ENZYMATIC HEXOSAMINIDATION OF LACTOSE

Abstract

The present disclosure relates to methods for producing human milk oligosaccharide (HMO) core structures using glycosidases from family GH20 hexosaminidases. In particular, the present disclosure provides methods for producing lacto-N-triose II (LNT II) and/or lacto-N-tetraose (LNT) by reacting glucosamine-oxazoline and/or lacto-N-biose-oxazoline with lactose catalysed by an enzyme of the glycoside hydrolase family 20 (GH20) according to the classification of the Carbohydrate-Active-Enzymes (CAZy) database. Specific optimized enzymes are identified to catalyse the reactions.

Claims

1. A method for producing lacto-N-triose II and/or lacto-N-tetraose comprising, the step of: (i) reacting glucosamine-oxazoline and/or lacto-N-biose-oxazoline with lactose catalysed by an enzyme of the glycoside hydrolase family 20 (GH20) to obtain lacto-N-triose II and/or lacto-N-tetraose.

2. Method according to claim 1, wherein step (i) is performed in an aqueous solution containing at least 50 g/L lactose, preferably at least 100 g/L lactose, particularly preferably at least 150 g/L lactose, most preferably at least 190 g/L lactose.

3. Method according to claim 1, wherein the method is performed under conditions that are free or essentially free of organic solvents.

4. Method according to claim 1, wherein the glucosamine-oxazoline and/or lacto-N-biose-oxazoline, respectively, is added to the lactose and the enzyme of the glycoside hydrolase family 20 (GH20) over a period of at least 20 minutes, preferably at least 60 minutes.

5. The method for producing lacto-N-triose II according to claim 1, wherein the enzyme of the glycoside hydrolase family 20 (GH20) is a β-N-acetylhexosaminidase, preferably β-N-acetylhexosaminidase of Bifidobacterium bifidum JCM1254 having an amino acid sequence of SEQ ID NO: 1 or an enzyme having an amino acid sequence identity of at least 70% to SEQ ID NO: 1 and having β-N-acetylhexosaminidase activity, or a β-N-acetylhexosaminidase enzyme comprising an amino acid sequence of SEQ ID NO: 2, or a β-N-acetylhexosaminidase enzyme comprising an amino acid sequence having an amino acid sequence identity of at least 70% to SEQ ID NO: 2.

6. Method for producing lacto-N-tetraose according to claim 1, wherein the enzyme of the glycoside hydrolase family 20 (GH20) is a lacto-N-biosidase, preferably lacto-N-biosidase of Bifidobacterium bifidum JCM1254 having an amino acid sequence of SEQ ID NO: 3 or an enzyme having an amino acid sequence identity of at least 70% to SEQ ID NO: 3 and having lacto-N-biosidase activity, or a lacto-N-biosidase enzyme comprising an amino acid sequence of SEQ ID NO: 4, or a lacto-N-biosidase enzyme comprising an amino acid sequence having an amino acid sequence identity of at least 70% to SEQ ID NO: 4.

7. Method according to claim 1, wherein the method further comprises the step of: (ii) deactivating the enzyme of the glycoside hydrolase family 20 (GH20) after the reacting step.

8. Method for producing lacto-N-triose II according to any-of-elaims claim 1 further comprising the step: (iii) adding a β-galactosidase or a galactosyl transferase and UDP-galactose, to the mixture of step (i) or, preferably to the mixture of step (ii), and optionally adding further lactose to obtain lacto-N-tetraose or lacto-N-neotetraose.

9. Use of an enzyme of the glycoside hydrolase family 20 (GH20) for producing lacto-N-triose II or lacto-N-tetraose from lactose.

10. Use according to claim 9, wherein glucosamine-oxazoline and/or lacto-N-biose-oxazoline are used as substrates.

11. Use The use according to claim 9 or 10, wherein the enzyme of the glycoside hydrolase family 20 (GH20) is used to produce lacto-N-triose II and/or lacto-N -tetraose by reacting glucosamine-oxazoline and/or lacto-N-biose-oxazoline.

12. An enzyme, comprising β-N-acetylhexosaminidase having an amino acid sequence identity of at least 70% to SEQ ID NO: 1 and carrying at least one mutation selected from the group of mutations consisting of: a mutation at position 746 of SEQ ID NO: 1; a mutation at position 827 of SEQ ID NO: 1, preferably carrying a glutamic acid or an alanine or a glutamine; a mutation at position 746 of SEQ ID NO: 1 and/or carrying a phenylalanine at a position counted as position 827 of SEQ ID NO: 1, or a β-N-acetylhexosaminidase comprising an amino acid sequence selected from the group consisting of: any-oUSEQ ID NO[[s]]: 5 to 8 or comprising an amino acid sequence having an amino acid sequence identity of at least 70% to any of SEQ ID NOs: 5 to 8.

13. A Lacto-N-biosidase having an amino acid sequence identity of at least 70% to SEQ ID NO: 3 and carrying at least one of the following substitutes selected from the group consisting of: a mutation at a position counted as position 320 of SEQ ID NO: 3, a mutation at a position counted as position 419 of SEQ ID NO: 3, a glutamic acid or an alanine at a position counted as position 320 of SEQ ID NO: 3, a phenylalanine at a position counted as position 419 of SEQ ID NO: 3; or At least one lacto-N-biosidase comprising an amino acid sequence selected from the group consisting of; SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11. and a sequence having an amino acid sequence identity of at least 70% to any of SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11.

14. The method according to claim 1, wherein the enzyme used is: a β-N-acetylhexosaminidase having an amino acid sequence identity of at least 70% to SEQ ID NO: and carrying at least one mutation selected from the group of mutations consisting of: a mutation at position 746 of SEQ ID NO: la mutation at position 827 of SEQ ID NO: 1, preferably carrying a glutamic acid or an alanine or a glutamine a mutation at a position 746 of SEQ ID NO: 1 and/or carrying a phenylalanine at a position counted as position 827 of SEQ ID NO: 1, or β-N-acetylhexosaminidase comprising an amino acid sequence selected from the group consisting of: SEQ ID No: 5 to 8 or comprising an amino acid sequence having an amino acid sequence identity of at least 70% to any of SEQ ID NOs: 5 to 8, or a Lacto-N-biosidase having an amino acid sequence identity of at least 70% to SEQ ID NO: 3 and carrying at least one of the following substitutes selected from the group consisting of; a mutation at a position counted as position 320 of SEQ ID NO: 3, a mutation at a position counted as position 419 of SEQ ID NO: 3, a glutamic acid or an alanine at a position counted as position 320 of SEQ ID NO: 3, a phenylalanine at a position counted as position 419 of SEQ ID NO: 3; or at least one lacto-N-biosidase comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO:10, and SEQ ID NO: 11.

15. Aqueous solution comprising an enzyme of the glycoside hydrolase family 20 (GH20) as defined in any of the preceding claims, lactose, preferably at least 50 g/L lactose, more preferably at least 100 g/L lactose, particularly preferably at least 150 g/L lactose, most preferably at least 190 g/L lactose and a) glucosamine-oxazoline and/or lacto-N-biose-oxazoline; and/or b) lacto-N-triose II and/or lacto-N-tetraose.

16. The method according to claim 1, wherein the enzyme used is: a β-N-acetylhexosaminidase having an amino acid sequence identity of at least 70% to SEQ ID NO: and carrying at least one mutation selected from the group of mutations consisting of: a mutation at position 746 of SEQ ID NO: 1a mutation at position 827 of SEQ ID NO: 1, preferably carrying a glutamic acid or an alanine or a glutamine; a mutation at position 746 of SEQ ID NO: 1 and/or carrying a phenylalanine at a position counted as position 827 of SEQ ID NO: 1, or a β-N-acetylhexosaminidase comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs: 5 to 8 or comprising an amino acid sequence having an amino acid sequence identity of at least 70% to any of SEQ ID NOs: 5 to 8, or a Lacto-N-biosidase having an amino acid sequence identity of at least 70% to SEQ ID NO: 3 and carrying at least one of the following substitutes selected from the group consisting of; a mutation at a position counted as position 320 of SEQ ID NO: 3, a mutation at a position counted as position 419 of SEQ ID NO: 3, a glutamic acid or an alanine at a position counted as position 320 of SEQ ID NO: 3, a phenylalanine at a position counted as position 419 of SEQ ID NO: 3; or at least one lacto-N-biosidase comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO:10, and SEQ ID NO: 11.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0108] FIG. 1: LNT II synthesis by wild-type Bbhl and mutants thereof. Time courses show synthesis from 60 mM GlcNAc-oxazoline using a 10-fold excess of lactose. (a) Wild-type, 0.23 μM; (b) D746E, 8.4 μM; (c) Y827F, 4 μM; (d) D746A, 18 μM; (e) D746Q, 9 pM. LNT II, filled circles; yield, open circles. (f) Trans-glycosylation versus primary hydrolysis. RTH is the ratio of the specific trans-glycosylation activity of GlcNAc-to-lactose transfer to the specific activity of non-productive GlcNAc-oxazoline hydrolysis (primary hydrolysis) under ‘synthesis conditions’. For the wild type and each mutant three bars are shown, the left bar indicates the specific trans-glycosylation activity; the middle bar indicates the maximum LNT II yield; the right bar indicates the RTH value.

[0109] FIG. 2: LNT II synthesis from GlcNAc-p-pNP by wild-type Bbhl and mutants thereof. Time courses show synthesis from 20 mM GlcNAc-β-pNP using a 20-fold excess of lactose in the presence of 20% DMSO. (a) Wild-type, 0.12 μM; (b) D746E, 8.4 μM; (c) D746A, 24 μM; (d) D746Q, 8.6 μM; (e) Y827F, 8 μM. LNT II, filled circles; yield, open circles; pNP released, open triangles. (f) TLC-analysis of LNT II synthesis using Bbhl enzymes. Reaction mixtures were 2-fold diluted. Carbohydrates were visualized by thymol-sulfuric acid reagent. pNP was detected by UV (254 nm). Lane 1, pNP; lane 2, lactose; lane 3, LNT II; lane 4, wild-type reaction after 1.5 h; lane 5, D746E reaction after 20 min; lane 6, Y975F reaction after 1.5 h; lane 7, D746A reaction after 8 h; lane 8, D746Q reaction after 6 h.

[0110] FIG. 3: HPLC analysis of LNT II synthesis from GlcNAc-oxazoline by wild-type Bbhl and mutants thereof. Overlay of HPLC-chromatograms showing maximum LNT II formation obtained by various enzyme variants of Bbhl. Reaction mixtures contained 60 mM GlcNAc-oxazoline, a 10-fold excess of lactose and 0.23-18 μM of the enzymes. Samples were 10-fold (D746Q) and 25-fold diluted (all other enzymes), respectively. UV-detection at 195 nm was used.

[0111] FIG. 4: TLC analysis of LNT II synthesis from GlcNAc-oxazoline by wild-type Bbhl and mutants thereof. 130 mM (D746E) or 60 mM (all other enzymes) GlcNAc-oxazoline, 600 mM lactose and 0.23-18 μM of the enzymes were used. Carbohydrates were visualized by thymol-sulfuric acid reagent. GlcNAc was also detected by UV (254 nm) (a) Lane 1, GlcNAc; lane 2, lactose; lane 3, LNT II; lane 4, wild-type reaction after 1 h, 1:10; lane 5, D746E reaction after 50 min, 1:50; lane 6, D746A reaction after 5 h, 1:25; lane 7, galactose; lane 8, glucose. (b) Lane 1, GlcNAc; lane 2, lactose; lane 3, LNT II; lane 4, Y827F reaction after 50 min, 1:9.

[0112] FIG. 5: LNT II synthesis from GlcNAc-oxazoline by wild-type Bbhl and the D746E mutant. Time courses show synthesis from 64 mM GlcNAc-oxazoline and 20 mM lactose. (a) Wild-type, 0.23 μM; (b) D746E, 8.4 μM. LNT II, filled circles; yield, open circles.

[0113] FIG. 6: LNT synthesis by wild-type LnbB and mutants thereof. Time courses show synthesis from 12 mM LNB-oxazoline using a 50-fold excess of lactose. (a) Wild-type, 0.5 μM; (b) D320E, 4 μM; (c) D320A, 20 μM; (d) Y419F, 4 μM. LNT , filled circles; yield, open circles; LNT II, filled triangles.

[0114] FIG. 7: LNT synthesis from LNB-β-pNP by wild-type LnbB and mutants thereof. Time courses show synthesis from 20 mM LNB-β-pNP in the presence of 15% DMSO, using a 30-fold excess of lactose. (a) Wild-type, 0.5 μM; (b) D320E, 10 μM; (c) D320A, 20 μM; (d) Y419F, 4 μμM. LNT, filled circles; yield, open circles; pNP released, open triangles. (e) TLC-analysis of LNT synthesis using LnbB enzymes. Reaction mixtures were 5-fold diluted. Carbohydrates were visualized by thymol-sulfuric acid reagent. Lane 1, LNB; lane 2, lactose; lane 3, LNT; lane 4, wild-type reaction after 10 min; lane 5, D320E reaction after 20 min; lane 6, Y419F reaction after 2.5 h; lane 7, D320A reaction after 21 h.

[0115] FIG. 8: HPLC analysis of LNT synthesis from LNB-oxazoline by wild-type LnbB and mutants thereof. Overlay of HPLC-chromatograms showing maximum LNT formation obtained by various enzyme variants of LnbB. Reaction mixtures contained 12 mM LNB-oxazoline, a 50-fold excess of lactose and 0.5-20 μM of the enzymes. Samples were 5-fold diluted. UV-detection at 195 nm was used.

[0116] FIG. 9: TLC analysis of LNT synthesis from LNB-oxazoline by wild-type LnbB and mutants thereof. 12 mM LNB-oxazoline, a 50-fold excess of lactose and 0.5-20 pM of the enzymes were used. Reaction mixtures were 5-fold diluted. Carbohydrates were visu-alized by thymol-sulfuric acid reagent. (a) Lane 1, GlcNAc; lane 2, LNB; lane 3, lactose; lane 4, LNT II; lane 5, LNT; lane 6, wild-type reaction after 10 min; lane 7, D320E reaction after 20 min; lane 8, Y419F reaction after 2.5 h; lane 9, D320A reaction after 21 h. (b) Lane 1, GlcNAc; lane 2, LNB; lane 3, LNT II; lane 4, LNT; lane 5, wild-type reaction after 0 min; lane 6, wild-type reaction after 10 min; lane 7, D320E reaction after 0 h; lane 8, D320E reaction after 1 h; lane 9, lactose.

[0117] FIG. 10: Synthesis of LNT by the LnbB D320E mutant. Reaction mixture contained 12 mM LNB-oxazoline, a 50-fold excess of lactose and 20 μM D320E. LNT , filled circles; yield, open circles; LNT II, filled triangles.

[0118] FIG. 11: Bulk synthesis of LNT II by the Bbhl D746E mutant. (a) Time-course of LNT II synthesis by the D746E variant (4 pM) using equimolar amounts of GlcNAc-oxazoline and lactose (600 mM). (b) Comparison of wild-type Bbhl (triangles) and D746E mutant (circles) for LNT2 synthesis, using a 2.4 fold excess of lactose over GlcNAc-oxazoline (255 mM) and 0.4 pM of enzyme. LNT II, filled symbols; yield, open symbols. Overlay of HPLC-UV traces (c) and HPLC-RI traces (d) used to evaluate the purity of LNT II produced on gram-scale. Note, the first peak in the HPLC-RI traces is the injection peak.

[0119] FIG. 12: LNT II synthesis with increasing GlcNAc-oxazoline concentration by the D746E mutant of Bbhl. Time courses show synthesis using varying concentrations of GlcNAc-oxazoline, 600 mM lactose and 4.2 μM D746E. GlcNAc-oxazoline concentration was: (a) 130 mM, (b) 260 mM, (c) 500 mM. LNT II, filled circles; yield, open circles. (d) TLC-analysis of LNT II using 500 mM of GlcNAc-oxazoline. Reaction mixtures were 50-fold diluted. Lane 1, GlcNAc; lane 2, lactose; lane 3, LNT II; lane 4, 0 h; lane 5, 2.5 min; lane 6, 30 min; lane 7, 5 h; lane 8, 23.5 h. (e) TLC-analysis of LNT II using 600 mM of GlcNAc-oxazoline. Reaction mixtures were 200-fold diluted. Lane 1, GlcNAc; lane 2, lactose; lane 3, LNT II; lane 4, 0 h; lane 5, 2.5 min; lane 6, 5 min; lane 7, 30 min; lane 8, 7 h; lane 9, 23.5 h. Carbohydrates were visualized by thymol-sulfuric acid reagent. GlcNAc was also detected by UV (254 nm).

[0120] FIG. 13: LNT II synthesis from GlcNAc-β-pNP by wild-type Bbhl and the D746E mutant. Time courses show synthesis from 100 mM GlcNAc-β-pNP and 600 mM lactose in the presence of 20% DMSO. (a) D746E, 8.4 μM; (b) wild-type, 0.12 μM. LNT II, filled circles; yield, open circles; pNP released, open triangles.

[0121] FIG. 14: .sup.1H NMR spectrum of isolated LNT II. LNT II was dissolved in D.sub.2O. Spectrum is in accordance with previously published data..sup.4

[0122] FIG. 15: .sup.13C NMR spectrum of isolated LNT II. LNT II was dissolved in D.sub.2O. Full spectrum and partial spectrum (inset) showing that LNT II (GlcNAc-β1,3-Gal-β1,4-Glc) was the only regioisomer formed (82 ppm). No other regioisomers could be detected. Spectrum is in accordance with previously published data..sup.4

[0123] FIG. 16: Global sequence alignment of the wildtype and mutant constructs of Bbhl and LnbB (SEQ ID NOs: 12-20) and corresponding sequences from other organ-isms (SEQ ID NOs 21-24). The alignment was performed with the program “MegAlign Pro” Version: 12.2.0 (82) Copyright © 2012-2015, DNASTAR, Inc.. The used algorithm was “MUSCLE” (Multiple Sequence Comparison by Log-Expectation). The aligned sequences are the following: Bbhl, wild type (wt), truncated construct including a His-tag (SEQ ID NO: 12), Bbhl, D746E mutant, truncated construct including a His-tag (SEQ ID NO: 13), Bbhl, D746A mutant, truncated construct including a His-tag (SEQ ID NO: 14), Bbhl, D746Q mutant, truncated construct including a His-tag (SEQ ID NO: 15), Bbhl, Y827F mutant, truncated construct including a His-tag (SEQ ID NO: 16), LnbB, wild type (wt), truncated construct including a His-tag (SEQ ID NO: 17), LnbB, D320E mutant, truncated construct including a His-tag (SEQ ID NO: 18), LnbB, D320A mutant, truncated construct including a His-tag (SEQ ID NO: 19), LnbB, Y419F mutant, truncated construct including a His-tag (SEQ ID NO: 20); Hex 1, from Actinomycetales bacterium, GenBank AKC34128.1 (SEQ ID NO: 21), Hex 2, from Bacteroidetes bacterium, GenBank AKC34129.1 (SEQ ID NO: 22), Chb, from Serratia marcescens, GenBank AAB03808.1 (SEQ ID NO: 23), SpHex, from Streptomyces plicatus, GenBank AAC38798.3 (SEQ ID NO: 24). The consensus sequence of the alignment is also shown (SEQ ID NO: 25).

EXAMPLES

Materials

[0124] Media components and chemicals were of reagent grade from Sigma Aldrich/Fluka (Austria/Germany), Roth (Karlsruhe, Germany) or Merck (Vienna, Austria). HisTrap FF 5 mL column was from GE Healthcare (Vienna, Austria). Minisart® NML syringe membrane filter (0.45 pm) and Vivaspin® Turbo 15 centrifugal concentrators (30 kDa, 50 kDa) were from Sartorius (Goettingen, Germany). GlcNAc, 2-hydroxybenzimidazole (purity 97%), dimethyl sulfone (purity 99.96%) and succinonitrile were from Sigma Aldrich (Austria/Germany). 4-nitrophenyl 2-acet-amido -2-deoxy-β-D-glucopyranoside (GlcNAc-β-pNP), 4-nitrophenyl 2-acetamido-2-deoxy-3-O-(β-D-galactopyranosyl) -β-D-glucopyranoside (LNB-β-pNP), lacto-N-triose II (LNT II), lacto-N-tetraose (LNT, purity ≥90%), lacto-N-biose (LNB) and α-D-galactose-1-phosphate dipotassium salt hydrate (Gal 1-P) were from Carbosynth (Compton, Berkshire, UK). Chromabond Flash FM 70/10C C18 ac adsorbent was von Macherey Nagel (Schoonebeek, Netherlands). Acetonitrile (HPLC gradient grade) was from Chem-Lab NV (Zedelgem, Belgium).

Example 1: Enzyme Preparation

[0125] Production of the enzymes (without signal peptide and transmembrane region/membrane anchor) and their purification were done according to protocols from literatures.sup., 6,26 Briefly, synthetic Bbhl genes (wild-type N-acetylhexosaminidase from B. bifidum JCM1254 (GenBank: AB504521.1, aa 33-1599).sup.5and D746E, D746A, D746Q, Y827F variants) and synthetic LnbB genes (wild-type lacto-N-biosidase from B. bifidum JCM1254 (GenBank: EU281545.1, aa 35-1064).sup.6 and D320E, D320A, Y419F variants) codon-optimized for E. coli expression were ligated into Ndel-Xhol-cut pET21b(+) and pET24b(+) plasmids, respectively (Bio-Cat GmbH, Heidelberg, Germany). Residue numbering of full length enzymes is used. All inserts were confirmed by DNA sequencing. Bbhl enzymes were expressed in E. coli BL21(DE3) at 25° C. for 20 h by induction with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG), using LB-medium supplemented with 115 mg L-1 ampicillin. LnbB enzymes were expressed in E. coli BL21(DE3) following an auto-induction protocol.sup.33 in LB medium with 50 μg mL.sup.−1, kanamycin, 25 mM Na.sub.2HPO.sub.4, 25 mM K.sub.2HPO.sub.4, 50 mM NH.sub.1Cl, 5 mM Na.sub.2SO.sub.4, 2 mM MgSO.sub.4, 0.5% glycerol, 0.05% glucose and 0.2% lactose at 110 rpm and 30° C. for 20 h..sup.34 Each enzyme was produced as C-terminal Hiss-tag fusion protein (SEQ ID NOs: 12 to 20) Enzyme purification was done by single-step Hiss-tag affinity chromatography. The enzyme preparations used were (almost) pure by the criterion of migration as single protein band in SDS PAGE.

Example 2: Protein purification by Hiss-tag Affinity Chromatography

[0126] For protein purification, cell pellet from 1 L cell culture was resuspended in 25-30 mL binding buffer (20 mM sodium phosphate, 150 mM NaCl, 15 mM imidazol, pH 7.4) and frozen at −20° C. overnight. 35 mL aliquots of thawed cell suspension were ultrasonicated on ice bath at 60% amplitude for 6 min (2 s pulse on and 4 s pulse off) using a Sonic Dismembrator (Ultrasonic Processor FB-505; Fisher Scientific, Austria) equipped with a 1.27 cm probe for cell disruption. Cell lysates were centrifuged at 4° C. and 21,130 g for 1 h (Eppendorf centrifuge 5424R) and filtered via 0.45 μm cellulose-acetate syringe filters. Target proteins were purified from the cell-free extract via their C-terminal His6-tag using an AktaPrime plus system (GE Healthcare, Germany) at 4° C. The cleared cell lysate was loaded onto a HisTrap FF 5 mL column (GE Healthcare, Austria) at a flow rate of 2 mL min.sup.−1. The column had been equilibrated with binding buffer. After a washing step of 15 column volumes (CVs), the enzyme was eluted with 300 mM imidazol within 6 CVs at a flow rate of 4 mL min.sup.−1. Target protein containing fractions were pooled. Eluted enzyme was concentrated and buffer exchanged to 20 mM sodium phosphate, 150 mM NaCl, pH 7.4 using Vivaspin® Turbo 15 centrifugal concentrators (30 kDa or 50 kDa, 3645 g, 4° C.). SDS PAGE was used to confirm purity of enzyme preparations. Protein concentrations were measured with a DeNovix SA-11+spectrophotometer (DeNovix Inc, US) at 280 nm. −40 mg of Bbhl and -120 mg of LnbB enzymes, respectively, were typically obtained per liter of culture medium. Purified enzymes were aliquoted and stored at −70° C.

Example 3: Preparation of 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI)

[0127] CDMBI was prepared as described previously,.sup.8 with the following modifications. CDMBI was prepared by chemical synthesis in 2 steps. For preparation of 1,3-dimethylbenzim-idazolone (DMBI), the N-atoms of 2-hydroxybenzimidazole were methylated by the action of Mel in the presence of KOH. The DMBI yield could be increased from 70%.sup.8to 92% by using KOH instead of NaOH, which was previously described in literature..sup.8 The reference experiment with NaOH as a base yielded 75% of DMBI.

[0128] Step 1: Preparation of 1,3-dimethylbenzimidazolone (DMBI). To a mixture of 50 g 2-hydroxybenzimidazole (1 equiv., 97% purity) and 216.55 g toluene (6.5 equiv.), 5.83 g Bu.sub.4NBr (0.05 equiv.) and 202.86 g KOH (40% w/w, 4 equiv.) were added. The reaction mixture was heated to 60° C. and 118.04 g Mel (2.3 equiv.) were added dropwise within 60 min at high stirring rate. The mixture was stirred for 4 days at 60° C. Note, the reaction time can be reduced to 21 h without any change in yield. After cooling the mixture to 45° C., the phases were separated. The organic layer was washed at 45° C. 3 times with 75 mL 1 N HCl, once with 75 mL saturated NaHCO.sub.3 and dried over Na.sub.2SO.sub.4. After phase separation, the solvent was removed at 50° C. and 150-5 mbar. 58.5 g of crude DMBI were obtained. The residue was recrystallized as follows: crude DMBI was taken up in 90 g acetone/n-heptane (3:1 v/v) at 65° C. The mixture was allowed to cool to room temperature for 16 h, then cooled to 5° C. and stirred for further 3 h. After filtration, the crystalline DMBI was washed once with 40 mL ice-cold n-heptane/acetone (3:1 v/v) and dried under nitrogen to give 53.7 g of DMBI as a white solid (92%) .sup.1H-NMR (700 MHz, CDCl.sub.3): δ=7.10 (m, 2H), 6.97 (m, 2H), 3.42 (s, 6H). .sup.13C-NMR (175 MHz, CDCl.sub.3): δ=27.14, 107.29, 121.17, 129.99, 154.63.

[0129] DMBI was converted to CDMBI in a yield of 54% by using oxalyl chloride. When compared to literature, an additional 1.1 equiv. of total oxalyl chloride was used, the reaction temperature was decreased from 80° C. to 70° C., and the reaction times were prolonged.

[0130] Step 2: Preparation of 2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI). To a solution of 34.4 g DMBI (1 equiv.) in 271 g toluene (13.9 equiv.), 80 g oxalyl chloride (3 equiv.) were added at 40 ° C. The mixture was heated to 70° C. After 5 d at 70° C. no precipitate was formed. Then, 40 g of oxalyl chloride (1.5 equiv.) were added, and the mixture was stirred at 70° C. overnight. The suspension was cooled to 0-5° C. within 3 h and stirred for further 3 h at this temperature. After filtration, the filter cake was washed with 70 mL of ice-cold toluene and dried in vacuo to give 24.8 g of CDMBI (54%). .sup.1H-NMR (500 MHz, D.sub.2O): δ=7.85 (m, 2H), 7.72 (m, 2H), 4.08 (s, 3H). .sup.13C-NMR (125 MHz, D.sub.2O): δ=35.28, 115.51, 130.08, 134.22, 143.45. In comparison to the protocol reported in literature (49% yield),.sup.8 an additional 1.1 equiv. of total oxalyl chloride was used, the reaction temperature was decreased from 80° C. to 70° C. and the reaction times were prolonged.

[0131] Then, CDMBI and Na.sub.3PO.sub.4were used for oxazoline formation from N-Acetylgucosa-min (GlcNAc) or lacto-N-biose (LNB)..sup.8

[0132] Overall, the CDMBI synthesis was significantly improved. The CDMBI yield over 2 steps could be increased from 34%.sup.8 to 50% by the modifications just described above.

Example 4: Preparation of lacto-N-biose (LNB)

[0133] LNB was synthesized from Gal 1-P and GlcNAc by lacto-N-biose phosphorylase (LNBP) from Bifidobacterium longum JCM 1217, previously described by Kitaoka and co-work-ers. .sup.8,10 LNBP production. Production of the LNBP and purification were done according to protocols from literature..sup.9 Briefly, synthetic LNBP gene (GenBank: AB181926.1, aa 20-2275) not codon-optimized for E. coli expression was ligated into Ndel-Xhol-cut pET30a(+) plasmid (GenScript, Piscataway, USA). Insert was confirmed by DNA sequencing. LNBP was expressed in E. coli BL21(DE3) at 30° C. for 20 h by induction with 0.5 mM isopropyl-β-D-thiogalactopyra-noside (IPTG), using LB-medium supplemented with 50 mg L.sup.−1 kanamycin. LNBP was produced as C-terminal Hiss-tag fusion protein. Enzyme purification was done by single-step Hiss-tag affinity chromatography (see above). The following buffers were used: binding buffer (20 mM MOPS, 500 mM NaCl, 15 mM imidazol, pH 7.4), elution buffer (20 mM MOPS, 500 mM NaCl, 300 mM imidazol, pH 7.4), storage buffer (20 mM MOPS, 150 mM NaCl, pH 7.5). −50 mg of LNBP was typically obtained per liter of culture medium. The enzyme preparation used was (almost) pure by the criterion of migration as single protein band in SDS PAGE.

[0134] Enzymatic synthesis of LNB. Reaction was performed in a total volume of 40 mL using 5.4 mmol Gal 1-P (1.82 g) and 1.8 mmol GlcNAc (0.40 g) dissolved in water. The pH was adjusted to 6.8 with 4 M HCl and the reaction was started by adding 0.05 mg mL.sup.−1 (0.6 μM) LNBP. The conversion was performed in a 50 mL Sarstedt tube (diameter 2.8 cm, height 11.5 cm) under magnetic stirring (stir bar: 18×5 mm; 500 rpm) at 37° C. For temperature control, the Sarstedt tube was placed in a water bath. The pH was constantly monitored and manually controlled by adding 4 M HCl (within first 1.5 h). Incubation was for 3.5 h. Samples were taken at certain times and analyzed by HPLC. The reaction yield was 92% (42 mM, 16 g L.sup.−1).

[0135] Downstream processing (DSP). Major task of the DSP was the removal of Gal 1-P (93 mM) from the LNB (42 mM). Only a small amount of GlcNAc (3 mM) was present. Gal 1-P was removed from the mixture by anion-exchange chromatography (AEC) after enzyme-removal by ultra-filtration (Vivaspin concentrators 30 kDa, 4000 rpm, 20° C.). AEC was performed at pH 7.5. To allow efficient removal of Gal 1-P by binding to the anion-exchange column, the filtrate was 8-fold diluted to an ionic strength of ˜3.6 mS cm.sup.−1 with ultra-pure water. LNB and remaining GlcNAc are not ionized at pH 7.5 and elute in the flow-through. AEC was performed on an ÄktaPrime plus system (GE Healthcare, Germany) at room temperature. A self-packed Proteus 20 mL FliQ column (100×16.0 mm, Generon, UK) containing about 15 mL of Toyopearl SuperQ-650M was applied. Ultra-pure water (mobile phase A) and 1 M potassium chloride in ultra-pure water (mobile phase B) were used for binding and elution, respectively. Column was equilibrated with mobile phase A at 4 mL min.sup.−1 (5 CVs). 40 mL sample were loaded at a flow rate of 2 mL min.sup.−1 using mobile phase A. LNB eluted together with GlcNAc within 5 CVs. Gal 1-P was eluted with mobile phase B at 4 mL min.sup.−1 (5 CVs). Detection was by conductivity. Complete removal of Gal 1-P from LNB was verified by TLC analysis. LNB containing fractions were pooled. Sample was concentrated under reduced pressure at 40° C., frozen in liquid nitrogen under rotary motion before freeze-drying overnight (Christ Alpha 1-4, B. Braun Biotech International, Melsungen, Germany). The final product (80% isolated yield) was analyzed by HPLC and its chemical identity confirmed by .sup.1H NMR. 5% (w/w) of GlcNAc were detected in the final product.

Example 5: Preparation of Sugar Oxazolines

[0136] GlcNAc- and LNB-oxazoline were prepared as described previously..sup.8 Briefly, CDMBI (3 equiv.) was used as dehydrative condensing agent and Na3PO4 (7.5 equiv.) as a base for oxazoline formation from GlcNAc (1 equiv.) or LNB (1 equiv.). N-acetyl-2-amino sugars were added and the resulting solution was cooled to 0-3° C. CDMBI (3 equiv.) was added to the solution in portions within 15 min, and the mixture was stirred for 1 h at the same temperature.

[0137] GlcNAc is easily available at low cost. Practical preparation of LNB in bulk quantities from sucrose and GlcNAc, using a one-pot four-enzyme reaction with lacto-N-biose phosphory-lase (LNBP) from Bifidobacterium bifidum as a key enzyme, was reported by Kitaoka and co-workers..sup.9,10 Analogously, LNB was synthesized from Gal1P and GlcNAc by LNBP. Anion-ex-change chromatography (AEC) and freeze-drying was used for preliminary downstream processing (DSP) (80% isolated yield, purity ≥90%). Initial downstream processing (DSP) of the sugar oxazolines included filtration (Chromabond Flash FM 70/10C C18 ac adsorbent (10 g per g of N-acetyl-2-amino sugar used)) and freeze-drying (lyophilized crude product). Product identities were unequivocally confirmed by .sup.1H NMR spectroscopy. NMR spectra were in accordance with published data..sup.8 The content of sugar oxazolines was determined by quantitative .sup.1H NMR spectroscopy with dimethyl sulfone or succinonitrile as internal standards. No DMBI was detectable, but the excess of salt remained in the lyophilized crude product. GlcNAc-oxazoline was obtained on 30 mmol scale in a yield of ˜60%. LNB-oxazoline was obtained on 0.5 mmol scale in a yield of 79%.

[0138] In order to ensure efficient trans-glycosylation from the sugar oxazolines in the next step, the lyophilized crude products were desalted. Desalting was established by extraction with acetonitrile (9.5 g per g lyophilized crude product, 1 h stirring at room temperature), which is scalable. After filtration, washing (2 * with acetonitrile (3 g per g lyophilized crude product)), concentration and drying in vacuo, GlcNAc-oxazoline was obtained on 3.5 mmol scale in a yield of ˜60%. LNB-oxazoline was obtained on 0.082 mmol scale in a yield of <10%. The yields were determined by HPLC analysis of the N-acetyl-2-amino sugars released (GlcNAc, LNB) after complete hydrolysis of the oxazoline ring.

Example 6: Bbhl and LnbB Activity Assays

[0139] Bbhl and LnbB activities were assayed in a total volume of 600 μL and 400 μL, respectively, using 50 mM sodium phosphate buffer, pH 7.5 (with oxazoline donor substrates) or pH 5.8 (with pNP-labelled donor substrates). Reaction mixture with sugar oxazoline donor substrate contained 60 mM GlcNAc-oxazoline or 12 mM LNB-oxazoline, 600 mM lactose and 0.23-18 μM enzyme. Reaction mixture with pNP-labelled donor substrate contained 20 mM GlcNAc-β-pNP or LNB-β-pNP, 15-20% DMSO, 400-600 mM lactose and 0.12-24 μM enzyme. Enzymatic conversion was carried out at 37° C. or 55° C. (as indicated in the text; see Table 1) and agitation rate of 650 rpm using a Thermomixer comfort (Eppendorf, Germany).

[0140] Reactions were stopped at certain times by heating for 10 min at 99° C. Precipitated protein was removed by centrifugation at 13,200 rpm for 10 min. Samples were analyzed by hydrophilic interaction liquid chromatography (HILIC-HPLC) and thin layer chromatography (TLC).

[0141] Specific activities were calculated from initial rates of product formation (trans-glyco-sylation) and product hydrolysis (secondary hydrolysis), respectively, obtained at 400-600 mM of lactose (synthesis conditions'). One unit (1 U) of trans-glycosylation activity was defined as the amount of enzyme that could transfer 1 μmol of N-acetyl-2-amino sugar (Bbhl: GlcNAc; LnbB: LNB) per min to lactose under the conditions described above. One unit (1 U) of secondary hydrolysis activity was defined as the amount of enzyme that could release 1 μmol of N-acetyl -2-amino sugar (Bbhl: GlcNAc; LnbB: LNB) per min from the product formed under the conditions described above. For R.sub.TH analysis, specific activities for total (productive and non-pro-ductive) GlcNAc-β-pNP or LNB-β-pNP hydrolysis (based on pNP released) and trans-glycosylation were calculated from initial rate data obtained at 400-600 of lactose. The difference gave the specific activity for non-productive donor substrate hydrolysis (primary hydrolysis). For the oxazoline substrates, the total donor hydrolysis could only be calculated based on initial rate data of release of N-acetyl-2-amino sugar (Bbhl: GlcNAc; LnbB: LNB) for the variants of Bbhl and LnbB having their catalytic Asp replaced by Glu. In all other cases, the R.sub.TH values were estimated based on endpoint measurements at maximum LNT II or LNT yield.

Example 7: Preparative-Scale Synthesis of LNT II

[0142] In order to allow bulk synthesis of LNT II, its synthesis from GlcNAc-oxazoline by the D746E glycosidase mutant of Bbhl had to be optimized with respect to the donor-to-acceptor ratio applied. Increasing concentrations of GlcNac-oxazoline (130-600 mM) were applied at a constant lactose concentration of 600 mM and the conversions compared at 37° C. (FIG. 11a, FIG. 12). The adjustment of the donor-to-acceptor ratio to 1 had no negative impact on the final yield (85-90%). FIG. 11a shows synthesis of ˜1 g of LNT II in a batch volume of only 3.6 mL under the optimized conditions. The initial LNT II production rate was 2190 g L.sup.−1 h.sup.−1. LNT II was obtained in excellent yield (85%) and concentration (281 g L.sup.−1, 515 mM) within 30 min of reaction. The STY of the biotransformation overall was 562 g L.sup.−1 h.sup.−1. The mass-based turnover number (g product formed per g enzyme added; TTNmass) reached a value of 388. Only marginal product hydrolysis was observed under the reaction conditions applied. The ratio of transglycosylation over secondary hydrolysis reached a value of ˜1800. When the D746E variant and the wild-type enzyme were assayed under exactly the same conditions (using one-tenth of the enzyme concentration compared to the bulk synthesis described above), one sees clearly the benefits of the new glycosidase, namely no secondary hydrolysis and doubling of the LNT II yield (FIG. 11b). The initial LNT II production rates of the two enzymes were comparable, but the conversion with the wild-type drastically slowed down already after 5 min while it remained constant over ˜0.5 h with the mutant. Note, 600 mM of each substrate was the upper concentration limit used in the reaction, allowing their full solubility. LNT II was obtained in 80% yield without any detectable hydrolysis of the product (FIG. 13a). Under these conditions, the wild-type yielded ˜50% of LNT II and showed also an improved trans-glycosylation to secondary hydrolysis ratio of ˜40 (FIG. 13b).

[0143] For DSP of LNT II, the reaction was stopped by heating when no further increase in product concentration was detected. The sample contained only 85 mM of GlcNAc and lactose next to 515 mM of LNT II. Therefore, a simple DSP, including centrifugation for enzyme removal and freeze-drying for water removal, was sufficient to isolate LNT II in a purity of ˜80% (based on LNT II content of the final product, FIGS. 11c,d). The main residual impurities were 5% (w/w) GlcNAc and 10% (w/w) lactose. If a higher product purity is required, nanofiltration could be used for removal of GlcNAc and lactose from the LNT .sup.36 About 1 g of LNT II was obtained as a white powder in ≥85% yield. LNT II was thus prepared from GlcNAc-oxazoline in 73% overall yield. Product identity was unequivocally confirmed by .sup.1H and .sup.13C NMR spectroscopy (FIGS. 14 and 15). NMR spectra were in accordance with published data..sup.5 LNT II was the only regioisomer detected (FIG. 15). Overlay of NMR spectra (.sup.13C, HSQC) of isolated LNT II (FIG. 15) and commercial standard showed exact match of the signal at 82 ppm, characteristic for β-GIcNAc linked to the C-3 position of the β-galactosyl residue of lactose.

[0144] The enzymatic conversion was carried out at pH 7.5 and 37° C. in a total volume of 3.6 mL, using equimolar amounts of GlcNAc-oxazoline and lactose (600 mM). Reaction was started by adding 0.73 mg mL.sup.−1 (4 μM) of Bbhl D746E variant. The conversion was performed in a 50 mL Sarstedt tube (diameter 2.8 cm, height 11.5 cm) under magnetic stirring (stir bar: 18×5 mm; 250 rpm). For temperature control, the Sarstedt tube was placed in a water bath. Samples were taken at certain times and analyzed by HPLC.

[0145] For downstream processing (DSP) of LNT II, enzyme was precipitated after 45 min reaction time by heating for 15 min at 99° C. Precipitated protein was removed by centrifugation at 13,200 rpm for 15 min. Sample was frozen at −70° C. and freeze-dried overnight (Christ Alpha 1-4, B. Braun Biotech International, Melsungen, Germany). The final product was analyzed by HPLC and its chemical identity confirmed by NMR.

Example 8: Synthesis of LNT II by Fed-Batch Addition of GIcNAc-oxazoline

[0146] 1.8 mmol GlcNAc-oxazoline was dissolved in 1.3 mL ice-cold water and added continuously over a period of 65 min to the reaction solution containing 2.1 mmol of lactose and 0.3 mg (0.4 βM) of Bbhl D746E in 2.3 mL of phosphate buffer pH 7.5. The enzymatic conversion was carried out at 37° C. under magnetic stirring. For temperature control, the reaction tube was placed in a water bath. Samples were taken at certain times and analyzed by HPLC. Formation of the product was demonstrated. The reaction was terminated after 4 h by heat deactivation of the enzyme.

Analytics

[0147] LNT II, LNT, GlcNAc, LNB, pNP and lactose were analyzed by HILIC-HPLC using a Luna® NH.sub.2 column (3 μm, 100 Å, 250×4.6 mm; Phenomenex, Germany). HPLC analysis was performed at 30° C. with a mobile phase of 75% acetonitrile and 25% water at an isocratic flow rate of 1 mL min.sup.1. UV-detection at 195 nm was used for quantification of LNT II, LNT, GlcNAc, LNB and pNP. For preparative synthesis of LNT II, lactose was monitored by refractive index (Rl) detection.

[0148] TLC was performed on silica gel 60 F254 aluminum sheet (Merck, Germany). The plate was developed in a solvent system of 1-butanol—acetic acid—water (2/1/1 by volume). TLC plates were analyzed under UV light (254 nm). Then carbohydrates were visualized by heating the plate after spraying it with thymol—sulfuric acid reagent.

[0149] LNT II, LNT, GlcNAc, LNB, pNP and lactose were used as authentic standards.

[0150] Varian (Agilent) INOVA 500-MHz NMR spectrometer (Agilent Technologies, Santa Clara, Calif., USA) and the VNMRJ 2.2D software were used for all NMR measurements. Dimethyl sulfone and succinonitrile were used as internal standards for quantitative .sup.1H NMR measurements. 19.08 mg of GlcNAc-oxazoline (lyophilized crude product) and 11.28 mg dimethyl sulfone were dissolved in D2O. 11.41 mg of LNB-oxazoline (lyophilized crude product) and 12.85 mg succinonitrile were dissolved in D.sub.2O. ˜200 mg of isolated LNT II were dissolved in 600 μL D20. Commercial LNT II standard (from Carbosynth; 65 mM) was dissolved in D.sub.2O -H.sub.2O (11.5:1 v/v). .sup.1H NMR spectra (499.98 MHz) were measured on a 5 mm indirect detection PFG-probe, while a 5 mm dual direct detection probe with z-gradients was used for .sup.13C NMR spectra (125.71 MHz). Standard pre-saturation sequence was used: relaxation delay 2 s; 90° proton pulse; 2.048 s acquisition time; spectral width 8 kHz; number of points 32 k. .sup.13C NMR spectra were recorded with the following pulse sequence: standard .sup.13C pulse sequence with 45° carbon pulse, relaxation delay 2 s, Waltz decoupling during acquisition, 2 s acquisition time. The HSQC spectrum was measured with 128 scans per increment and adiabatic carbon 180° pulses. Mnova 9.0 was used for evaluation of spectra.

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