Enzymatic method for preparation of UDP-GlcNAc

Abstract

The present invention relates to an enzyme-catalyzed process for producing UDP-N-acetyl-α-D-glucosamine (UDP-GlcNAc) from low-cost substrates uridine monophosphate and N-acetyl-D glucosamine in a single reaction mixture with immobilized or preferably co-immobilized enzymes. Uridine may be used as starting material instead of uridine monophosphate as well. Further, the process may be adapted to produce GlcNAcylated molecules and biomolecules including saccharides, particularly human milk oligosaccharides (HMO), proteins, peptides, glycoproteins, particularly antibodies, or glycopeptides, and bioconjugates, particularly carbohydrate conjugate vaccines and antibody-drug conjugates.

Claims

1. A method for producing uridine 5′-diphospho-N-acetyl-α-D-glucosamine comprising: ##STR00003## providing a solution comprising (i) uridine monophosphate and N-acetyl-D-glucosamine represented by the following formulae ##STR00004## (ii) polyphosphate, and adenosine triphosphate; and providing a set of enzymes comprising a glucose-1-phosphate uridylyltransferase, an N-acetylhexosamine kinase, a polyphosphate kinase, and a uridine monophosphate kinase; A) producing uridine 5′-diphospho-N-acetyl-α-D-glucosamine from uridine monophosphate and N-acetylglucosamine in the presence of the set of enzymes, polyphosphate, and adenosine triphosphate, wherein the set of enzymes is covalently or adsorptively immobilized on a reusable, mechanically stable solid support.

2. The method according to claim 1, wherein the set of enzymes is co-immobilized on the solid support.

3. The method according to claim 1, wherein the set of enzymes is covalently immobilized on a reusable, mechanically stable solid support.

4. The method according to claim 1, wherein the solid support is composed of beads or resins comprising a polymer with epoxide functional groups, with amino epoxide functional groups, with ethylenediamine functional groups, with amino C2 functional groups, with amino C6 functional groups, with anionic/amino C6 spacer functional groups.

5. The method according to claim 1, wherein the solid support is a polymer functionalized with epoxy groups.

6. The method according to claim 1, wherein the set of enzymes further comprises a pyrophosphatase.

7. The method according to claim 1, wherein the set of enzymes is directly co-immobilized on a solid support from fermentation broth, crude cell lysate, purified cell lysate or cell homogenate.

8. The method according to claim 1, wherein the set of enzymes further comprises a one-domain polyphosphate kinase 2 and/or wherein the set of enzymes further comprises a two-domain polyphosphate kinase 2.

9. The method according to claim 1, wherein the concentration of adenosine triphosphate in the solution provided in A) is in the range of 0.001 moles to 0.9 moles per mole N-acetyl-D-glucosamine.

10. The method according to claim 1, wherein the concentration of uridine monophosphate and N-acetyl-D-glucosamine in the solution provided in A) is in the range of 0.2 mM to 15,000 mM.

11. The method according to claim 1, wherein the uridine 5′-diphospho-N-acetyl-α-D-glucosamine is produced in a single reaction mixture.

12. The method according to claim 1, wherein the uridine monophosphate in A) is obtained from (i) uridine, adenosine triphosphate and a uridine kinase; or (ii) uracil, 5-phospho-α-D-ribose 1-diphosphate and an uracil phosphoribosyltransferase; or (iii) from orotic acid, 5-phospho-α-D-ribose 1-diphosphate, an orotate phosphoribosyltransferase and a UMP transferase.

13. The method according to claim 1, further comprising producing a GlcNAcylated saccharide, a GlcNAcylated glycopeptide, a GlcNAcylated glycoprotein, a GlcNAcylated protein, a GlcNAcylated peptide, a GlcNAcylated bioconjugate or a GlcNAcylated small molecule from uridine 5′-diphospho-N-acetylglucosamine and a saccharide, glycopeptide, glycoprotein, protein, peptide, bioconjugate or small molecule by forming an O-glycosidic bond between uridine 5′-diphosphoN-acetylglucosamine and an available hydroxyl group of the saccharide, glycopeptide, glycoprotein, protein, peptide, bioconjugate or small molecule in the presence of an N-acetylglucosaminyltransferase.

14. The method according to claim 13, wherein the saccharide, glycopeptide, glycoprotein, protein, peptide, bioconjugate or small molecule is an antibody or a monoclonal antibody; or a human milk oligosaccharide or a bioconjugate.

15. The method according to claim 13, further comprising recycling of uridine diphosphate formed from the producing a GlcNAcylated saccharide, a GlcNAcylated glycopeptide, a GlcNAcylated glycoprotein, a GlcNAcylated protein, a GlcNAcylated peptide, a GlcNAcylated bioconjugate or a GlcNAcylated small molecule from uridine 5′-diphospho-N-acetylglucosamine and a saccharide, glycopeptide, glycoprotein, protein, peptide, bioconjugate or small molecule by forming an O-glycosidic bond between uridine 5′-diphospho-N-acetylglucosamine and an available hydroxyl group of the saccharide, glycopeptide, glycoprotein, protein, peptide, bioconjugate or small molecule in the presence of an N-acetylglucosaminyltransferase to obtain uridine triphosphate.

16. The method according to claim 13, wherein the saccharide, glycopeptide, glycoprotein, protein, peptide, bioconjugate or small molecule is a carbohydrate conjugate vaccine or an antibody drug conjugate.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: shows the multi-enzyme cascade through which UDP-N-acetyl-α-D-glucosamine is enzymatically synthesized from low-cost substrates N-acetyl-D-glucosamine, polyphosphate and UMP. The reaction cascade consists of (a) the formation of N-acetylglucosamine-1-phosphate (GlcNAc-1P) from N-acetyl-D-glucosamine and ATP, (b) the formation of uridine triphosphate (UTP) from UMP and polyphosphate, and (c) the reaction of N-acetylglucosamine 1-phosphate with uridine triphosphate to UDP-N-acetyl-α-D-glucosamine. Optionally an inorganic diphosphatase (PmPpa) can added to the reaction cascade in order to hydrolyze pyrophosphate PPi which inhibits the enzyme glucose 1-phosphate uridylyltransferase. The cascade can also be extended by adding a 1 D-PPK2 to assist the conversion of ADP to ATP. Also, the cascade can be extended by adding a 2D-PPK2 in order to activate phosphorylation of AMP to ADP. Moreover, the cascade can be extended by adding a 1 D-PPK2 and a 2DPPK2 in order to inhibit frequent hydrolysis of adenosine phosphates.

(2) FIG. 2: shows an exemplary reaction scheme of the inventive method for producing UDP-N-acetyl-α-D-glucosamine starting from uridine or uracil and 5-phospho-α-D-ribose 1-diphosphate. The formation of UMP from uridine is catalyzed by uridine kinase and the formation of UMP from uracil is catalyzed by uracil phosphoribosyltransferase.

(3) FIG. 3: shows the comparison of the productivity for the synthesis of UDP-GlcNAc with separately immobilized enzymes and co-immobilization of the set of enzymes. Co-immobilization results in much higher productivity.

(4) FIG. 4 shows results of the solid support screening of the UDP-GlcNAc synthesis in a first cycle. Productivities were measured by HPAEC-UV.

(5) FIG. 5 shows results of the solid support screening of the UDP-GlcNAc synthesis in a second cycle. Productivities were measured by HPAEC-UV.

(6) FIG. 6 shows results of the UDP-GlcNAc synthesis with co-immobilized enzymes on ECR8285 resin—a methacrylate resin functionalized with both butyl and epoxy groups—in nine cycles. Productivities were measured by HPAEC-UV.

(7) FIG. 7 shows results of the UDP-GlcNAc synthesis with co-immobilized enzymes on Eupergit CM resin—a methacrylate/acrylamide resin functionalized with epoxy groups—in nine cycles. Productivities were measured by HPAEC-UV.

(8) FIG. 8 shows results of the UDP-GlcNAc synthesis with co-immobilized enzymes on EC-HFA resin—a methacrylate resin functionalized with amino epoxy groups—in nine cycles. Productivities were measured by HPAEC-UV.

(9) FIG. 9 shows results of the UDP-GlcNAc synthesis with co-immobilized enzymes on ECR8215F resin—a methacrylate resin functionalized with epoxy groups—in nine cycles. Productivities were measured by HPAEC-UV.

(10) FIG. 10 shows results of the UDP-GlcNAc synthesis with co-immobilized enzymes on EC-HFA 403/M resin—a methacrylate resin functionalized with amino epoxy groups—in nine cycles. Productivities were measured by HPAEC-UV.

(11) FIG. 11 shows results of the UDP-GlcNAc synthesis with co-immobilized enzymes on ECR8209F resin—a methacrylate resin functionalized with epoxy groups—in nine cycles. Productivities were measured by HPAEC-UV.

(12) FIG. 12 shows A) a diagram of the amount of protein bound to epoxy solid support determined by quantifying the protein in the supernatant after immobilization of several solid supports. Standard BCA protein quantification protocols were followed; B) a diagram of the amount of protein bound to ionic and adsorption solid support determined by quantifying the protein in the supernatant after immobilization of several solid supports. Standard BCA protein quantification protocols were followed; and C) a diagram of the amount of protein bound to glutaraldehyde activated solid support determined by quantifying the protein in the supernatant after immobilization of several solid supports. Standard BCA protein quantification protocols were followed.

(13) FIG. 13 shows A) HPAEC-UV chromatogram of the quantification of UDP-GlcNAc (and other reactants). The chromatogram shown is from a reaction catalyzed by the enzyme cascade immobilized on Eupergit CM; B) HPAEC-UV chromatograms of the quantification of UDP-GlcNAc (and other reactants). The chromatogram shown is from a reaction catalyzed by the enzyme cascade immobilized on IB-ADS1, IB-CAT, ECR1504, IB-ANI1; C) HPAEC-UV chromatogram of the quantification of UDP-GlcNAc (and other reactants). The chromatogram shown is from a reaction catalyzed by the enzyme cascade immobilized on ECR8315F active by glutaraldehyde.

(14) FIG. 14 shows results of the UDP-GlcNAc synthesis with co-immobilized enzymes on EC-EP resin—a methacrylate resin functionalized with epoxy groups—in 20 cycles in 3 series. Productivities were measured by HPAEC-UV.

(15) FIG. 15 shows results of the UDP-GlcNAc synthesis with co-immobilized enzymes on EP403/M resin—a methacrylate resin functionalized with epoxy groups—in 20 cycles in 3 series. Productivities were measured by HPAEC-UV.

(16) FIG. 16 shows results of the UDP-GlcNAc synthesis with co-immobilized enzymes on COV1 resin—a polyacrylic resin functionalized with butyl/epoxy groups—in 10 cycles in 3 series. Productivities were measured by HPAEC-UV.

(17) FIG. 17 shows results of the UDP-GlcNAc synthesis with co-immobilized enzymes on COV2 resin—a polyacrylic resin functionalized with epoxy groups—in cycles in 3 series. Productivities were measured by HPAEC-UV.

(18) FIG. 18 shows results of the UDP-GlcNAc synthesis with co-immobilized enzymes on COV3 resin—a polyacrylic resin functionalized with epoxy groups—in cycles in 3 series. Productivities were measured by HPAEC-UV.

(19) FIG. 19 shows results of the UDP-GlcNAc synthesis with co-immobilized enzymes on Eupergit CM resin—an acrylic resin functionalized with epoxy groups—in 20 cycles. Productivities were measured by HPAEC-UV.

(20) FIG. 20 shows results of the UDP-GlcNAc synthesis with co-immobilized enzymes on ECR8215F resin—a methacrylate resin functionalized with epoxy groups—in 20 cycles. Productivities were measured by HPAEC-UV.

(21) FIG. 21 shows results of the UDP-GlcNAc synthesis with co-immobilized enzymes on ECR8204F resin—a methacrylate resin functionalized with epoxy groups—in 20 cycles. Productivities were measured by HPAEC-UV.

(22) FIG. 22 shows results of the UDP-GlcNAc synthesis with co-immobilized enzymes on ECR8209F resin—a methacrylate resin functionalized with epoxy groups—in 20 cycles. Productivities were measured by HPAEC-UV.

(23) FIG. 23 shows results of the UDP-GlcNAc synthesis with co-immobilized enzymes on ECR8285F resin—a methacrylate resin functionalized with butyl/epoxy groups—in 20 cycles. Productivities were measured by HPAEC-UV.

(24) FIG. 24 shows results of the UDP-GlcNAc synthesis with co-immobilized enzymes on EP403/S resin—a polymethacrylate resin functionalized with epoxy groups—in 20 cycles. Productivities were measured by HPAEC-UV.

(25) FIG. 25 shows results of the UDP-GlcNAc synthesis with co-immobilized enzymes on EP400/SS resin—a polymethacrylate resin functionalized with epoxy groups—in 20 cycles. Productivities were measured by HPAEC-UV.

(26) FIG. 26 shows results of the UDP-GlcNAc synthesis with co-immobilized enzymes on EC-HFA 403/M resin—a polymethacrylate resin functionalized with amino epoxy groups—in 20 cycles. Productivities were measured by HPAEC-UV.

(27) FIG. 27 show results of the UDP-GlcNAc synthesis with co-immobilized enzymes on EC-HFA 403/S resin—a polymethacrylate resin functionalized with amino epoxy groups—in 20 cycles. Productivities were measured by HPAEC-UV.

(28) FIG. 28 shows results of the UDP-GlcNAc synthesis with co-immobilized enzymes on EC-HFA/S resin—a polymethacrylate resin functionalized with amino epoxy groups—in 20 cycles. Productivities were measured by HPAEC-UV.

(29) FIG. 29 shows results of the UDP-GlcNAc synthesis with co-immobilized enzymes on EC-HFA/M resin—a polymethacrylate resin functionalized with amino epoxy groups—in 20 cycles. Productivities were measured by HPAEC-UV.

(30) FIG. 30 shows results of the UDP-GlcNAc synthesis with co-immobilized enzymes on Immobead 150P resin—a copolymer of methacrylate resin functionalized with epoxy groups—in 20 cycles. Productivities were measured by HPAEC-UV.

(31) FIG. 31 shows a workflow scheme for the complete UDP-GlcNAc cascade starting from mixing the biomasses containing the overexpressed enzymes to carrying out the synthesis reaction of UDP-GlcNAc on a solid support. The workflow is also suitable for screening various solid supports for enzyme immobilization.

(32) FIG. 32 shows exemplary GlcNAc human milk saccharides.

(33) FIG. 33 shows the activity of EP403/S, EC-EP/M and Ni-NTA beads up to 20 cycles for the synthesis of UDP-GlcNAc. Ni-NTA experiments were carried out in triplicates.

(34) FIG. 34 shows intermediates and product formed in the UDP-GlcNAc cascade of Example 5. (A) UDP-GlcNAc; (B) UMP, UDP and UTP; (C) ADP and ATP. The experiments were carried out in triplicate; error bars represent standard deviation.

(35) FIG. 35 shows educts, intermediates and product formed in the UDP-GlcNAc scale-up experiment of Example 5. (A) uridine; (B) UDP-GlcNAc; (C) UMP; (D) UDP; (E) UTP; (F) ATP; and (G) ADP.

(36) FIG. 36 shows relative total amount of protein bound to each bead. The experiments were carried out in triplicate; errors bar represent standard deviation.

(37) FIG. 37 shows chromatograms of reaction products for the inventive UDP-GlcNAc synthesis on each tested solid support bead.

(38) FIG. 38 shows the results of the activity test on different beads. The experiments were carried out in triplicate, except for Relizyme HFA 403/S and Relizyme HFA 403/M, of which average of three consecutive cycles are shown; errors bar represent standard deviation.

(39) FIG. 39 shows the results of the activity test on each selected bead over 10 consecutive reaction cycle.

(40) The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims.

EXAMPLES

Abbreviations and Acronyms

(41) ADP adenosine 5′-diphosphate

(42) AMP adenosine 5′-monophosphate

(43) ATP adenosine 5-triphosphate

(44) dH.sub.2O deionized water

(45) NahK N-acetylhexosamine kinase

(46) UDP uridine 5′-diphosphate

(47) UMP uridine 5′-monophosphate

(48) UTP uridine 5-triphosphate

(49) GlcNAc N-acetyl-D-glucosamine

(50) PolyP polyphosphate

(51) PPi pyrophosphate

(52) Pi phosphate

(53) PPK2 polyphosphate kinase 2

(54) PPK3 polyphosphate kinase 3

(55) 1 D-PPK2 1-domain polyphosphate kinase 2

(56) 2D-PPK2 2-domain polyphosphate kinase 2

(57) GalU glucose 1-phosphate uridylyltransferase

(58) URA6 uridine monophosphate kinase

(59) UPP uracil phosphoribosyltransferase

(60) PmPpA Pasteurella multocida inorganic pyrophosphatase

(61) Chemicals & Reagents

(62) Unless otherwise stated, all chemicals and reagents were acquired from Sigma-Aldrich, and were of the highest purity available. Solid supports were obtained from Resindion, ChiralVision, Röhm GmbH & Co. KG and micromod GmbH.

Example 1: Preparation of Enzymes

(63) The engineered cell-free synthetic metabolic pathway consists of five enzymes (FIG. 1) all produced by E. coli BL21 Gold (DE3). Enzymes were chosen according to literature: NahK (EC 2.7.1.162) from Bifidobacterium longum to phosphorylate GlcNAc; GalU (EC 2.7.7.9) from E. coli K-12 MG1655 as a GlcNAc-1P uridylyltransferase; URA6 (EC 2.7.4.14) from Arabidopsis thaliana for in situ regeneration of UDP from UMP; PPK3 (EC 2.7.4.1) from Ruegeria pomeroyi for in situ recovery of energy carriers, ADP and UDP, to their tri-phosphate conjugates; and PmPpA (EC 3.6.1.1) from Pasteurella multocida Pm70 for the decomposition of GalU inhibiting pyrophosphate. Details of all enzymes used are given in Table 1 below.

(64) TABLE-US-00001 TABLE 1 Enzymes used in this example Enzyme Abbreviation EC class Origin SEQ ID glucose 1-phosphate GalU 2.7.7.9 E. coli K-12 MG1655 SEQ ID 4 uridylyltransferase N-acetylhexosamine NahK EC Bifidobacterium longum SEQ ID 1 1-kinase 2.7.1.162 Polyphosphate kinase 3 PPK3 2.7.4.1 Ruegeria pomeroyi SEQ ID 3 Uridine monophosphate URA6 2.7.4.14 Arabidopsis thaliana SEQ ID 2 kinase Inorganic diphosphatase PmPpa 3.6.1.1 Pasteurella multocida Pm70 SEQ ID 5 1-domain polyphosphate 1D-PPK2 2.7.4.1 Pseudomonas aeruginosa SEQ ID 6 kinase 2 2-domain polyphosphate 2D-PPK2 2.7.4.1 Pseudomonas aeruginosa SEQ ID 7 kinase 2

(65) Transformation, Cultivation, Expression

(66) For all gene expressions E. coli BL21 Gold (DE3) was used as a host organism.

(67) Gene Expression

(68) Plasmids and Stock Cultures

(69) Stock solutions of all E. coli cultures carrying the plasmids (pET28a with kanamycin resistance) with the gene sequences were available from earlier studies [1,2]. The stock solutions contained 50% glycerol and were kept at −20° C.

(70) The gene and corresponding protein sequences were obtained from the UniProt database: PmPpA (P57918), NahK (E4R3E3), GalU (P0AEP3), PPK3 (Q5LSN8), and URA6 (004905). Gene Designer 2.0 software (Gene Designer, DNA 2.0, Menlo Park, Calif.) was used for optimizing the codon usage of nucleotide sequences for expression in E. coli. The resulting sequences were synthesized de novo and cloned by GeneArt™ (Thermo Fisher Scientific, Regensburg, Germany). The following restriction sites for subcloning into vector pET-28a(+) were used: NcoI and XhoI for GalU, NahK and PmPpA (enzymes carrying a C-terminal hexahistidin-tag (His-tag)), NdeI and XhoI with PPK3 and URA6 (for an N-terminal His-tag). After transformation of the plasmids into E. coli, the DNA was isolated and the accuracy of the constructs was checked by gene sequencing (Eurofins Genomics, Ebersberg, Germany).

(71) Enzyme Expression

(72) For heterologous gene expression, aliquots were removed from the stock solutions and spread on LB agar plates containing the according antibiotic. The plates were cultivated overnight at 37° C. Single cultures were used to inoculate precultures (containing 50 μg/mL kanamycin) in shaker flasks with baffles. Cultures were typically grown to an OD.sub.600 of about 4.2. Main expression cultures containing 50 μg/mL kanamycin were typically inoculated with 1% preculture and cultivated at 37° C. to an OD.sub.600 of around 0.6-0.8. The temperature was then changed to 16-20° C. and the expression was induced with typically 0.4 mM IPTG. After, typically, 20 h, the culture were harvest typically by 6000×g for 30 min at 4° C. Media used were TB media except for GalU (LB media) (see table 2).

(73) TABLE-US-00002 TABLE 2 Media used in this Example Media Content Luria-Bertani 10 g tryptone (LB) 5 g yeast extract 5 g NaCl in 1 L dH.sub.2O Terrific broth 24 g yeast extract (TB) 12 g tryptone 5 g glycerol 89 mM Phosphate buffer (added after autoclaving) in 1 L dH.sub.2O

(74) Enzyme Purification

(75) The plasmids pET28a and pET100/D-TOPO harbor a N-terminal His6-tag and the enzyme are, thus, purified with Ion metal affinity chromatography using the ÄKTAstart system and HisTrap High-Performance or Fast-Flow columns (1 mL column volume) from GE Healthcare. For the purification of enzymes the cells were lysed by sonication in lysis buffer (50 mM HEPES (pH 7.5), 10 mM Mg.sup.2+, 300 mM NaCl, 10 mM imidazole and 5% glycerol).

(76) Imidazole (500 mM) was used as eluent in isocratic elutions (50 mM HEPES (pH 7.5), 10 mM Mg.sup.2+, 300 mM NaCl, 500 mM imidazole and 5% glycerol). Standard conditions as recommended by the manufactures were used. After purification the enzyme concentrations were tested by BCA assays and evaluated by SDS-gels.

Example 2: Heterogeneous Preparation of UDP-N-Acetyl-α-D-Glucosamine

(77) Measurements

(78) High-performance anion exchange chromatography (HPAEC) with UV (260 nm) and pulsed amperometric detection (PAD) was utilized to measure concentrations of reactants. For analyte separation and quantification a step gradient elution method was developed and validated chromatographic separation was performed at a system flow of 0.5 mL/min using a non-porous pellicular column CarboPac PA1 (250×2 mm). The HPAEC system (ICS5000) as well as all columns, components and software were purchased from Thermo Scientific (Waltham, USA).

(79) Experiment A

(80) A wide range of commercially available solid supports (see Table 3) were tested for the co-immobilization of the enzymes used in the inventive UDP-N-acetyl-α-D-glucosamine synthesis (see FIG. 1) and their effect on the synthesis of UDP-N-acetyl-α-D-glucosamine was evaluated.

(81) TABLE-US-00003 TABLE 3 Table of solid supports tested in Experiment A Pore Particle Mass diameter size Functional Solid support (mg) Matrix (nm) (μm) group EC-EP 120 polymethacrylate 10-20 200-500 epoxy EP403/M 90 polymethacrylate 40-60 200-500 epoxy IB-COV1 93 polyacrylic 150-300 butyl, epoxy IB-COV2 92 polyacrylic 150-300 epoxy IB-COV3 98 polyacrylic 300-700 epoxy Eupergit ® CM 102 acrylic  50-300 epoxy ECR8215F 92 methacrylate 120-180 150-300 epoxy ECR8204F 94 methacrylate 30-60 150-300 epoxy ECR8209F 98 methacrylate  60-120 150-300 epoxy ECR8285 90 methacrylate 40-60  250-1000 butyl, epoxy EC-HFA 120 HFA403/M 121 ECR8806F 112 methacrylate 50-70 150-300 octadecyl ECR1091M 101 Macroporous divinylbenzene  95-120 300-710 — ECR1030F 108 DVB/methacrylic polymer 22-34 150-300 — ECR1504 104 styrene  300-1200 tert. amine ECR1604 108 styrene  300-1200 quart. amine

(82) To test the multi-enzyme cascade on various enzyme loaded beads, a given mass (see Table 3) of each resin was added to a 2 mL low-binding tube. After approx. 2 h of incubation with lysis buffer (see Table 4), the supernatant was removed [equilibration step]. Afterwards, 0.5 mL of cell lysate were added to each tube and incubated overnight (approx. 12 h) at 4° C. After incubation, beads were washed (3 times) and blocking buffer (2 M glycine) was added. Beads were incubated for 24 h at room temperature with the blocking buffer. Afterwards, the blocking buffer was removed and beads were washed with lysis buffer three times.

(83) TABLE-US-00004 TABLE 4 Buffers used for the immobilization of biocatalysts in Experiment A Buffers Conc. in mM/% Immobilization/Lysis Blocking Buffer HEPES 200  125 MgCl.sub.2  50  25 NaCl 300  150 Glycerol  5%   3% Glycine 3000

(84) 200 μL of the feed solution (see Table 5) containing substrates was transferred to each tube containing the beads. The reactions were carried out for around 17 h at 30° C. and under shaking (600 rpm). The UDP-N-acetyl-α-D-glucosamine concentrations were then measured by HPAEC-UV/PAD. The results are shown in FIG. 4.

(85) TABLE-US-00005 TABLE 5 Concentration of reactants in the feed solution of Experiment A Substrate Conc. (mM) UMP 11 ATP 17 GlcNac 12 PolyP.sub.25 14 HEPES 80 MgCl.sub.2 60

(86) In order to evaluate the re-usability of the beads—after the first cycle-supernatant were removed and the beads were washed with Lysis buffer once. Afterwards, 200 μL of feed solution was added to the beads. The reactions were carried out for around 10 h at 30° C. and under shaking (600 rpm). The results are depicted in FIG. 5. It is shown that enzymes co-immobilized on several commercially available beads are useful for re-usability and provide mechanically robust beads with co-immobilized enzymes.

(87) After the second cycle, certain beads were selected to evaluate their further re-usability. The results are shown in FIG. 6 to FIG. 11.

(88) Experiment B

(89) Enzyme Immobilization

(90) Immobilized enzymes can often be separated from solutions and reused. Moreover, they may exhibit higher activity and can be used for a wide range of processes, such as continuous synthesis in packed bed reactors. A wide range of commercially available solid supports were tested for the co-immobilization of the UDP-GlcNAc multi-enzyme cascade.

(91) TABLE-US-00006 TABLE 6 Table of solid supports tested in Experiment B (epoxy) Oxiran Pore Particle content Solid diameter size (μm/g Bonding Functional Support Matrix (nm) (μm) wet) type group SEPABEAD Polymeth- 10-20 200-500 144 covalent epoxy S EC-EP/M acrylate RELIZYME Polymeth- 40-60 200-500 56 covalent epoxy EP403/M acrylate SEPABEAD Polymeth- 10-20 200-500 77 covalent amino- S EC-HFA/M acrylate epoxy RELIZYME Polymeth- 40-60 200-500 30 covalent amino- HFA403/M acrylate epoxy RELIZYME Polymeth- 40-60 100-300 59 covalent amino- HFA403/S acrylate epoxy SEPABEAD Polymeth- 10-20 100-300 91 covalent amino- S EC-HFA/S acrylate epoxy RELIZYME Polymeth- 40-60 100-300 66 covalent epoxy EP403/S acrylate RELISORB Polymeth-  80-100  50-150 Min. 100 covalent epoxy EP400/SS acrylate μm/g dry Eupergit ® acrylic  50-300 0.75 covalent epoxy CM Lifetech ™ methacrylate 120-180 150-300 covalent epoxy ECR8215F Lifetech ™ methacrylate 30-60 150-300 covalent epoxy ECR8204F Lifetech ™ methacrylate  60-120 150-300 covalent epoxy ECR8209F Lifetech ™ methacrylate 40-60  250-1000 covalent butyl, ECR8285 epoxy Lifetech ™ methacrylate 50-60 150-300 covalent epoxy ECR8206F Lifetech ™ methacrylate 120-180 300-710 covalent epoxy ECR8215M Lifetech ™ methacrylate 30-60 300-710 covalent epoxy ECR8204M Lifetech ™ methacrylate  60-120 300-710 covalent epoxy ECR8209M Lifetech ™ methacrylate 50-60 300-710 covalent epoxy ECR8206M Imm150P Copolym. Of 150-500 methacrylate IB-COV1 polyacrylic 150-300 covalent butyl, epoxy IB-COV2 polyacrylic 150-300 covalent epoxide IB-COV3 polyacrylic 300-700 covalent epoxide

(92) TABLE-US-00007 TABLE 7 Table of solid supports tested in Experiment B (other) Pore Particle Solid diameter size Bonding Functional Support Matrix (nm) (μm) type group Lifetech ™ methacrylate  30-60 150-300 covalent or Amino C2 ECR8305F ionic Lifetech ™ methacrylate  60-120 150-300 covalent or Amino C2 ECR8309F ionic Lifetech ™ methacrylate 120-180 150-300 covalent or Amino C2 ECR8315F ionic Lifetech ™ methacrylate 120-180 150-300 covalent or Amino C6 ECR8415F ionic Lifetech ™ methacrylate  60-120 150-300 covalent or Amino C6 ECR8409F ionic Lifetech ™ methacrylate  30-60 150-300 covalent or Amino C6 ECR8404F ionic Lifetech ™ methacrylate  30-60 300-710 covalent or Amino C2 ECR8305M ionic Lifetech ™ methacrylate  60-120 300-710 covalent or Amino C2 ECR8309M ionic Lifetech ™ methacrylate 120-180 300-710 covalent or Amino C2 ECR8315M ionic Lifetech ™ methacrylate 120-180 300-710 covalent or Amino C6 ECR8415M ionic Lifetech ™ methacrylate  60-120 300-710 covalent or Amino C6 ECR8409M ionic Lifetech ™ methacrylate  30-60 300-710 covalent or Amino C6 ECR8404M ionic Lifetech ™ methacrylate  30-60 300-710 covalent or Amino C2 ECR8305M ionic Lifetech ™ methacrylate  30-60 150-710 covalent or Amino C2 ECR8305 ionic Lifetech ™ methacrylate  60-120 150-710 covalent or Amino C2 ECR8309 ionic Lifetech ™ methacrylate 120-180 150-710 covalent or Amino C2 ECR8315 ionic Lifetech ™ methacrylate 120-180 150-710 covalent or Amino C6 ECR8415 ionic Lifetech ™ methacrylate  60-120 150-710 covalent or Amino C6 ECR8409 ionic Lifetech ™ methacrylate  30-60 150-710 covalent or Amino C6 ECR8404 ionic Lifetech ™ methacrylate  30-60 150-710 covalent or Amino C2 ECR8305 ionic SEPABEADS Polymethacrylate  10-20 200-500 covalent or ethylamino EC-EA/M ionic SEPABEADS Polymethacrylate  10-20 100-300 covalent or ethylamino EC-EA/S ionic RELIZYME Polymethacrylate  40-60 100-300 covalent or ethylamino EA403/S ionic RELIZYME Polymethacrylate  40-60 200-500 covalent or ethylamino EA403/M ionic SEPABEADS Polymethacrylate  10-20 100-300 covalent or hexylamino EC-HA/S ionic RELIZYME Polymethacrylate  40-60 100-300 covalent or hexylamino HA403/S ionic SEPABEADS Polymethacrylate  10-20 200-500 covalent or hexylamino EC-HA/M ionic RELIZYME Polymethacrylate  40-60 200-500 covalent or hexylamino HA403/M ionic Lifetech ™ methacrylate  50-70 300-710 adsorption octadecyl ECR8806M Lifetech ™ methacrylate  35-45 300-710 adsorption octadecyl ECR8804M Lifetech ™ methacrylate  50-70 150-300 adsorption octadecyl ECR8806F Lifetech ™ methacrylate  35-45 150-300 adsorption octadecyl ECR8804F Lifetech ™ methacrylate  50-70 150-710 adsorption octadecyl ECR8806 Lifetech ™ methacrylate  35-45 150-710 adsorption octadecyl ECR8804 IB-ADS-1 Polyacrylic 71% pore 300-700 adsorption Alkyl volume IB-ADS-2 Styrene 75% pore 150-300 adsorption Phenyl volume IB-ADS-3 Methacrylate 58% pore 150-300 adsorption Octadecyl volume IB-ADS-4 Styrene 58% pore 300-700 adsorption Styrene, volume methyl SEPABEADS Polymethacrylate  10-20 200-500 adsorption butyl EC-BU RELIZYME Polymethacrylate  40-60 100-300 adsorption butyl BU403 SEPABEADS Polymethacrylate  10-20 200-500 adsorption Octyl EC-OC RELIZYME Polymethacrylate  40-60 100-300 adsorption octyl OC403 SEPABEADS Polymethacrylate  10-20 200-500 adsorption Octadecyl EC-OD RELIZYME Polymethacrylate  40-60 100-300 adsorption octadecyl OD403 Lifetech ™ Macroporous  90-110 300-710 adsorption — ECR1090M divinylbenzene Lifetech ™ Macroporous  95-120 300-710 adsorption — ECR1091M divinylbenzene Lifetech ™ Macroporous  90-110 150-300 adsorption — ECR1090F divinylbenzene Lifetech ™ Macroporous  95-120 150-300 adsorption — ECR1091F divinylbenzene Lifetech ™ Macroporous  90-110 150-710 adsorption — ECR1090 divinylbenzene Lifetech ™ Macroporous  95-120 150-710 adsorption — ECR1091 divinylbenzene Lifetech ™ DVB/methacrylic  22-34 150-300 adsorption — ECR1030F polymer Lifetech ™ DVB/methacrylic  20-30 150-300 adsorption — ECR1030M polymer Lifetech ™ DVB/methacrylic  60-75 150-300 adsorption — ECR1061M polymer Lifetech ™ DVB/methacrylic  20-30 150-710 adsorption — ECR1030M polymer Lifetech ™ DVB/methacrylic  60-75 150-710 adsorption — ECR1061M polymer Lifetech ™ styrene 300-1200 ionic tert. amine ECR1504 Lifetech ™ styrene 300-1200 ionic tert. amine ECR1508 Lifetech ™ styrene 300-1200 ionic quat. amine ECR1604 Lifetech ™ styrene 300-1200 ionic quat. amine ECR1640 IB-CAT-1 styrene 54% pore 300-700 cationic, Sulphonic volume strong IB-ANI-1 polyacrylic 78% pore 150-300 anionic primary volume amine IB-ANI-2 polystyrene 55% pore 630 anionic, tertiary volume weak amine IB-ANI-3 polystyrene 72% pore 800 anionic, quat. volume weak Ammon. IB-ANI-4 polystyrene 62% pore 690 anionic, quat. volume strong Ammon. SEPABEADS Polymethacrylate  10-20 100-300 1,2-diol EC-HG/S RELIZYME Polymethacrylate  40-60 100-300 1,2-diol HG403/S SEPABEADS Polymethacrylate  10-20 200-500 1,2-diol EC-HG/M RELIZYME Polymethacrylate  40-60 200-500 1,2-diol HG403/M

(93) The solid supports are here divided into three groups depending on their immobilization mechanism: a) epoxy (including amino-epoxy) supports, b) ionic & adsorption supports and c) glutaraldehyde activated supports. In addition, three different solid support to protein ratios were tested for each solid support to find the optimal ratios: series 1, series 2 and series 3 (see Table 8-Table 11).

(94) TABLE-US-00008 TABLE 8 Tested protein stock solution to solid support ratio Series 1 2 3 Protein stock solution 1:12 1:35 1:45 to solid support ratio (mass) Volume 500 μL 500 μL 700 μL

(95) TABLE-US-00009 TABLE 9 Selection of tested epoxy (including amino-epoxy) supports Mass (mg) Resin Series 1 Series 2 Series 3 EC-EP 104 268 478 EP403/M 107 267 484 IB-COV1 102 256 442 IB-COV2 110 271 440 IB-COV3 123 247 438 Eupergit ® CM 81 156 269 ECR8215F 97 251 466 ECR8204F 96 246 468 ECR8209F 104 285 450 ECR8285 102 253 450 EP403/S 102 248 449 EP400/SS 93 248 453 EC-HFA/M 101 275 460 HFA403/M 100 253 465 HFA403/S 92 259 470 EC-HFA/S 117 286 504 Imm150P 105 266 425

(96) TABLE-US-00010 TABLE 10 Selection of tested ionic & adsorption supports Mass (mg) Resin Series 1 Series 2 Series 3 8409F 96 253 422 8315F 93 264 470 8309F 106 268 443 1030F 92 244 481 1509 108 245 438 8806F 103 266 434 8415F 92 252 443 1091M 95 259 475 1604 102 273 465 EC-EA/M 95 239 429 EC-HA 99 272 438 EA403/S 101 251 478 ADS-1 111 263 471 ADS-2 106 253 503 ADS-3 108 265 457 ADS-4 99 246 499 CAT-1 106 243 445 ANI-1 97 276 444 ANI-2 122 303 466 ANI-3 106 253 460 ANI-4 117 256 453

(97) TABLE-US-00011 TABLE 11 Selection of tested glutaraldehyde activated supports Mass (mg) Resin Series 1 Series 2 Series 3 8409F 95 252 434 8315F 102 247 469 8309F 96 247 492 8415F 97 248 474 EC-EA/M 113 253 476 EC-HA 99 250 436 EA403/S 104 242 461

(98) The following protocol was followed for the experiment: Biomass containing the overexpressed enzymes were mixed together [see table 13, step 1] and centrifuged 6000×g for 30 min at 4° C. [step 2]. The cell pellets were resuspended in immobilization/lysis buffer to a volume of 150 mL (see table 12) [step 3]. Cells were lysed by sonication [step 4]. After sonication the slurry was centrifuged 12 000×g for 45 min at 4° C. [step 5] to remove cell debris, followed by filtration through 1.2 μm and 0.8 μm filters. After centrifugation, the supernatant was removed and kept on ice. The total protein concentration of the supernatant (protein stock solution) was 14.5 (+/−0.5) mg/mL. A given mass of each immobilizer was added to a 2 mL low-binding tube. Amino-functionalized supports were activated with glutaraldehyde by incubation in activation buffer for 1 hour to generate glutaraldehyde activated supports (group c)). The solid supports were washed two times with washing buffer A (for epoxy supports and glutaraldehyde supports) and washing buffer B (for ionic & adsorption supports) and equilibrated for 1 hour with immobilization/lysis buffer. Afterwards, cell lysate was added to each tube and incubated overnight (˜36 h) at 4° C. [step 6]. The supports with the immobilized enzymes were washed (3 times) with washing buffer [step 7]. In addition the epoxy supports were incubated with blocking buffer (2 M glycine) for 24 h [step 8]. Afterwards, the blocking buffer was discarded and the supports were washed with washing buffer A three times.

(99) TABLE-US-00012 TABLE 12 Buffers used for the immobilization of biocatalysts. Buffers Conc in mM/ Immobili- Washing Washing Blocking Activation % zation/Lysis Buffer A Buffer B Buffer buffer HEPES 250 400 125  125 250 MgCl.sub.2  50  50  25  25  50 NaCl 300 600 150  150 300 Glycerol  5%  5%  3%   3%   5% Glycine 3000 Glutaraldehyde 2.5%

(100) TABLE-US-00013 TABLE 13 Composition of the biomass mixtures used in step 1. Enzyme Biomass (gr) NahK 10.52 GalU 5.17 URA6 6.25 PPK3 7.44 PmPpa 3.54 1d-PPK2 1.48 2d-PPK2 2.67

(101) The amount of protein bound to solid support was determined by quantifying the protein in the supernatant after immobilization. Standard BCA protein quantification protocols were followed. The results for several resins (see Tables 9-11) are shown in FIGS. 12A-12C.

(102) Reactions

(103) To test the multi-enzyme cascade—on various supports immobilized-, feed solution (see table 14) containing substrates was transferred to the tubes containing the biocatalysts. To keep a volume of feed to mass of solid support ratio of 1, the following feed volumes were added: 100 μl (series 1), 250 μL (series 2) and 500 μL (series 3). The reactions were carried out for around 20-25 h at 30° C. and shaking in a rotating mixer (8 rpm). To evaluate the reactions, the supernatant was removed and the UDP-GlcNAc concentrations were then measured by HPAEC-UV/PAD. For the quantification by HAPEC-UV/PAD an aliquot of 3 μl was diluted with 100 μl deionized water and then injected. Example chromatograms are shown in FIG. 13A-C. The solid supports were washed with 1 mL deionized water 2 times before starting the next reaction.

(104) TABLE-US-00014 TABLE 14 Concentration of reactants in the feed solution. Substrate Conc. (mM) UMP 5 ATP 4 GlcNac 5 PolyP.sub.25 10 HEPES 100 MgCl.sub.2 50

(105) Results

(106) Enzyme Immobilization

(107) The results of the reaction are shown in FIGS. 14-30. It should be noted that finding the optimal solid support is always down to experimental trial and error as insufficient knowledge about the immobilization of enzymes exist to predict the optimal solid support [3].

(108) The surprising finding was that the multi-enzyme cascade showed activity when co-immobilized on a wide range of epoxy supports. The epoxy supports that were tested and showed activity varied in support matrix, particle size, pore size and oxiran content. Other solid supports where enzymes are immobilized by hydrophobic adsorption, ionic interaction or covalent crosslinking with glutaraldehyde showed very little to no activity implying that at least one of the five key enzymes is little active to inactive. Moreover, the multi-enzyme cascade was active on epoxy supports when a large range of different rations of proteins to solid supports where used. For the synthesis of UDP-GlcNAc, many of the epoxy supports loaded with the enzymes could be used in more than 20 reaction cycles without re-immobilizing the enzymes on the supports.

(109) TABLE-US-00015 TABLE 15 Tested Epoxy supports. (+) indicating the multi-enzyme cascade was active or (−) inactive. Mass (mg) Resin Series 1 Series 2 Series 3 EC-EP + + + EP403/M + + + IB-COV1 + + + IB-COV2 + + + IB-COV3 + + + Eupergit ® CM + + + ECR8215F + + + ECR8204F + + + ECR8209F + + + ECR8285 + + + EP403/S + + + EP400/SS + + + EC-HFA/M + + + HFA403/M + + + HFA403/S + + + EC-HFA/S + + + Imm150P + + +

(110) TABLE-US-00016 TABLE 16 Tested Ionic & Adsorption supports: (+) indicating the multi-enzyme cascade was active or (−) inactive. Mass (mg) Resin Series 1 Series 2 Series 3 8409F − − − 8315F − − − 8309F − − − 1030F − − − 1504 − − − 8806F + + + 8415F − − − 1091M + + + 1604 − − − EC-EA/M − − − EC-HA − − − EA403/S − − − ADS-1 + + + ADS-2 + + + ADS-3 − − − ADS-4 − − − CAT-1 − − + ANI-1 − − − ANI-2 − − − ANI-3 − − − ANI-4 − − −

(111) TABLE-US-00017 TABLE 17 Tested Glutaraldehyde activited supports. (+) indicating the multi-enzyme cascade was active or (−) inactive. Glutaraldehyde activated supports are supports with amine-reactive groups that were activated by glutaraldehyde to generate covalent binding between protein and solid support. Mass (mg) Resin Series 1 Series 2 Series 3 8409F + − − 8315F + − − 8309F − − − 8415F − − − EC-EA/M + − − EC-HA + − − EA403/S + − −

Example 3: Coupling of the Cascade

(112) The cascade can be coupled to GlcNAc-transferases (EC 2.7.1.X) to transfer GlcNAc to acceptor molecules. Acceptor molecules can be for example monoclonal antibodies. For the coupling soluble GlcNAc-transferase can be added, a GlcNAc-transferase can be co-immobilized on the same support and/or the GlcNAc-transferase can be immobilized on an additional support and then be added to reaction.

Example 4: Synthesis of UDP-GlcNAc by a Multi-Enzyme Cascade Immobilized on Ni-NTA Solid Supports

(113) Enzymes of the UDP-GlcNAc synthesis pathway were recombinantly produced in E. coli as detailed before. The bio mass was mixed as detailed in Table 18A and homogenized for 8 minutes at 800-1000 psi in 150 mL lysis buffer (see Table 18B). The cell lysate was centrifuged (7000×g, 45 min) and the supernatant containing the enzymes was filtered (1.8 μm filter). A total protein concentration of 10 mg/mL was determined. To prepare the immobilization 500 μL of the Ni-NTA bead slurry were transferred each to 2 mL Eppendorf tubes and equilibrated with lysis buffer containing additionally 10 mM imidazole. Immobilization on Ni-NTA was carried out by incubating 1.5 mL lysate with the preequilibrated beads in immobilization buffer (lysis buffer plus 10 mM imidazole). After immobilization the beads were washed three times with washing buffer (see Table 18C).

(114) TABLE-US-00018 TABLE 18 A. Biomass mix used for the immobilization. B. Lysis buffer. C. Washing buffer A. B. C. Mass of Lysis Conc. Wash. Conc. Enzyme biomass (g) buffer (mM) buffer (mM) NahK 6.14 HEPES 250 HEPES 400 URA6 4.13 MgCl.sub.2 50 MgCl.sub.2 50 GalU 13.63 NaCl 300 NaCl 600 PPK3 11.92 glycerol 5% glycerol 5% PmPpA 1.08

(115) A reaction cycle was carried out to assess the activity of the beads. Each of the reactions was carried out for 20-25 hours at 30° C. and shaking at 600 rpm. To start a reaction 250 μL of the feed solution was added to the washed beads (Table 19). In between the experiments the supernatant was removed and the beads were washed 2 times with 1 mL deionized water.

(116) TABLE-US-00019 TABLE 19 Feed solution for UDP-GlcNAc synthesis. Substrate Conc. (mM) UMP 5 ATP 4 GlcNAc 5 PolyP.sub.25 10 HEPES 100 MgCl.sub.2 50

(117) The UDP-GlcNAc cascade immobilized on Ni-NTA beads shows decreasing activity for nine reactions (see FIG. 33). Some residual activity is detectable in reaction number 10 and 11 but is negligible compared to the activity on epoxy beads. In summary, the cascade immobilized on a wide range of solid supports with epoxy functional groups shows extended activity in comparison to Ni-NTA beads. Consequently, epoxy beads can be reused more often and are, hence, more economical.

Example 5: Synthesis of UDP-GlcNAc from Uridine and GlcNAc

(118) The cascade for synthesis of UDP-GlcNAc from uridine is shown in FIG. 2. The cascade contains six enzymes and seven reactions. Uridine (Uri), N-acetyl-glucosamine (GlcNAc), polyphosphate (PolyP.sub.n) are used as the main substrates, including catalytic amount of adenine triphosphate (ATP).

(119) Recombinant Production of Enzymes

(120) The list of the plasmids used in this study is shown in Table 20. LOBSTR E. coli competent cells (Kerafast, US) were used as the expression host. Cells were transformed based on heat-shock protocol. The fermentation carried out in TB media supplement with 1.5 mM MgSO.sub.4 and corresponding antibiotic. The cells were cultivated at 37° C. until OD.sub.600 of 0.8-1.0 was observed. Afterwards, induction was carried out with 0.4 mM IPTG, followed by 20-24 h cultivation at 16° C.

(121) At the end of the cultivation, cells were harvested by centrifugation (7000×g, 20 minutes) and cell pellets were resuspended in lysis buffer (50 mM MOPS buffer, 300 mM NaCl, 10 mM MgCl.sub.2, 10 mM imidazole and 5% glycerol at pH 7.4) and were disrupted by high-pressure homogenizer (Maximator, Germany) (3 times passage at 800-1000 psi). The His-tag purification was performed based on immobilized metal affinity chromatography with ÄKTA start instrument (GE Health care Life Sciences, Uppsala, Sweden) in combination with 1 mL or 5 mL HisTrap HP (GE Health care Life Sciences, Sweden) columns. The binding buffer contains: 50 mM MOPS buffer, 300 mM NaCl, 10 mM MgCl.sub.2, 10 mM imidazole and 5% glycerol at pH 7.4. And the elution buffer consists of 50 mM MOPS buffer, 300 mM NaCl, 10 mM MgCl.sub.2, 250 mM imidazole and 5% glycerol at pH 7.4.

(122) In order to remove imidazole from elution buffer and concentrate the enzyme solution, buffer exchange performed with Amicon® Ultra-15 Centrifugal Filter Unit—3 KDa MW cutoff (Merck, Germany). The exchange buffer contained: 50 mM MOPS buffer, 300 mM NaCl, 10 mM MgCl.sub.2, 5% glycerol at pH 7.4. Afterwards, the retentate solution (concentrated enzyme) was mixed 1:1 with glycerol to have the final enzyme solution in 50% glycerol and enzymes were stored at −20° C.

(123) TABLE-US-00020 TABLE 20 Enzymes used in this example, their origin, and expression plasmid Uniprot. SEQ ID Gene Abbr. Enzyme No. Origin Plasmid No Nahk NahK N-acetylhexosamine E8MF12 Bifidobacterium pET-28a(+) 1 1 kinase longum glmu GLMU UTP-GlcNAc- Q9CK29 Pasteurella pET-15b 8 1-phosphate multocida uridylyl transferase ppa PmPpa Inorganic P57918 Pasteurella pET-28a(+) 5 diphosphatase multocida udk UDK uridine/cytidine P0A8F4 Escherichia pET-28a(+) 9 kinase coli (strain K12) UMK3 URA6 UMP/CMP O04905 Arabidopsis pACYCDuet 2 kinase thaliana SPO1727 PPK3 NDP kinase/ Q5LSN8 Ruegeria pACYCDuet 3 polyP.sub.n kinase pomeroyi LgtA β1,3Glc β-1,3-N- NAcT acetylglucosamine Q51115 Neisseria pMAL-c4X 10 transferase meningitidis

(124) Experiment A: Synthesis of UDP-GlcNAc with Purified Enzymes

(125) Reactions were conducted at 200 μL, 37° C. and 550 rpm. The reaction conditions were as follows: UDK, 0.07 μg/μL; URA6/PPK3, 0.11 μg/μL; NAHK, 0.18 μg/μL; GLMU, 0.2 μg/μL; PmPpa, 0.05 μg/μL; uridine, 68 mM; GlcNAc, 68 mM; ATP, 2.1 mM; PolyP.sub.n, 21 mM; Tris-HCl (pH, 8.5), 150 mM; MgCl.sub.2, 75 mM. The successful production of UDP-GlcNAc and concentration of the cascade intermediates are shown in FIG. 34. UDP-GlcNAc was produced in quantitative yield which results in a final concentration of ˜40 g/L

(126) Experiment B: Large-Scale Synthesis of UDP-GlcNAc

(127) For preparation of cell lysate for synthesis of UDP-GlcNAc the following biomasses were mixed: UDK, 6.65 g; URA6/PPK3, 9.26 g; NAHK, 11.23 g; GLMU, 6.9 g; PmPpa, 4.94 g in 200 mL of 50 mM HEPES buffer (pH 8.1), 400 mM NaCl, and 5% glycerol. The mixture was passed three times through a high-pressure homogenizer. Cell-free extract was centrifuged at 11,000×g for 45 min. Afterwards, preliminary experiments were carried out on a small scale (200 μL) to find a suitable amount of lysate for the synthesis. The findings based on 200 μL synthesis was directly used for 4 liter scale synthesis which correlate to a 20,000× scaling factor.

(128) To carry out a 4-liter large scale experiment, a seven-liter single wall glass autoclavable bioreactor (Applikon, Netherlands), equipped with two pitched-blade impellers was selected to carry out the large-scale production.

(129) The synthesis conditions were as follows: 200 mM Tris-HCl (pH 8.5), 62 mM uridine, 62 GlcNAc, 1.6 mM ATP, 18 mM PolyP.sub.n, 75 mM MgCl.sub.2, and total protein load of 0.5 g/L in the form of cell lysate. The reaction was carried out at 37° C. room and 120 rpm. To understand the effect of scale-up on the performance of the cascade, a parallel 200 μL experiment was carried out. The time course of cascade intermediates is shown in FIG. 35.

(130) Experiment C: Synthesis of UDP-GlcNAc with Immobilized Enzymes

(131) For making the process closer to future industrial application, immobilization was carried out by using cell lysate containing all the necessary enzymes (as described above). The cell lysate solution was the same as used in 4-L scale synthesis of UDP-GlcNAc. The list of the beads used in this study as a support for co-immobilization of enzyme are described in Table 21.

(132) TABLE-US-00021 TABLE 21 Solid support beads used in this experiment for co-immobilization of enzymes Oxiran Pore size content Bead Matrix (nm) Size (μm) (μmol/gwet) Relizyme EP 112/S epoxy/polymethacrylate  40-60 100-300 115 Relizyme EP 112/M epoxy/polymethacrylate  40-60 200-500 112 Relizyme EP 113/S epoxy/polymethacrylate  40-60 100-300  87 Relizyme EP 113/M epoxy/polymethacrylate  40-60 200-500  94 Relizyme HFA 403/S epoxy/polymethacrylate  40-60 100-300  43 Relizyme HFA 403/M epoxy/polymethacrylate  40-60 200-500  47 Relizyme EP 403/S epoxy/polymethacrylate  40-60 100-300  60 Relizyme EP 403/M epoxy/polymethacrylate  40-60 200-500  56 ECR 8204F epoxy/methacrylate  30-60 150-300 ECR 8204M epoxy/methacrylate  30-60 300-710 ECR 8215F epoxy/methacrylate 120-180 150-300 ECR 8215M epoxy/methacrylate 120-180 300-710 ECR 8285 epoxy/butyl methacrylate  40-60 250-1000 ECR 8209F epoxy/methacrylate  60-120 150-300 ECR 8209M epoxy/methacrylate  60-120 300-710

(133) On average, 200 mg of beads (Table 21) were transferred to a new 2 mL Eppendorf tube, followed by addition of 0.6 mL cell lysate solution containing enzymes for synthesis of UDP-GlcNAc. The ratio of beads (mass) over total protein was approximately 20. After 24 h of incubation at room temperature with interval rotational mixing (˜every 6 h), the enzyme containing solution was removed. Afterwards, beads were washed three times with washing buffer containing high concentration of salt (200 mM Tris-HCl (pH 8.5) and 600 mM NaCl) to remove weakly bound proteins. Afterwards, beads were incubated for 24 h in storage buffer (200 mM Tris-HCl (pH 8.5) and 300 mM NaCl) to block the uncoupled binding sites. The percentage of bound protein is illustrated in FIG. 36.

(134) The feed solution for evaluating the activity of immobilized enzymes contained: 200 mM Tris-HCl (pH 8.5), 75 mM MgCl.sub.2, 25 mM uridine, 25 mM GlcNAc, 5 mM ATP, 10 mM PolyP.sub.n. 250 μL of feed solution added to beads and incubated at 37° C. and 600 rpm for 24 h. To confirm that all six enzymes bind in their active form to the solid support, the reaction with each solid support bead was monitored. The chromatogram of the reaction with each bead is shown in FIG. 37. The results of the activity test of each bead are summarized in FIG. 38. Therefore, the following beads were selected as good performing beads: Relizyme EP 113/M, ECR 8204F, ECR 8204M, ECR 8215M, ECR 8209F and ECR 8209M.

(135) To evaluate one of the most important factors in using immobilized enzymes—stability in various cycles—the activity of aforementioned beads were evaluated in different cycles. In each cycle, 250 μL of feed solution (200 mM Tris-HCl (pH 8.5), 75 mM MgCl.sub.2, 25 mM uridine, 25 mM GlcNAc, 5 mM ATP, 10 mM PolyP.sub.n) were added to each vial containing beads and incubated at 600 rpm and 37° C. for 24 h. Afterwards, liquids were removed and beads were washed with water twice to avoid any carry over from previous cycles. The activity of each bead in 10 cycles is shown in FIG. 39. The tested beads Relizyme EP 113/M, ECR 8204F, ECR 8204M, ECR 8215M, ECR 8209F and ECR 8209M have been proven to be active for 10 consecutive cycles without losing activity significantly. On average, in each cycle, UDP-GlcNAc is accumulated in the supernatant with a concentration of ˜7 g/L.

(136) Experiment D: Coupling of UDP-GlcNAc Cascade to ß-1,3-N-Acetyl-Glucosamine Transferase

(137) In this experiment, the reaction cascade for synthesis of UDP-GlcNAc from uridine and GlcNAc (as shown in FIG. 2) was coupled to a ß-1,3-N-acetylglucosamine transferase (ß1,3GlcNAcT) in order to synthesize lacto-N-triose (LNT II) in a single pot.

(138) The experimental conditions were as follows: 200 mM Tris-HCl (pH 8.5), 30 mM lactose, 5 mM uridine, 40 mM GlcNAc, 1.1 mM ATP, 12 mM PolyP.sub.n, 50 mM MgCl.sub.2 and the following enzymes: UDK (0.06 μg/μL), URA6/PPK3 (0.11 μg/μL), NAHK (0.14 μg/μL), GLMU (0.21 μg/μL), PmPpa (0.04 μg/μL), B1,3GlcNAcT (0.06 μg/μL) with final volume of 250 μL. After 48 h of incubation at 30° C., LNT II was produced with a final concentration of 4.7 mM (2.5 g/L). 1. Mahour, R., et al., Establishment of a five-enzyme cell-free cascade for the synthesis of uridine diphosphate N-acetylglucosamine. Journal of Biotechnology, 2018. 283: p. 120-129. 2. Rexer, T. F. T., et al., One pot synthesis of GDP-mannose by a multi-enzyme cascade for enzymatic assembly of lipid-linked oligosaccharides. Biotechnology and Bioengineering, 2018. 115(1): p. 192-205. 3. Liese, A. and L. Hilterhaus, Evaluation of immobilized enzymes for industrial applications. Chemical Society reviews, 2013. 42(15): p. 6236-6249.