Method for the production of nucleotide sugars with enzyme cascades in a continuously fed membrane reactor

Abstract

The present invention relates to a continuous method for the production of activated sugars, comprising the method steps of: a) introducing an enzyme cascade comprising enzymes dissolved in a solvent, into a reaction volume (14); b) introducing the reactants phosphate and saccharide dissolved in a solvent into the reaction volume (14) through a reactant stream, the reactants being used to produce the activated sugar; c) reacting the reactants using the enzyme cascade to form the activated sugar; d) passing formed reaction solution comprising the formed activated sugar and the enzyme cascade to a separation membrane (32), wherein the reaction solution is divided into a first fluid stream and a second fluid stream, the first fluid stream containing the enzyme cascade and being depleted of the activated sugar, and the second fluid stream containing activated sugar and being depleted of the enzyme cascade; e) recycling the first fluid stream into the reaction volume (14); wherein, f) the amount of reactant introduced into the reaction volume (14) in method step a) is adjusted as a function of the amount of activated sugar contained in the second fluid stream; such that, g) the molar ratio of the reactant to the activated sugar ranges from 0.8: 1.2 to 1.2:0.8, preferably being 1:1.

Description

[0001] Process for producing nucleotide sugars using enzyme cascades in a continuously fed membrane reactor

[0002] The present invention relates to a process for producing activated sugars. In particular, the present invention relates to a process by which activated sugars, such as nucleotide sugars and / or sugar-1-phosphates, can be produced in a particularly efficient manner. The present invention further relates to a reactor for carrying out such a process.

[0003] Enzyme cascades for the production of nucleotide sugars are usually based on the synthesis branch of the "salvage pathway." Monosaccharides are phosphorylated with a kinase at the anomeric CI atom of the sugar, consuming ATP. In the next step, the sugar is activated with a pyrophosphorylase with the acceptor nucleotide, such as UTP, CTP, or GTP, with the cleavage of pyrophosphate (PPi). For an efficient enzyme cascade, the PPi is then cleaved with the help of a pyrophosphatase to shift the cascade reaction toward the product. State-of-the-art enzyme cascades were used for the synthesis of nucleotide sugars in batch processes (one-pot processes) or in repetitive batch processes.

[0004] EP 0 827 549 A1 relates to a process for the enzymatic galactosylation of monosaccharides and oligosaccharides with in situ regeneration of the nucleotide sugar (or the nucleoside diphosphate sugar) in the presence of sucrose synthase, beta-1-4-galactosyltransferase and uridine diphosphate-glucose-4'-epimerase (UDP-glucose-4 1 - epimerase). This process is carried out as a batch process.

[0005] EP 0 650 520 and DE 4 221 595 CI describe a process for the production of nucleotide sugars, such as UDP-galactose and UDP-glucuronic acid, based on a specially purified sucrose synthase. However, this publication does not focus on the production of nucleotide sugars and does not describe a design for this in a continuously fed membrane reactor.

[0006] EP 0 767 239 and DE 195 37 217 A1 relate to processes for the enzymatic production of nucleotide-6-deoxy-D-xylo-4-hexulose, which are characterized in that nucleoside monophosphate, phosphoenolpyruvate, adenosine triphosphate, and sucrose as substrates, and pyruvate kinase, nucleoside monophosphate kinase, sucrose synthase, and deoxythymide in-D-glucose-4,6-dehyratase as enzymes, are simultaneously incubated in a buffer solution. This document also does not address the use of a continuously fed membrane reactor for the production of nucleotide sugars.

[0007] DE 10 2018 116 200 A1 describes a process for producing a monosaccharide 1-nucleoside diphosphate in which a monosaccharide, a monosaccharide 1-phosphate kinase, a nucleoside triphosphate-sugar 1-phosphate nucleosidyltransferase, a pyrophosphatase, and at least one nucleoside triphosphate or a deoxynucleoside triphosphate and a nucleoside triphosphate are brought into contact in an aqueous solution in a reaction vessel. The specificity of the monosaccharide 1-phosphate kinase allows phosphorylation of the monosaccharide, and the specificity of the nucleoside triphosphate-sugar 1-phosphate nucleosidyltransferase allows the transfer of a nucleoside monophosphate unit to a monosaccharide 1-phosphate of the monosaccharide.The monosaccharide 1-nucleoside diphosphate is removed by means of an ultra- and / or nanofiltration membrane in such a way that the total volume is maintained or restored, the enzymes remain and at least one nucleoside triphosphate or the deoxynucleoside triphosphate and the nucleoside triphosphate and the monosaccharide are reintroduced.

[0008] However, the solutions known from the state of the art may still have potential for improvement, in particular with regard to efficient process control or high yield.

[0009] It is therefore the object of the present invention to at least partially overcome at least one disadvantage of the prior art. In particular, it is an object of the present invention to provide a solution that allows a process for producing an activated sugar with particularly high efficiency or with a particularly high yield.

[0010] The object is achieved according to the invention by a method having the features of claim 1. The object is further achieved by a reactor having the features of claim 11 and by a use having the features of claim 13. Preferred embodiments of the invention are disclosed in the subclaims, in the description and in the figure, wherein further features described or shown in the subclaims or in the description or the figure can represent an object of the invention individually or in any combination, unless the context clearly indicates the opposite.

[0011] The present invention relates to a continuous process for producing activated sugars, comprising the process steps: a) introducing an enzyme cascade comprising enzymes dissolved in a solvent into a reaction volume; b) introducing reactants dissolved in a solvent into a reaction volume through a reactant stream, wherein the reactants serve to produce the activated sugar, in particular wherein the reactants comprise a saccharide reactant and a phosphate; c) reacting the reactants using the enzyme cascade to form the activated sugar;d) feeding the reaction solution formed, comprising the activated sugar formed and the enzyme cascade, to a separation membrane, the reaction solution being divided into a first fluid stream and a second fluid stream, the first fluid stream comprising the enzyme cascade and being depleted in activated sugar, and the second fluid stream comprising activated sugar and being depleted in the enzyme cascade; e) returning the first fluid stream to the reaction volume, f) adjusting the amount of reactant introduced into the reaction volume in process step a) as a function of the amount of activated sugar contained in the second fluid stream, such that g) the molar ratio of the reactant to the activated sugar is in a ratio of 0.8:1.2 to 1.2:0.8, preferably 1:1;

[0012] A process described above for producing an activated sugar has significant advantages over the prior art solutions, in particular with regard to particularly good efficiency and advantageous yield.

[0013] According to the invention, activated sugars are thus produced. For the purposes of the present invention, activated sugars are understood to mean, in particular, sugars that have at least one activated position relative to the saccharide starting material and are thus accessible for a reaction that is not possible with the structure of the saccharide starting material. Examples of activated sugars according to the invention are, in particular, nucleotide sugars, also referred to as nucleotide-activated sugars, or sugar-1-phosphates. A sugar-1-phosphate is an intermediate in the production of the nucleotide sugar.

[0014] Nucleotide sugars are considered cost-intensive substrates and thus a bottleneck for the large-scale application of Leloir glycosyltransferases, for example, as a standard process in synthetic carbohydrate chemistry. Nucleotide sugars are used, for example, in in vitro glycoengineering. In the pharmaceutical industry, nucleotide sugars are used for the modification of antibodies or in therapy. Furthermore, nucleotide sugars are used in the food industry and research. Accordingly, improved efficiency in the production of nucleotide sugars, or activated sugars in general, is a major advantage.

[0015] In order to produce activated sugars, the process according to the invention basically comprises reacting a reactant with an enzyme cascade, wherein the enzyme cascade is adaptable in a manner apparent to the person skilled in the art to the reaction to be carried out and in particular to the reactants and the activated sugar to be produced.

[0016] The following should be noted: According to the current state of the art, enzyme cascades for the production of nucleotide sugars are often based on the synthesis branch of the so-called "salvage pathway." Monosaccharides, for example, are phosphorylated with a kinase at the anomeric CI atom of the sugar, consuming ATP. In the next step, the sugar is activated with a pyrophosphorylase with the acceptor nucleotide, such as UTP, CTP, or GTP, with the cleavage of pyrophosphate (PPi). For an efficient enzyme cascade, the PPi is then cleaved with the help of a pyrophosphatase to shift the cascade reaction toward the product. The reaction can be terminated at the sugar 1-phosphate produced, because this product already represents a valuable activated sugar.According to the invention, to produce the activated sugar, an enzyme cascade comprising enzymes dissolved in a solvent is first introduced into a reaction volume according to process step a), and further, according to process step b), the reactants phosphate and saccharide dissolved in a solvent are introduced into a reaction volume via a reactant stream, wherein the reactants serve to produce the activated sugar and can therefore be configured, for example, as a monosaccharide and a phosphate, as described in more detail below. Preferably, the enzyme cascade comprising enzymes dissolved in a solvent is initially introduced into the reaction volume, and the reactants are then added, as described in more detail below. In principle, however, any order of addition of enzymes and reactants is encompassed by the present invention.For example, the phosphate can be added to the reaction volume together with the saccharide or the enzymes, or individually. The solvent of the enzyme cascade and the solvent of the reactant can be the same and include, for example, water. For example, the solvents can consist of water.

[0017] The enzyme cascade can be selected, for example, within the scope of the reaction to be carried out. In particular, if a monosaccharide is used as the reactant in the production of a nucleotide sugar, a monosaccharide 1-phosphate kinase, a nucleoside monophosphate kinase, a nucleoside diphosphate kinase, a nucleoside triphosphate-sugar-1-phosphate nucleosidyltransferase, a pyrophosphatase and a nucleoside triphosphate, or a deoxynucleoside triphosphate and a nucleoside triphosphate are placed in a reaction vessel in a solution, such as an aqueous solution. The nucleoside triphosphate-sugar-1-phosphate nucleosidyltransferase can also be referred to as a nucleoside diphosphate-sugar pyrophosphorylase. A nucleotide sugar synthetase is also possible. Bringing the enzyme cascade into contact with the reactants produces a preferably aqueous reaction mixture.The monosaccharide 1-phosphate kinase is preferably selected such that the specificity of the monosaccharide 1-phosphate kinase allows phosphorylation of the monosaccharide by means of the deoxynucleoside triphosphate or one of the at least one nucleoside triphosphate. Furthermore, the nucleoside triphosphate, or one of the at least one nucleoside triphosphate, is selected such that the specificity of the nucleoside triphosphate-sugar 1-phosphate nucleosidyltransferase allows the transfer of a nucleoside monophosphate unit to a monosaccharide 1-phosphate of the monosaccharide.

[0018] When using other reactants, the enzymes may need to be adapted, as will be readily understood by those skilled in the art.

[0019] In some embodiments, the nucleoside triphosphate sugar l-phosphate nucleosidyltransferase is a UDP-sugar pyrophosphorylase from Hordeum vulgare (barley). In some embodiments, the nucleoside triphosphate sugar l-phosphate nucleosidyltransferase is the enzyme encoded by the RNA sequence with GenBank accession number AK366137.1 from Hordeum vulgare subsp. vulgare, version 1 of the sequence dated March 18, 2011, version 2 of the database entry dated May 23, 2011 (noted: PLN dated May 20, 2011). This is the protein from Hordeum vulgare with Swissprot / Uniprot accession number F2DQG9, version 1 of the sequence dated May 31, 2011, version 19 of the database entry dated May 23, 2018. In some embodiments, the nucleoside triphosphate sugar-l-phosphate nucleosidyltransferase is a protein whose sequence is substantially identical to the sequence of the preceding Swissprot / Uniprot accession number F2DQG9.

[0020] In some embodiments, the nucleoside triphosphate sugar-l-phosphate nucleosidyltransferase is a D-galactose-l-phosphotransferase (galactokinase) from Bifidobacterium longum. This enzyme demonstrated broad substrate acceptance in specificity studies. For example, two suitable N-acetylhexosamine-l-kinases from Bifidobacterium longum subsp. infantis, strain ATCC 15697 and strain ATCC 55813, were cloned and characterized by Li et al. (Li, Y., et al., Molecules 2011, 16, 6396-6407).

[0021] In some embodiments, the nucleoside triphosphate sugar-l-phosphate nucleosidyltransferase is the enzyme from Bifidobacterium longum subsp. infantis (strain ATCC 15697) with Swissprot / Uniprot accession number B7GUI0, version 1 of the sequence dated February 10, 2009, version 79 of the database entry dated May 23, 2018. In some embodiments, the nucleoside triphosphate sugar-l-phosphate nucleosidyltransferase is a protein whose sequence is substantially identical to the sequence of Swissprot / Uniprot accession number B7GUI0. In some embodiments, the nucleoside triphosphate sugar-l-phosphate nucleosidyltransferase is a galactokinase with Swissprot / Uniprot accession number C5E862 from Bifidobacterium longum subsp. infantis CCUG 52486, version 1 of the sequence dated July 28, 2009, version 50 of the database entry dated October 25, 2017. As described by Li et al. (Li, L. et al., Carbohydrate Research (2012) 355, 35-39, which reveal the sequence of a corresponding galactokinase from Bifidobacterium longum subsp. infantis, report that this enzyme shows broad substrate specificity with respect to the monosaccharide and to the nucleoside triphosphate or deoxynucleoside triphosphate.

[0022] In some embodiments, the nucleoside triphosphate sugar-l-phosphate nucleosidyltransferase is the enzyme UTP-glucose-1-phosphate uridylyltransferase from Hordeum vulgare with Swissprot / Uniprot accession number Q43772, version 1 of the sequence dated July 15, 1999, version 74 of the database entry dated May 23, 2018. In some embodiments, the nucleoside triphosphate sugar-l-phosphate nucleosidyltransferase is a protein whose sequence is substantially identical to the sequence of the preceding Swissprot / Uniprot accession number Q43772. In some embodiments, the nucleoside triphosphate sugar-l-phosphate nucleosidyltransferase is the enzyme UTP-glucose-1-phosphate uri dylyltransferase from Hordeum vulgare with Swissprot / Uniprot accession number Q43772, version 1 of the sequence dated July 15, 1999, version 74 of the database entry dated May 23, 2018.In some embodiments, the nucleoside triphosphate sugar-l-phosphate nucleosidyltransferase is a protein whose sequence is substantially identical to the sequence of the preceding Swissprot / Uniprot accession number Q43772.

[0023] In some embodiments, the nucleoside triphosphate sugar-l-phosphate nucleosidyltransferase is a human UDP-GalNAc pyrophosphorylase. In some embodiments, the nucleoside triphosphate sugar-l-phosphate nucleosidyltransferase is the human enzyme UDP-κ-acetylhexosamine pyrophosphorylase with Swissprot / Uniprot accession number Ql 6222, version 3 of the sequence dated July 5, 2005, version 174 of the database entry dated November 22, 2017. In some embodiments, the nucleoside triphosphate sugar-l-phosphate nucleosidyltransferase is a protein whose sequence is substantially identical to the sequence of the preceding Swissprot / Uniprot accession number Ql 6222. In some embodiments, the nucleoside triphosphate sugar-l-phosphate nucleosidyltransferase is the human enzyme UTP-glucose-l-phosphate uridylyltransferase with Swissprot / Uniprot accession number Q168512, version 5 of the sequence dated January 23, 2007, version 160 of the database entry dated May 23, 2018.In some embodiments, the nucleoside triphosphate sugar-l-phosphate nucleosidyltransferase is a protein whose sequence is substantially identical to the sequence of Swissprot / Uniprot accession number Q168512. In some embodiments, the nucleoside triphosphate sugar-l-phosphate nucleosidyltransferase is the human protein with Swissprot / Uniprot accession number Q3KQV9, version 2 of the sequence dated March 18, 2008, version 97 of the database entry dated November 22, 2017. In some embodiments, the nucleoside triphosphate sugar-l-phosphate nucleosidyltransferase is a protein whose sequence is substantially identical to the sequence of Swissprot / Uniprot accession number Q3KQV9. In some embodiments, the nucleoside triphosphate sugar-l-phosphate nucleosidyltransferase is a human enzyme named UDP-7V-acetylhexosamine pyrophosphorylase with Swissprot / Uniprot accession number Ql 6222, version 3 of the sequence dated 5.July 2005, version 175 of the database entry dated June 20, 2018. In some embodiments, this is isoform AGX1 (identifier Ql 6222-2) of the database entry with the Swissprot / Uniprot accession number Ql 6222. In some embodiments, this is isoform AGX2 (identifier Ql 6222-1) of the database entry with the Swissprot / Uniprot accession number Ql 6222. In some embodiments, this is isoform AGX3 (identifier Ql 6222-3) of the database entry with the Swissprot / Uniprot accession number Ql 6222. Expression of the AGX1 isoform with a tag was described by Bourgeaux et al. described (Bourgeaux, V., et al., Bioorganic & Medicinal Chemistry Letters (2005) 15, 5459-5462).

[0024] In some embodiments, the nucleoside triphosphate sugar-l-phosphate nucleosidyltransferase is the murine enzyme UDP-7V-acetylhexosamine pyrophosphorylase having Swissprot / Uniprot accession number Q91YN5, version 1 of the sequence dated December 1, 2001, version 125 of the database entry dated August 30, 2017. In some embodiments, the nucleoside triphosphate sugar-l-phosphate nucleosidyltransferase is a protein whose sequence is substantially identical to the sequence of the preceding Swissprot / Uniprot accession number Q91YN5.

[0025] In some embodiments, the nucleoside triphosphate sugar-l-phosphate nucleosidyltransferase is a UDP-sugar pyrophosphorylase from Arabidopsis thaliana (thale cress, sweet cress), which was cloned and characterized by Liu et al. (Liu, J., et al., Bioorganic & Medicinal Chemistry Letters (2013) 23, 3764-3768). For example, the protein with Swissprot / Uniprot accession number Q9C5I1, version 1 of the sequence dated June 1, 2001, version 102 of the database entry dated April 25, 2018, or the protein with Swissprot / Uniprot accession number 064765, version 1 of the sequence dated August 1, 1998, version 127 of the database entry dated April 25, 2018, may be used. In some embodiments, the nucleoside triphosphate sugar-l-phosphate nucleosidyltransferase is a protein whose sequence is substantially identical to the sequence of the preceding Swissprot / Uniprot accession number 064765. A protein as described by Liu et al.(2013, supra) can also be used in a process disclosed here.

[0026] In some embodiments, the nucleoside triphosphate sugar-l-phosphate nucleosidyltransferase is the enzyme mannose-1-phosphate guanylyltransferase from E. coli with Swissprot / Uniprot accession number P37741, version 1 of the sequence dated October 1, 1994, version 91 of the database entry dated May 23, 2018. In some embodiments, the nucleoside triphosphate sugar-l-phosphate nucleosidyltransferase is a protein whose sequence is substantially identical to the sequence of the preceding Swissprot / Uniprot accession number P37741. In some embodiments, the nucleoside triphosphate sugar-l-phosphate nucleosidyltransferase is the enzyme mannose-1-phosphate guanylyltransferase from Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720) with Swissprot / Uniprot accession number P26404, version 1 of the sequence dated August 1, 1992, version 123 of the database entry dated March 28, 2018.

[0027] In some embodiments, the monosaccharide 1-phosphate kinase is a galactokinase from coli. In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyltransferase is a galactokinase encoded by the nucleic acid from Escherichia coli strain TB182A with GenBank accession number EU903918.1, version 1 of the sequence dated April 30, 2009. In some embodiments, the monosaccharide 1-phosphate kinase is a protein whose sequence is substantially identical to the sequence of the protein encoded by the nucleic acid sequence with GenBank accession number EU903918.1. In some embodiments, the monosaccharide 1-phosphate kinase is an N-acetylhexosamine 1-kinase from Bifidobacterium longum. In some embodiments, the nucleoside triphosphate sugar 1-phosphate nucleosidyltransferase is the N-acetylhexosamine 1-kinase from Bifidobacterium longum subsp.Longum with Swissprot / Uniprot accession number E8MF12, version 1 of the sequence dated April 15, 2011, version 31 of the database entry dated February 28, 2018. In some embodiments, the nucleoside triphosphate sugar-l-phosphate nucleosidyltransferase is a protein whose sequence is substantially identical to the sequence of Swissprot / Uniprot accession number E8MF12.

[0028] In some embodiments, the monosaccharide 1-phosphate kinase is the enzyme galactokinase from Lactococcus lactis, for example one of the enzymes described by Grossiord et al. (Grossiord, BP, et al., Journal of Bacteriology (2003) 185, 3, 870-878). The galactokinase can be the enzyme from Lactococcus lactis subsp. cremoris (strain MG1363), with Swissprot / Uniprot accession number Q9S6S2, version 1 of the sequence dated May 1, 2000, version 108 of the database entry dated October 25, 2017, or the enzyme from Lactococcus lactis subsp. lactis (strain IL1403), with Swissprot / Uniprot accession number Q9R7D7, version 1 of the sequence dated May 1, 2000, version 123 of the database entry dated June 20, 2018. In some embodiments, the monosaccharide 1-phosphate kinase is a protein whose sequence is substantially identical to the sequence of one of the preceding Swissprot / Uniprot accession numbers Q9S6S2 or Q9R7D7.

[0029] In typical embodiments, monosaccharide 1-phosphate kinase and nucleoside triphosphate sugar 1-phosphate nucleosidyltransferase are two different enzymes. In some embodiments, both catalytic functions can be performed by the same enzyme. Thus, in some embodiments, monosaccharide 1-phosphate kinase and nucleoside triphosphate sugar 1-phosphate nucleosidyltransferase are the same enzyme.

[0030] Numerous pyrophosphatases are known that can be used in a method and use described here. An example of a pyrophosphatase is the enzyme from S. cerevisiae with the Swissprot / Uniprot accession number P00817, version 4 of the sequence dated July 24, 2007, and version 193 of the database entry dated May 23, 2018. Another example of a pyrophosphatase is the enzyme from Pasteurella multocida, which was used by Muthana (Muthana, MM, et al., Chem. Commun. (2012) 48, 2728-2730). For example, the enzyme from Pasteurella multocida may be the enzyme with Swissprot / Uniprot accession number Q9CKF5, version 1 of the sequence dated June 1, 2001, version 89 of the database entry dated June 20, 2018. In some embodiments, the pyrophosphatase is a protein whose sequence is substantially identical to one of the sequences of the preceding Swissprot / Uniprot accession numbers P00817 or Q9CKF5.Another example of a pyrophosphatase is the enzyme from E. coli used by Li et al. (Li, L., et al. Org. Lett. (2013) 15, 21, 5528-5530). The pyrophosphatase can, for example, be the enzyme with the Swissprot / Uniprot accession number P0A7A9, version 2 of the sequence dated January 23, 2007, version 113 of the database entry dated March 28, 2018. In some embodiments, the pyrophosphatase can be a protein whose sequence is substantially identical to the sequence of the preceding Swissprot / Uniprot accession number P0A7A9.

[0031] In some embodiments, the monosaccharide employed is λ-acetylgalactosamine. As shown by Bourgeaux et al. (Bourgeaux, V., et al., Bioorganic & Medicinal Chemistry Letters (2005) 15, 5459-5462), a porcine 7λ-acetylgalactosamine kinase can be used as the monosaccharide 1-phosphate kinase for this monosaccharide. Bourgeaux et al. describe the cloning and expression of the enzyme. This enzyme can be, for example, a porcine enzyme designated galactokinase with the Swissprot / Uniprot accession number A0A287BB25, version 1 of the sequence dated November 22, 2017, version 5 of the database entry dated March 28, 2018. In some embodiments, the galactokinase is a protein whose sequence is substantially identical to the sequence of the preceding Swissprot / Uniprot accession number A0A287BB25.For example, human UDP-N-acetylhexosamine pyrophosphorylase with the Swissprot / Uniprot accession number Q16222 (supra) can be used as nucleoside triphosphate sugar-l-phosphate nucleosidyltransferase (Bourgeaux et al., 2005, supra).

[0032] Furthermore, the reactants are added to the reaction volume. The reactants can be chosen in principle, as long as they are suitable for producing the desired activated sugar.

[0033] In principle, a wide variety of phosphates can be used as starting materials. For example, a nucleoside monophosphate or a nucleoside triphosphate can be used.

[0034] The nucleoside triphosphate can, for example, comprise adenosine triphosphate (ATP). In principle, adenosine triphosphate (ATP), guanosine triphosphate (GTP), uridine triphosphate (UTP), particularly for sugar kinases, and especially for pyrophosphorylases, preferably adenosine triphosphate (ATP), guanosine triphosphate (GTP), uridine triphosphate (UTP), cytidine triphosphate (CTP), deoxythymidine triphosphate (dTTP), and deoxyuridine triphosphate (dUTP).

[0035] Deoxynucleoside triphosphate can, for example, include deoxyadenosine triphosphate (dATP). In principle, deoxythymidine triphosphate (dTTP) and deoxyuridine triphosphate (dUTP) can be used for pyrophosphorylases.

[0036] In particular, the reactants may comprise, for example, a saccharide reactant. In one embodiment, the saccharide reactant may comprise a monosaccharide. The monosaccharide in question may, in principle, be a D-monosaccharide or an L-monosaccharide.

[0037] The monosaccharide in question can be a hexose or a pentose, including a monodeoxyhexose or a monodeoxypentose. Examples of hexoses include glucose (Glc) and glucosamine (2-amino-2-deoxyglucose, GICNH2). Hexoses also include α-acetylglucosamine (2-acetamido-2-deoxyglucose, GlcNAc). Hexoses also include galactose (Gal) and galactosamine (2-amino-2-deoxygalactose, GalNdE). N-acetylgalactosamine (2-acetamido-2-deoxygalactose, GalNAc) is also an example of a hexose. Two other examples of hexoses are mannose (Man) and mannosamine (2-amino-2-deoxymannose, ManbdHE). Hexoses also include α-acetylmannosamine (2-acetamido-2-deoxymannose; ManNAc). Another example of a hexose is glucuronic acid (GlcA). Hexoses also include iduronic acid (IdoA) and galacturonic acid (GalA). Another example of a hexose is 6-deoxygalactose, called fucose.

[0038] Pentoses include ribose and xylose. Arabinose (Arb) is also an example of a pentose. The monosaccharide can be unsubstituted or substituted with one or more groups. Possible substituents include, but are not limited to, an amino group or an amido group. An acylamido group is also an example of a suitable substituent. Other examples of possible substituents are an O-sulfate group or an A-sulfate group (sulfamate).

[0039] Also suitable as corresponding saccharide starting materials are nonulonic acids, such as N-acetylneuraminic acid (Neu5Ac), which is a C9 sugar and belongs to the substance class of sialic acids or nonulonic acids. As already stated above, in some embodiments the monosaccharide may contain several different monosaccharides or consist of several different monosaccharides. For example, the monosaccharide may contain λ-acetyl-galactosamine and λ-acetyl-glucosamine. In some embodiments, the monosaccharide may contain fucose, arabinose, mannose, and galactose. In some embodiments, the monosaccharide may contain glucuronic acid and galacturonic acid. In some such embodiments, the monosaccharide 1-phosphate kinase contains a plurality of monosaccharide 1-phosphate kinases.In some such embodiments, the nucleoside triphosphate-sugar-1-phosphate nucleosidyltransferase contains a plurality of nucleoside triphosphate-sugar-1-phosphate nucleosidyltransferases. In further embodiments, the monosaccharide C9 may contain sugar. In these embodiments, the nucleotidyltransferase contains a plurality of nucleotidyltransferases.

[0040] However, the use of a starting material is in no way limited to monosaccharides. Alternatively or in addition to monosaccharides, the starting materials can also comprise other saccharides. Examples include disaccharides in basically any form. The disaccharides possible according to the invention include, but are not limited to, sucrose, maltose, and lactose.

[0041] Furthermore, as indicated above, derivatives of monosaccharides and / or disaccharides are also generally included among the possible starting materials. For the purposes of the present invention, derivatives are understood to mean, in particular, deoxy-, amino-, amino-functionalized, azido-, halogenated derivatives, and others which bear substituents on the saccharide structure. Suitable substituents include, but are not limited to, an amino group or an amido group. An acylamido group is also an example of a suitable substituent. Further examples of possible substituents are an O-sulfate group or a TV-sulfate group (sulfamate). According to process step c), the reactant is further reacted using phosphates and the enzyme cascade to form the activated sugar. In principle, the parameters customary for this reaction can be used.

[0042] Depending on the enzyme used, the reaction can be carried out within the entire range in which the solvent, such as in particular water, optionally after the addition of salts, is in a liquid state. When using thermostable enzymes, it is possible to work in ranges close to the boiling point. In some embodiments, the process or use can be carried out in a temperature range from about 30°C to about 95°C, including, for example, a temperature range from about 35°C to about 95°C. In some embodiments, the process or use can be carried out in a temperature range from about 18°C ​​to about 70°C, including, for example, a temperature range from about 22°C to about 45°C. In some embodiments, the reaction by means of the enzymes used can take place in a temperature range from about 20°C to about 38°C; for example, a range from 27°C to 35°C can be selected.In some embodiments, a temperature range of 28°C to 32°C can be selected, including a temperature of 30°C ± 1°C or ± 0.5°C. It has further been observed that, with a possibly lower reaction rate, reaction conditions close to the freezing point of water—optionally after the addition of salts, see above—enable a sufficiently effective conversion in the protein quantities usable here. In some embodiments, the process or use can be carried out in a temperature range of about 2°C to about 28°C, including, for example, a temperature range of about 4°C to about 18°C.

[0043] It may further be preferred that the reactant, such as the monosaccharide, be present in the reactor in process step c) at a concentration ranging from 1 mM to 5000 mM. The advantages of the present invention are particularly efficiently achieved within this concentration range.

[0044] For an effective reaction, it may also be advantageous for enzymes to be present in the reactor during process step c) at a concentration ranging from 0.1 mg / mL to 1000 mg / mL. In principle, as explained in more detail below, the enzyme concentration can be selected depending on the concentration of the reactants. The aforementioned concentration also refers to any enzyme present in the enzyme cascade.

[0045] The enzymes used are typically used in a non-immobilized form. The enzymes used are typically used in a non-crosslinked form. The enzymes used are usually present in aqueous solution as soluble, free protein.

[0046] With regard to a particularly efficient reaction, it may also be advantageous to use a pH value in a range of > 7 to < 9 in process step c).

[0047] Typically, the aqueous solution contains one or more buffer compounds such as trisaminomethane (Tris), 2-(4-(2-hydroxyethyl)-l-piperazinyl)-ethanesulfonic acid (HEPES), 4-(2-hydroxyethyl)-piperazine-l-propanesulfonic acid (HEPPS), an acetate, or a phosphate.

[0048] The activated sugar produced in the process according to the invention can, in one embodiment, be a nucleotide sugar. In particular, in one embodiment, it can be provided that the activated sugar is at least 90 mol% a nucleotide sugar. This can be achieved in a manner known per se by providing the appropriate enzymes as described above. Accordingly, in this embodiment, the process thus serves to produce a nucleotide sugar. A nucleotide sugar can, for example, be a UDP-sugar, i.e., a nucleotide sugar whose nucleoside diphosphate group is UDP. Examples of a UDP-sugar include, but are not limited to, UDP-Glc, UDP-GlcNAc, UDP-GlcNH2, UDP-GlcA, UDP-IdoA, UDP-GalA, UDP-Gal, UDP-GalNAc, UDP-GalNFE, UDP-Man, UDP-ManNAc, and UDP-ManNFE. The UDP sugar may be unsubstituted or contain substituents as described above.

[0049] Alternatively, it can also be provided that the activated sugar produced in the process according to the invention is a sugar-1-phosphate. In particular, it can be provided in one embodiment that the activated sugar is at least 90 mol% sugar-1-phosphate. This embodiment can also be implemented in a simple manner by not adding the enzymes that promote the reaction of the sugar-1-phosphate to the nucleotide sugar to the reaction solution. Accordingly, the reaction is terminated upon production of the sugar-1-phosphate. The sugar-1-phosphate formed can be configured in a manner that is immediately recognizable to the person skilled in the art, depending on the selected starting material.

[0050] In some embodiments, one or more additional cofactors are used that are necessary for the function of one or more enzymes used. This can, in particular, be a magnesium salt that forms a complex with a nucleoside triphosphate and / or a deoxynucleoside triphosphate. For a number of the enzymes usable in a method and use disclosed herein, a manganese salt can also be used as a cofactor.

[0051] According to process step d), the process according to the invention further provides for conveying the reaction solution formed, comprising the activated sugar formed and the enzyme cascade, to a separation membrane, wherein the reaction solution is divided into a first fluid stream and a second fluid stream, wherein the first fluid stream comprises the enzyme cascade and is depleted in activated sugar, and wherein the second fluid stream comprises activated sugar and is depleted in the enzyme cascade. In this process step, the reaction solution formed during the reaction is thus conveyed to the separation membrane in order to allow separation between the product formed and the enzymes or the enzyme cascade. In particular, in process step d), the first fluid stream is a retentate stream and the second fluid stream is a permeate stream.

[0052] For this purpose, it may be preferable to perform tangential flow filtration through the separation membrane. This allows for a particularly defined and reproducible separation of the product, i.e., activated sugar, and the enzymes required for the reaction. A particularly efficient separation of the resulting components can be achieved by using a separation membrane with a molecular weight cutoff of at least 1 kDa. Particularly when separating activated sugars from the enzymes required for their production, such a membrane offers advantages in terms of efficient separation and good flow, i.e., good separation per unit time.

[0053] Finally, according to process step e), the first fluid stream, i.e. the fluid stream comprising the enzyme cascade and depleted of activated sugar, is returned to the reaction volume. Thus, the fluid stream from which the product or activated sugar was removed is returned to the reaction volume. This results in the enzymes being recycled and, accordingly, being recycled for the reaction. This reaction step, in particular, can lead to a particularly effective reaction or a particularly high yield. In parallel, further reactants are added to the reaction volume. Accordingly, further reactants are present, which can in turn be converted into activated sugar using the recycled enzyme cascade. The reactants are added with particular consideration of the removed product, as described in more detail below.

[0054] In the present invention, activated sugars, such as nucleotide sugars or sugar-1-phosphates, are thus produced continuously using enzyme cascades over a long period of time. Cross-flow filtration is used to continuously separate enzymes and products at a membrane. The enzymes of a nucleotide sugar enzyme cascade or an enzyme cascade for the production of sugar-1-phosphates are dissolved and are continuously flushed through a membrane and recirculated.

[0055] Parallel to this step, the substrate solution is pumped into the reaction volume or reaction vessel by means of a feed or inlet, particularly in the same ratio to the filtration volume. This maintains a constant volume in the reaction chamber, feeds the substrate into the reactor at a constant concentration, and continuously removes the product.

[0056] Accordingly, the amount of reactants introduced into the reaction volume in process step a) is adjusted depending on the amount of activated sugar contained in the second fluid stream. Thus, the removed product can be replaced by new reactant in a desired proportion, or even completely or in a larger proportion.

[0057] More specifically, the molar ratio of the added reactant to the generated activated sugar is 0.8:1.2 to 1.2:0.8, preferably 1:1. The continuous process described here enables the production of activated sugars with stable product formation at low technical expense. The described process leads to multigram quantities of nucleotide sugar within one day, even on a laboratory scale. The effective use of the biocatalysts leads to high key figures – such as the space-time yield (STY, g L' 1 h' 1) and catalytic productivity (TTN, g product per g enzyme) as well as an efficient product formation rate (gh' 1 ) which are of industrial relevance for bioprocesses.

[0058] In the described continuously fed process, for example, the reactor volume is reduced to 60 mL and the amount of biocatalyst is significantly reduced. Crude product solutions of >1 L are obtained in the process. This procedure represents a significant improvement in the production process and results in the stable production of activated sugars on a gram scale. In the continuously fed membrane reactor, constant substrate and product concentrations prevail due to the simultaneous addition of substrate solution and product removal (filtration) of the product solution. Thus, a so-called steady state prevails, which leads to a particularly advantageous utilization of enzyme kinetics, as these are constantly used for the continuous production of activated sugars.

[0059] Such advantages were not possible in the prior art.

[0060] According to the state of the art, enzyme cascades were mostly used in batch processes (one-pot processes) or in repeated batch processes (repetitive batch) for the synthesis of nucleotide sugars.

[0061] Previous enzyme cascades for the production of nucleotide sugars are therefore based on batch processes, which result in the complex and expensive provision of recombinant enzymes. Under comparable conditions to synthesis in a continuous reactor, batch processes require significantly higher amounts of biocatalyst for the same product volume and at a constant enzyme concentration, resulting in significant effort in the production of the recombinant enzymes. In addition, catalytic limitations due to product and substrate inhibition can occur in batch processes, which can reduce conversion rates or increase production times. Immobilization techniques for the reuse of enzymes are usually complex, expensive, and reduce catalytic activity.Enzyme and membrane reactors described for nucleotide sugar synthesis either utilize immobilized enzyme or rely on continuous flow through the reactor, without recirculation of unreacted substrate. In repetitive batch processes, the stepwise separation and reuse of dissolved enzyme using filtration units leads to significant productivity losses, as no new substrate can be converted during the volume reduction through filtration. Furthermore, the filtration time extends the overall production time.

[0062] In particular, the invention offers advantages over repetitive batch processes, such as those described in DE 10 2018 116 200 A1. In such a process, a quasi-continuous process takes place in which a batch reaction mixture is continuously passed over a membrane and the filtrate (monosaccharide, nucleotides, magnesium, and product) is recycled into the batch. This continues until the reactants have been converted as completely as possible. In this case, the recycling is interrupted, the filtrate is collected as a product solution, and a new batch, which still contains all the enzymes, is started by adding the new reactants. A steady state is therefore not reached. Each batch starts with high reactant concentrations and is subject to limitations such as substrate excess and product inhibition. The added value of the present invention lies in the effective use of the recombinant enzymes.This leads to cost savings in the production process compared to other described processes. Furthermore, the system is easy to acquire and robust for the rapid production of activated sugars on a multigram scale. Furthermore, the use of the continuously fed membrane reactor separates the enzymes directly from the product, simplifying further purification of the activated sugars. These factors combined offer a significantly improved reaction process, particularly in terms of efficiency and yield, and thus cost savings, which were previously unknown and impossible.

[0063] However, producing suitable enzyme quantities for large-scale synthesis is costly and time-consuming. Maximizing enzyme productivity in terms of the mass-based turnover number (TTNmass, g enzyme per g product) as well as the specific product performance (space-time yield, STY) is therefore a challenge in order to exploit the full potential of enzyme cascades. This can be effectively achieved according to the invention.

[0064] For further advantages and technical features of the process, reference is made to the description of the reactor system, the use, the figures and the use and vice versa.

[0065] The invention further relates to a reactor system for producing an activated sugar, at least comprising a reactor for forming the activated sugar from a reactant using an enzyme cascade; a reactant feed for feeding the reactants into the reactor; a membrane unit with a separation membrane, wherein the membrane unit is connected to the reactor by a fluid line for separating a product stream comprising the activated sugar formed and the enzyme cascade through the separation membrane to form a retentate stream and a permeate stream; a retentate guide for discharging the retentate stream from the separation membrane; and a permeate guide for discharging the permeate stream from the separation membrane; wherein the permeate guide or the retentate guide, preferably the retentate guide, leads into the reactor.

[0066] The reactor can, in principle, be designed in a suitable manner. The reaction chamber should be designed to withstand the prevailing conditions or remain inert under these conditions. The corresponding supply and discharge lines can be formed by conventional liquid lines, so that they can also withstand the prevailing conditions or remain inert under these conditions. Furthermore, appropriate conveying units, such as pumps, can be provided to convey the fluids or liquid streams.

[0067] The membrane unit may in particular comprise a separation membrane comprising a membrane with a molecular weight cutoff of at least 1 kDa.

[0068] The reactor system described above is thus particularly designed to carry out a process as described above in greater detail. Accordingly, the advantages described above can be achieved using a reactor system in particular by passing the enzyme cascade back into the reaction volume after separation from the product stream, whereby in particular a so-called steady state can be achieved in the reaction vessel. In detail, it is possible to form an activated sugar, such as in particular a nucleotide sugar or a sugar-1-phosphate, with particularly high efficiency and a particularly high yield. Due to the continuous reaction, larger amounts of substrate can be converted over a longer period of time, in contrast to the repetitive batch process, because excess substrate inhibition or product inhibition do not have a limiting effect.In addition, the continuous discharge of the product in the process allows for a higher volume / product turnover during the process.

[0069] Accordingly, a further object of the present invention is the use of a process as described above or of a reactor system as described above for producing an activated sugar, in particular a phosphate-1-sugar or a nucleotide sugar.

[0070] For further advantages and technical features of the reactor system and its use, reference is made to the description of the process, the figures and the description of the figures, and vice versa.

[0071] The invention is explained below by way of example with reference to the attached drawing, wherein the features shown below can represent an aspect of the invention both individually and in combination, and wherein the invention is not limited to the following drawing, the following description and the following embodiments.

[0072] They show:

[0073] Fig. 1 schematically shows a reactor system for producing an activated sugar; and

[0074] Fig. 2 is a diagram illustrating an exemplary embodiment of a method according to the invention.

[0075] Figure 1 shows a reactor system 10 for producing an activated sugar according to the present invention. According to Figure 1, the reactor system 10 comprises a reactor 12 for forming the activated sugar from a reactant using an enzyme cascade. For this purpose, the reactor 12 comprises a reaction volume 14 into which the

[0076] Reaction partners can be inserted and react to form the activated sugar.

[0077] An enzyme cascade can initially be introduced into reactor 12, which serves to form the activated sugar from a reactant. The reactants, such as a monosaccharide and a phosphate, can be stored in a storage container 18 and pumped into reactor 12 through a reactant feed 20 using a reactant pump 22. Here, a reaction can then proceed under suitable conditions, and the activated sugar can be formed. For this purpose, for example, a heat exchanger 24 can be provided, with which the reactor 12 can be brought to a suitable temperature.

[0078] Through a product discharge 26 as a fluid line, the reaction solution containing the enzyme cascade and the resulting product can be conveyed, for example, by a pump 28, such as a membrane pump, into a membrane unit 30. The membrane unit 30 comprises a separation membrane 32, with which the reaction solution can be filtered and divided or separated into a permeate and a retentate stream.

[0079] In the embodiment according to Figure 1, the retentate stream flows through a retentate line 34 to divert the retentate stream s from the separation membrane 32 back into the reactor 12. A pressure sensor 36 can be provided in the retentate line 34, which can measure the pressure of the retentate stream s and, if necessary, increase or reduce the force of the pump 28. Accordingly, the enzyme cascade can be fed back into the reactor 12 and, with fresh reactants, can again undergo a reaction to form activated sugar. Furthermore, a permeate guide 38 is provided, which carries the permeate stream, which according to Figure 1 contains the activated sugar formed, away from the membrane unit 30. According to Figure 1, the permeate stream can be fed into a collecting tank 40 and collected there. For temperature control, such as cooling, of the permeate, a temperature control unit 42 can be provided, which can cool the contents of the collecting tank 40.

[0080] The described reactor system 10 can be used to produce activated sugars, such as nucleotide sugars, in a continuously fed (continuous-fed-batch) membrane reactor on a gram scale. The products and enzymes are continuously separated at the separation membrane 32, also known as cross-flow filtration. The enzymes of the enzyme cascade are in solution and are continuously flushed through the separation membrane 32 and then returned. In parallel, the substrate solution is pumped into the reactor 12 using the reactant feed 20 in the same ratio to the filtration volume. This results in a constant volume in the reaction volume 14; the substrate is added at a constant concentration, and the product is removed (steady state). The disclosed process thus enables stable production of activated sugars with optimal utilization of enzyme kinetics in the steady-state process.The effective use results in the space-time yield (STY), the catalytic productivity (TTN) and the product formation rate being in the industrially relevant range.

[0081] In detail, a continuous process for producing activated sugars can be carried out, comprising the process steps: a) introducing an enzyme cascade comprising enzymes dissolved in a solvent into a reaction volume 14; b) introducing reactants dissolved in a solvent into the reaction volume 14 through a reactant stream, wherein the reactants serve to produce the activated sugar; c) reacting the reactants using the enzyme cascade to form the activated sugar; d) passing the reaction solution formed, comprising the activated sugar formed and the enzyme cascade, to a separation membrane 32, wherein the reaction solution is divided into a first fluid stream and a second fluid stream, wherein the first fluid stream comprises the enzyme cascade and is depleted in activated sugar, and wherein the second fluid stream comprises activated sugar and is depleted in the enzyme cascade;e) returning the first fluid stream to the reaction volume 14;

[0082] An exemplary concrete embodiment is described below. The reactions proceed according to the following reaction scheme: In the reaction scheme of the CMP-Neu5 Ac synthesis reaction shown, the following abbreviations were used: / ACM PK: Escherichia coli CMP kinase; ö'cCDPK: Saccharomyces cerevisiae CDP kinase.

[0083] In the described embodiment, a cascade was additionally developed using the reaction cascade for CMP-Neu5Ac synthesis, in which the nucleoside triphosphate is obtained from a nucleoside monophosphate and ATP. The experimental setup included a reaction cascade for the synthesis of CMP-Neu5Ac from CMP and Neu5Ac. In this experiment, 0.72 mg / mL AmCSS, 0.66 mg / mL / ACM PK, 0.021 mg / mL &CDPK, and δ'cPPase (Roche) at a concentration of 5 pg / mL were used. The membrane reactor consisted of a 10 kDa MWCO Vivaflow 50R cross-flow filtration module (Ml-10) (Sarotrius). A Watson Marlow 101U pump served as the feed pump or reactant pump 22, and the reaction took place in a heated 100 mL beaker as reactor 12. The Vivaflow 50R membrane, which was used as separation membrane 32, was operated using a Masterflex Easy Load pump 28.Before the start of the experiment, the filtration module or membrane unit 30 was rinsed with 500 mL of MQ H2O to remove any residues from the manufacturing process, such as glycerides, from the separation membrane 32. Before the enzyme addition, the reactor 12 was cycled for 10 minutes with 50 mL of a priming solution (10 mM MgCl, 100 mM HEPES pH 8, 0.2 mM DTT) over the separation membrane 32. The enzymes were then added, and the solution was recycled for another 5 minutes. The inflow of the feed or reactants was then synchronized with the outflow of the filtrate at approximately 2.2–3 mL / min. The feed was a solution containing 10 mM CMP, 20 mM ATP, 10 mM Neu5Ac, 30 mM MgCh, 0.2 mM DTT, and 100 mM HEPES (pH 8). Three production runs were performed with the same enzyme batch for 4.5 h, 4 h, and 4.8 h, respectively, and a sample was taken hourly from the filtrate, for example, from collection container 40. The enzyme was stored overnight at 4 °C between production runs.

[0084] The result is shown in Figure 2, where the X-axis represents time in hours, the Y1 axis represents the concentration of CMP-Neu5Ac in mM, and the Y2 axis represents product growth in g. Curve A further describes the respective parameters in reactor 12, curve B represents the respective parameters in the filtrate, and curve C represents the respective parameters of the product. When considering the product concentration curve, it can be seen that a nearly constant concentration of CMP-Neu5Ac between 9.84 mM and 10.71 mM was maintained in reactor 12 over an entire process period of 14.3 h. As the filtrate volume increased, the product quantity also increased steadily up to a final quantity of 8.2 g. With the amount of enzyme used, reactor 12 achieved a product conversion rate (TTN) of 91 gp / gr and a nearly constant space-time yield (STY) of 4.6 g. P The 1 h' 1.

[0085] Thus, the synthesis of CMP-Neu5Ac showed that it is also possible to produce CMP-Neu5Ac from CMP and Neu5Ac on a multigram scale and with high efficiency using reactor 12.

Claims

Patent claims 1. A continuous process for producing activated sugars, comprising the process steps: a) introducing an enzyme cascade comprising enzymes dissolved in a solvent into a reaction volume (14); b) introducing reactants dissolved in a solvent into the reaction volume (14) through a reactant stream, wherein the reactants serve to produce the activated sugar; c) reacting the reactants using the enzyme cascade to form the activated sugar; d) passing the reaction solution formed, comprising the activated sugar formed and the enzyme cascade, to a separation membrane (32), wherein the reaction solution is divided into a first fluid stream and a second fluid stream, wherein the first fluid stream comprises the enzyme cascade and is depleted in activated sugar, and wherein the second fluid stream comprises activated sugar and is depleted in the enzyme cascade;e) returning the first fluid stream to the reaction volume (14), wherein f) the amount of reactant introduced into the reaction volume (14) in process step a) is adjusted as a function of the amount of activated sugar contained in the second fluid stream such that g) the molar ratio of the reactant to the activated sugar is in a ratio of 0.8:1.2 to 1.2:0.8, preferably 1:1; 2. The process according to claim 1, characterized in that the reactants comprise a saccharide reactant and a phosphate.

3. Process according to one of claims 1 or 2, characterized in that the activated sugar is at least 90 mol% a nucleotide sugar.

4. Process according to one of claims 1 or 2, characterized in that the activated sugar is at least 90 mol% a sugar-1-phosphate.

5. Method according to one of claims 1 to 4, characterized in that a tangential flow filtration is carried out through the separation membrane (32).

6. Method according to one of claims 1 to 5, characterized in that a separation membrane (32) with a molecular weight cutoff of at least 1 kDa is used.

7. The method according to any one of claims 1 to 6, characterized in that the reactant in the reactor (12) in process step c) is present in a concentration in a range from 1 mM to 5000 mM.

8. The method according to any one of claims 1 to 7, characterized in that in process step c) enzymes are present in the reactor in a concentration in a range from 0.1 mg / mL to 1000 mg / mL.

9. Process according to one of claims 1 to 8, characterized in that in process step c) a pH value in a range of > 7 to < 9 is used.

10. The process according to any one of claims 1 to 9, characterized in that in process step d) the first fluid stream is a retentate stream and that the second fluid stream is a permeate stream.

11. A reactor system (10) for producing an activated sugar, comprising at least one reactor (12) for forming the activated sugar from a reactant using an enzyme cascade; a reactant feed (20) for feeding the reactants into the reactor (12); a membrane unit (30) with a separation membrane (32), wherein the membrane unit (30) is connected to the reactor (12) by a fluid line for separating a product stream comprising formed activated sugar and the enzyme cascade through the separation membrane (32) to form a retentate stream and a permeate stream; a retentate guide for discharging the retentate stream from the separation membrane (32); and a permeate guide for discharging the permeate stream from the separation membrane (32); wherein the permeate guide or the retentate guide leads into the reactor (12).

12. Reactor system (10) according to claim 13, characterized in that the retentate guide leads into the reactor (12).

13. Use of a method according to one of claims 1 to 10 or of a reactor system (10) according to one of claims 11 or 12 for producing an activated sugar, in particular a phosphate-1-sugar or a nucleotide sugar.

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