 5, 6, 7, 8, 9, 10 volumes of solvent relative to the product volume

[0304] c. Salt: 10-200 mM sodium acetate, 10-200 mM potassium acetate

[0305] d. Incubation temperature: -20℃ to 4℃

[0306] e. Incubation time: 2-24 hours

[0307] 2. Removal of enzymes by filtration (ultrafiltration, tangential flow filtration, diafiltration) or other adsorption methods.

[0308] 3. Separate the precipitate (galactose-UDP) and the solute by filtration, centrifugation, decantation or related techniques.

[0309] 4. Dry the Galactose-UDP by spray drying, freeze drying, vacuum or general evaporation methods.

[0310] Example 3. Enzyme variants for producing UDP-galactose

[0311] The following enzymes were used in the preparation of UDP-galactose.

[0312] 1. Enzyme that converts galactose-1-phosphate and UTP to galactose-UDP

[0313] 1.1.UDP-sugar pyrophosphorylase (USP) EC2.7.7.64

[0314] 1.1.1.BlUSP (Bifidobacterium longum)

[0315] 1.1.2.EcUSP (Escherichia coli)

[0316] 1.1.3.SpUSP (Streptococcus pneumoniae)

[0317] 1.2. UTP-glucose 1-phosphate uridyltransferase (GalU) EC2.7.7.9

[0318] 1.2.1SpGalU (Streptococcus pneumoniae)

[0319] 2. Galactokinase (GalK) EC2.7.1.6

[0320] 2.1BiGalK (Bifidobacterium infantis)

[0321] 2.2SpGalK (Streptococcus pneumoniae)

[0322] 2.3EcGalK (Escherichia coli)

[0323] 2.4LgGalK (LgGallinium)

[0324] 3. Inorganic pyrophosphatase (Ppa) EC3.6.1.1

[0325] 3.1PmPpa (Pasteurella multocida)

[0326] 3.2EcPpa (Escherichia coli)

[0327] 4. Phosphate monoester hydrolase EC3.1.3.

[0328] 4.1 Alkaline phosphatase EC3.1.3.1

[0329] 4.1.1 Calf Intestinal Alkaline Phosphatase (CIAP)

[0330] 4.2 Acid phosphatase EC3.1.3.2

[0331] 4.3 5'-nucleotidase EC3.1.3.5

[0332] Example 4. Flow Synthesis of UDP-Galactose

[0333] 4a. Reagents and Materials:

[0334] Galactose was synthesized using the following chemical reagents: UTP (uridine 5'-triphosphate trisodium salt; Carbosynth: NU03863), ATP (adenosine 5'-triphosphate disodium salt hydrate; Carbosynth: NA00135), D-(+)-galactose (Thermo Scientific: A1281330), ethanol (200 proof, Fisher BioReagents, BP2818500), sodium dihydrogen phosphate dihydrate (Fisher Chemical, S381-3), sodium dihydrogen phosphate anhydrous (Fisher Chemical, S375-500) and MgCl2 (magnesium chloride; Macron Fine Chemicals: 595804). All water was from BarnStead Nanopure water purifier (Thermo Scientific, 18.5 MOhm-cm).

[0335] Enzymes BiGalK (Bifidobacterium infantis galactokinase), BlUSP (Bifidobacterium longum UDP-sugar pyrophosphorylase) and PmPpa (Pasteurella multocida inorganic pyrophosphatase) were produced recombinantly in Escherichia coli (BL21 / DE3). Enzymes were purified from soluble lysates by affinity chromatography (NiNTA) and buffer exchanged by dialysis against 50mM Tris pH7.5. Enzymes were supplemented with 10% (w / w) glycerol and stored frozen at -80°C. Plasmids for protein expression were produced by custom synthetic inserts and spliced ​​into commercial pET28a vectors (Novagen) by Genewiz or Genscript. CIAP was purchased from New England Biolabs (M0525S).

[0336] 4b. Flow Synthesis of Galactose-UDP:

[0337] This example describes a process in which D-galactose (Gal), uridine-5'-triphosphate (UTP), and adenosine-5'-triphosphate (ATP) are converted into galactose-UDP (synonymously referred to as: UDP-galactose, UDP-Gal; Gal-UDP, galactose-uridine-5'-diphosphate), adenosine-5'-diphosphate (ADP), and inorganic phosphate (orthophosphate) ( Figure 1A ).

[0338] BNCs were formed from 5.0 mg BiGalK, 2.0 mg BlUSP and 1.0 mg PmPpa in a 100 ml volume of pH 9 water and 25 ml pH 9 nanoparticle solution (390 ug / ml). BlUSP, PmPpa and BiGalK each had a C-terminal affinity tag consisting of a (HR) 4 sequence (enzyme-GGGGS-HRHRHRHRP*). The scaffold BNCs were formed by gradually adding 1 g of a strontium ferrite scaffold (Powdertech, catalog number S20) and mixing under overhead stirring (500-1000 rpm). The pH was then gradually reduced to pH 6.0 over one hour. After another hour of incubation, the supernatant was removed and the scaffold BNCs were washed with water. The immobilization yield was determined by the Bradford assay. The BNCs were transferred and loaded into a column (length 2", ID 1 / 16"), which constituted the "flow reactor 1". A syringe pump (New Era Pump Systems) then delivered a reagent stream (100 ml: 25 mM galactose, 30 mM ATP, 30 mM UTP and 10 mM MgCl2 in 25 mM phosphate pH 6.8, 37°C) at 0.5 ml / hr to provide galactose-UDP at 95% conversion (HPLC). The product was then delivered directly through "Flow Reactor 2" consisting of BNCs with 5.0 mg CIAP (immobilized as described above) at 0.5 ml / hr without prior purification by a syringe pump (New Era Pump Systems).

[0339] The flow-through product from the second flow reactor was concentrated approximately 10-fold using tangential flow filtration (nanofiltration) and supplemented with 200 mM sodium acetate. Galactose-UDP was precipitated by adding five volumes of ethanol and incubating the solution at 4°C for 18 hours. The galactose-UDP precipitate was recovered by centrifugation and spray-dried at room temperature.

[0340] Example 5. Tab5 cloning and expression

[0341] Tab5 plasmid and protein sequence

[0342] Cloning and expression of Tab5, an alkaline phosphatase from the psychrophilic strain TAB5 (Uniprot: Q9KWY4). The codon-optimized Tab5 gene was synthesized by GenScript and cloned into the pET30a(+) vector using the Nde1 and Xho1 restriction sites, while placing a 6His tag at the N-terminus. The final expressible Tab5 sequence (SEQ ID NO: 1) is:

[0343] MHHHHHHGGGGSVLVKNEPQLKTPKNVILLISDGAGLSQISSTFYFKEGTPNYTQFKNIGLIKTSSSREDVTDSASGATAFSCGIKTYNAAIGVADDSTAVKSIVEIAALNNIKTGVVATSSITHATPASFYAHALNRGLEEEIAMDMTESDLDFFAGGGLNYFTKRKDKKDVLAILKGNQFT INTTGLTDFSSIASNRKMGFLLADEAMPTMEKGRGNFLSAATDLAIQFLSKDNSAFFIMSEGSQIDWGGHANNASYLISEINDFDDAIGTALAFAKKDGNTLVIVTSDHETGGFTLAAKKNKREDGSEYSDYTEIGPTFSTGGHSATLIPVFAYGPGSEEFIGIYENNEIFHKILKVTKWNQ*.

[0344] After transformation into BL21(DE3) competent cells, cultures were grown in TB medium at 37°C, induced with 0.5 mM IPTG at 18°C, and grown for 16 hours.

[0345] Purification of Tab5. After expression, cells were collected by centrifugation and lysed using ultrasound with a lysis buffer containing 20mM tris pH8.0, 150mM NaCl, 1mM MgCl2, 10mM imidazole, 10% glycerol, and 0.1% TritonX-100. The lysate was filtered and the enzyme (Tab5) was purified using IMAC NiNTA affinity chromatography. The loading buffer consisted of 20mM tris pH8.0, 150mM NaCl, 1mM MgCl2, 10mM imidazole, and 10% glycerol. The washing buffer consisted of 20mM tris pH8.0, 300mM NaCl, 1mM MgCl2, 25mM imidazole, and 10% glycerol, and the elution buffer consisted of 20mM tris pH8.0, 300mM NaCl, 1mM MgCl2, 500mM imidazole, and 10% glycerol. After elution, the enzyme was dialyzed against 10 mM tris pH 7.4, 1 mM MgCl2, 10 μM ZnCl2 and 10% glycerol. 117 mg Tab5 was collected from 1 L of expression, and the purity was >95% ( Figure 4 ).

[0346] We investigated the downstream processing steps of flow reactor 2 ( Figure 1A) were used for feasibility studies. In-house purified Tab5 (500 μg / ml) was added to 20 mM ADP in either of two buffers, and the reactions were performed at 37°C and 25°C, respectively:

[0347] Buffer 1: 250 mM sodium phosphate, pH 6.0.

[0348] Buffer 2: 50 mM Bis-Tris-propane HCl, pH 6.0, 1 mM MgCl2, 0.1 mM ZnCl2 (New England Biolabs (NEB) Antarctic Phosphatase Buffer).

[0349] The conversion of ADP to AMP and adenosine at the 4 and 20 hour time points was analyzed using HPLC (Agilent 1100, diode array detector, λ=254 nm; DNA-Pac Bio LC IEX column, buffer A: 25 mM Tris pH 7.5, buffer B: 25 mM Tris pH 7.5 in 1 M LiCl). Figure 5 The percent conversion of ADP to AMP and the qualitative conversion values ​​to adenosine are shown.

[0350] All publications and patent documents disclosed or mentioned herein are incorporated by reference in their entirety.

[0351] 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 converting adenosine diphosphate into adenosine, comprising the step of contacting a first composition comprising adenosine diphosphate and sugar nucleotides with a phosphatase at an acidic pH to convert the first composition into a second composition comprising adenosine, phosphate and the sugar nucleotides.

2. The method of claim 1 further comprising an initial step of producing the first composition by contacting a sugar, nucleotide triphosphates and adenosine triphosphate with a kinase, a nucleotide sugar pyrophosphorylase and an inorganic pyrophosphorylase.

3. The method of claim 1 or claim 2, wherein the phosphatase is alkaline phosphatase or acid phosphatase. The method of claim 3 , wherein the phosphatase is Antarctic phosphatase.

5. The method of claim 4, wherein the Antarctic phosphatase is a bacterial Antarctic phosphatase.

6. The method of claim 5, wherein the bacterial Antarctic phosphatase has Tab5 activity and has at least 90% sequence identity to SEQ ID NO:

1. The method according to claim 6 , wherein the Tab5 has the sequence of SEQ ID NO:

1.

8. The method according to any one of claims 1 to 7, wherein the acidic pH is 6.9 or below.

9. The method of claim 8, wherein the acidic pH is from 5.0 to pH 6.

9.

10. The method of any one of claims 1-9, comprising separating the sugar nucleotide and the adenosine.

11. The method of claim 10, wherein the separation is performed by selectively precipitating the sugar nucleotides in the presence of an antisolvent.

12. The method according to any one of claims 1 to 11, further comprising the following steps: The second composition is concentrated to obtain a concentrate, a salt is added to the concentrate to obtain a salified concentrate, an anti-solvent is added to the salified concentrate to provide a precipitation solution, the precipitation solution is separated to obtain a filtrate and a precipitate, and the precipitate is dried to obtain the sugar nucleotide.

13. The method of any one of claims 1-12, further comprising an initial step of removing the phosphatase, the kinase, the nucleotide sugar pyrophosphorylase, and the inorganic pyrophosphorylase from the first composition or the second composition.

14. The method according to claim 13, wherein the removing is performed by filtration or adsorption methods.

15. The method of claim 14, wherein the filtration is ultrafiltration, tangential flow filtration or diafiltration.

16. The method of any one of claims 12-15, wherein said concentrating to obtain said concentrate is about 2 to about 100 times.

17. The method according to claim 16, wherein the concentration is performed by tangential flow nanofiltration.

18. The method of any one of claims 12-17, wherein the salt is an ammonium salt, a lithium salt, a sodium salt, or a potassium salt.

19. The method of claim 18, wherein the sodium salt is sodium acetate or the potassium salt is potassium acetate.

20. The method of claim 19, wherein the sodium acetate is present at about 10 mM to about 200 mM.

21. The method of claim 19, wherein the potassium acetate is present at about 10 mM to about 200 mM.

22. The method of any one of claims 11-21, wherein the anti-solvent is an alcohol.

23. The method of claim 22, wherein the alcohol is ethanol, methanol or isopropanol.

24. The method of claim 23, wherein the alcohol is ethanol.

25. The method of any one of claims 11-24, wherein the anti-solvent has a solvent volume of 2, 3, 4, 5, 6, 7, 8, 9 or 10 volumes of solvent relative to the volume of the salified concentrate.

26. The method of any one of claims 11-25, further comprising an incubation step, wherein the antisolvent and the salted concentrate are incubated.

27. The method of claim 26, wherein the temperature of the incubation step is from about -20°C to about 4°C.

28. The method of claim 26 or claim 27, wherein the incubation step has an incubation time of about 2 hours to about 24 hours.

29. The method according to any one of claims 10-28, wherein the separation is performed by a physical separation method.

30. The method of claim 29, wherein the physical separation method is filtration, centrifugation or decantation.

31. The method according to any one of claims 12 to 30, wherein the drying is performed by spray drying, freeze drying, vacuum drying or evaporative drying.

32. The method of claim 31 , wherein the drying is performed by spray drying.

33. The method of any one of claims 1-32, wherein the nucleotide is uridine 5'-diphosphate (UDP) and the sugar is D-glucose, D-galactose, D-fructose, L-rhamnose, D-mannose, D-ribose, D-xylose, L-arabinose, D-xylose, L-fucose, D-apiose, D-glucuronic acid, D-galacturonic acid, N-acetyl-D-glucosamine, or N-acetyl-D-galactosamine.

34. The method of any one of claims 1-32, wherein the nucleotide is adenosine 5'-diphosphate (ADP) and the sugar is D-glucose, D-galactose, D-fructose, D-mannose, D-ribose, L-arabinose, D-xylose, or D-glucuronic acid.

35. The method of any one of claims 1-32, wherein the nucleotide is guanosine 5'-diphosphate (GDP) and the sugar is D-glucose, D-galactose, D-mannose, L-rhamnose, D-ribose, D-xylose, L-arabinose or L-fucose.

36. The method of any one of claims 1-32, wherein the nucleotide is cytidine 5'-monophosphate (CMP) and the sugar is N-acetylneuraminic acid.

37. The method of any one of claims 1-32, wherein the nucleotide is thymidine 5'-diphosphate (TDP) and the sugar is D-glucose or D-galacturonic acid.

38. The method of any one of claims 1-37, wherein the phosphatase, the kinase, the nucleotide sugar pyrophosphorylase, or the inorganic pyrophosphorylase is a free enzyme.

39. The method of any one of claims 1-37, wherein the phosphatase, the kinase, the nucleotide sugar pyrophosphorylase, or the inorganic pyrophosphorylase is an immobilized enzyme.

40. The method of claim 39, wherein the immobilized enzyme is immobilized via an N-terminal tag or a C-terminal tag.

41. The method of claim 40, wherein the N-terminal tag or the C-terminal tag comprises poly-arginine, poly-lysine, or histidine-arginine repeats.

42. The method of claim 39, wherein the immobilized enzyme is immobilized by an iron oxide material.

43. The method of claim 42, wherein the iron oxide material is hematite, magnetite or strontium ferrite.

44. A method according to claim 42 or claim 43, wherein the immobilized enzyme is a type A scaffold BNC.

45. A method according to claim 42 or claim 43, wherein the immobilized enzyme is a Type B scaffold BNC.

46. ​​The method of any one of claims 2-45, wherein the sugar nucleotide pyrophosphorylase is uridine 5'-diphosphate-sugar-pyrophosphorylase (UDP-sugar pyrophosphorylase).

47. The method of claim 46, wherein the nucleotide sugar pyrophosphorylase is UDP-sugar pyrophosphorylase (USP) or UTP-glucose 1-phosphate uridyltransferase (GalU).

48. The method of claim 47, wherein the UDP-sugar pyrophosphorylase is BlUSP, EcUSP or SpUSP.

49. The method of claim 47, wherein the UTP-glucose 1-phosphate uridyl transferase is SpGalU.

50. The method of any one of claims 2-49, wherein the kinase is galactokinase.

51. The method of claim 50, wherein the galactokinase is a galactokinase from Bifidobacterium infantis (BiGalK), a galactokinase from Streptococcus pneumoniae (SpGalK), a galactokinase from Escherichia coli (EcGalK), or a galactokinase from Leminorella grimontii (LgGalK).

52. The method of any one of claims 2-51, wherein the inorganic pyrophosphatase is an inorganic pyrophosphatase from Pasteurella multocida (PmPpa) or an inorganic pyrophosphatase from Escherichia coli (EcPpa).

53. The method of any one of claims 1-52, wherein the sugar nucleotide is UDP-galactose.

54. The method of claim 53, wherein the UDP-galactose has a purity of about or at least 97%, about or at least 98%, or about or at least 99%.

55. The method of any one of claims 1-54, wherein the method occurs in a single reaction vessel.

56. The method of any one of claims 1-55, wherein the method is performed under batch, flow, semi-continuous or continuous flow conditions.

57. The method of claim 56, wherein the method is performed in a continuous flow reactor.

58. The method of any one of claims 1-57, wherein the method does not comprise a chromatography method or a chromatographic purification method.

59. The method of any one of claims 1-58, wherein the method is a cGMP production method.

60. A sugar nucleotide prepared by the method of any one of claims 1-59.

61. UDP-galactose prepared by the method of any one of claims 1-33 or claims 38-59.

62. A compound prepared by the method of any one of claims 1-59, wherein the compound is obtained from non-animal based plant material.

63. A machine configured for use in the method of any one of claims 1-59.

64. A machine according to claim 63, wherein the method is carried out in one or two reaction vessels.

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