Preparative and kinetic assessment of β-1,4- and β-1,3-glucan phosphorylases informs access to human milk oligosaccharide fragments and analogues thereof.

Abstract The enzymatic synthesis of oligosaccharides depends on the availability of suitable enzymes, which remains a limitation. Without recourse to enzyme engineering or evolution approaches, herein we demonstrate the ability of wild‐type cellodextrin phosphorylase (CDP: β‐1,4‐glucan linkage‐dependent) and laminaridextrin phosphorylase (Pro_7066: β‐1,3‐glucan linkage‐dependent) to tolerate a number of sugar‐1‐ phosphate substrates, albeit with reduced kinetic efficiency. In spite of catalytic efficiencies of <1 % of the natural reactions, we demonstrate the utility of given phosphorylase–sugar phosphate pairs to access new‐to‐nature fragments of human milk oligosaccharides, or analogues thereof, in multi‐milligram quantities.


General methods
All chemicals including sugar-1-phosphates were purchased from Sigma-Aldrich (UK) and used as received, unless otherwise stated. Oligosaccharide substrates for the enzymatic reactions were purchased from Carbosynth Limited (Berkshire UK) or Megazyme (Wicklow, Ireland), unless otherwise noted. Milli-Q H2O was used for the preparation of aqueous buffers. All reagents and solvents used for analytical applications were of analytical quality. TLC was performed on Silica Gel 60 F254 (Merck). Compounds were visualised by spraying TLC with orcinol solution (20 mg/ml orcinol monohydrate in EtOH-H2SO4-H2O 75:10:5, v/v/v), followed by heating.
Product characterisation. Reaction products were characterised by TLC, MALDI-ToF MS on a Bruker Autoflex Speed spectrometer using flexControl -autoflex TOF/TOF and flexAnalysis software or by low resolution ESI MS on an Advion Expression Compact Mass Spectrometer (CMS). High-resolution ESI MS were obtained on a Waters Synapt G2-Si mass spectrometer. 1 H, 13 C-DEPT, 2D-COSY and 2D-HSQC NMR spectra were recorded on Bruker Avance III 400 MHz spectrometer equipped with a broadband BBFO probe or on Bruker Neo 600 MHz spectrometer equipped with a TCI cryoprobe. Chemical shifts (δ) are reported in parts per million (ppm) using residual solvent signal for referencing. Colorimetric assays were performed in NUNC 96 plates on a BMG Labtech FLUOStar Omega microplate reader equipped with suitable absorbance filters.
Sample preparation for analysis of products of enzymatic reactions by MALDI ToF. Reaction mixture (5 µl) was diluted with Milli-Q H2O (5 µl), TMD-8 (Sigma) mixed bed resin was added, followed by incubation at room temp. for 5 min. Samples were typically mixed with equal volume of2,5-dihydroxybenzoic acid (DHB) matrix (10 mg/ml in 30% acetonitrile + 0.1% TFA in H2O) and spotted on a target plate (Bruker MTP 384 Polished Steel TF Target), then analysed in positive mode.
HRMS analysis procedure. Samples were diluted into 50% methanol-0.1% formic acid and infused into the mass spectrometer at 5-10 μl/min using a Harvard Apparatus syringe pump. The mass spectrometer was controlled by Masslynx 4.1 software (Waters). It was operated in high-resolution positive ion mode and calibrated using sodium formate. The sample was analysed for 2 min with 1 s MS scan time over the range of 50-1200 m/z (or as appropriate) with 3.5 kV capillary voltage, 40 V cone voltage, 100 °C cone temperature. Leu-enkephalin peptide (1 ng/ml, Waters) was infused at 10 μl/min as a lock mass (m/z 556.2766) and measured every 10 s. Spectra were generated in Masslynx 4.1 by combining several scans, and peaks were centred using automatic peak detection with lock mass correction.
Purification techniques. Ion exchange chromatography was performed using Bioscale™ Mini Macro-Prep cartridges (Bio-Rad, UK), High Q for neutral oligosaccharides and High S for glucosamine-containing compounds respectively, and a step-gradient from 0 to 1 M ammonium bicarbonate buffer (pH 9.4). Product containing fractions were combined and reduced to dryness. The residue was co-evaporated repeatedly with methanol to remove residual ammonium bicarbonate. Gel Filtration Chromatography was performed with a Perkin Elmer series 200 HPLC, equipped with a refractive index detector and a fraction collector, using as stationary phase a Toyopearl TSK-HW40S column (100 cm × 2.2 cm) at 40 °C. Products were eluted with Milli-Q H2O at a flow-rate of 0.5 ml/min and collected in 2 mL fractions. If necessary, further purification was carried out by solid phase extraction using C-18 Sep-Pak cartridges (Waters) with a gradient elution of acetonitrile against Milli-Q H2O. Other purification methods are specified in appropriate sections below.

Recombinant protein production
A recombinant plasmid (pET15b) containing the CDP gene from Ruminiclostridium thermocellum YM4 strain (GenBank accession number BAB71818) was transformed into E. coli BL21 (DE3) cells and the transformant grown according to the literature. 1 Briefly, transformant in 1 L of LB medium containing appropriate antibiotic (carbenicillin, 100 μg/ml) was incubated at 37 °C with gentle shaking until an OD600 of about 0.6. Heterologous protein expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) at 1 mM final concentration, followed by incubation at 30 °C for 4 hours at 180 rpm. Afterwards, cells were harvested by centrifugation (4000 × g, 4 °C, 20 min), and then re-suspended in lysis buffer (50 mM HEPES, pH 7.5, 100 mM NaCl, 1 × Complete™ EDTA-free Protease Inhibitor Cocktail Tablet (Roche), 0.02 mg/ml DNaseI). The cells were lysed by sonication (Vibra-Cell™ Ultrasonic liquid processor) and the cell debris removed by centrifugation (20 000 × g, 4 °C, 30 min). Proteins in the supernatant were purified at 4 °C using an ÄKTA pure FPLC system (GE Healthcare). The supernatant was passed through a HisTrap TM HP column (5 ml, GE healthcare), preequilibrated with buffer A (50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 20 mM imidazole). Unbound proteins were washed with five column volumes of buffer A, followed by elution with buffer B (50 mM Tris-HCl (pH 8), 500 mM NaCl, 500 mM imidazole). Further purification was achieved by gel filtration chromatography using a Superdex S200 16/600 column (GE Healthcare) eluted with 50 mM HEPES (pH 7.5) and 150 mM NaCl, at the flow rate of 1 ml/min. CDP comprising fractions were pulled together and concentrated using Amicon Ultra-15 Centrifugal Filter Units (30,000 MWCO). A recombinant plasmid (pET28a) containing laminarin phosphorylase (Pro_7066) was kindly supplied by Prozomix limited, which was transformed into E. coli BL21 (DE3) cells, and the transformant grown according to the literature. 2 Briefly, 1 L of transformant in LB medium containing appropriate antibiotic (kanamycin, 50 μg/ml) was incubated at 22 °C overnight at 180 rpm. Heterologous protein expression was induced by adding IPTG to a final concentration of 0.2 mM, and further incubated at 18 °C overnight at 180 rpm. Harvesting of cells and purifications were achieved as previously described for CDP. Concentration of both proteins was determined by NanoDrop TM spectrophotometer (Thermo Fisher Scientific, UK). Concentrated enzymes were divided into aliquots and stored at -80°C until required.

Phosphorylase activity assay
Optimization of enzymatic reaction conditions and kinetic measurements were performed measuring the inorganic phosphate (Pi) released from the sugar-1-phosphate in the phosphorylase synthetic reaction. Released Pi was measured with a colorimetric assay modified by De Groeve et al. 3 Briefly, 25 µl of reaction mixture containing 200 mM sodium molybdate (Na2MoO4 · 2H2O) were added to 75 µl of a colour solution (0.24% [w/v] sodium ascorbate dissolved in 0.1 N HCl solution) and the reaction incubated at room temp. until colour developed. The colour reaction was stopped by adding 75 µl of stop solution (2% [w/v] sodium citrate tribasic dihydrate in 2% [v/v] acetic acid). Absorbance at 620 nm (A620) was measured using NUNC 96 well plates on a BMG Labtech FLUOStar Omega microplate reader.

Optimization of enzymatic reactions for the synthesis of terminal galactoside oligosaccharides
Optimum pH and temperature for the synthetic enzymatic reactions of CDP and Pro_7066 with galactose-1phosphate (Gal1P) were established carrying out the colorimetric assay measuring the inorganic phosphate (Pi) released in the reaction (see section above). The enzymatic reactions were set up in 100 µl of reaction cocktail containing acceptor (8 mM, cellobiose (1) for Pro_7066 and laminaribiose (4) for CDP, respectively), donor (25 mM), enzymes (0.1 mg/ml) and buffer (50 mM) (all concentrations are final concentrations). Sodium citrate (pH 3 to 6), HEPES (pH 7 and 8) and Tris-HCl (pH 9) at 50 mM were screened for determining optimum pH of both phosphorylases. The enzymatic reactions were tested at three different temperatures (22, 37 and 55 °C) to establish the optimum temperature. All reactions were incubated over 24 h and released Pi was periodically measured by colorimetric assay (see Section 3). A phosphate standard curve was made using Na2HPO4 ranging from 0.5 to 2.5 mM for calculating released Pi from above reactions. Reactions were performed in 100 µl of reaction cocktail containing acceptor (8 mM, cellobiose (1) for Pro_7066 and laminaribiose (4) for CDP, respectively), Gal1P (25 mM), enzymes (0.16 mg/ml) and buffer (50 mM). Sodium citrate (pH 3 to 6), HEPES (pH 7 and 8) and Tris-HCl (pH 9) at 50 mM were used for determining optimum pH (all concentrations are final concentrations). In control reactions, enzymes were heat killed prior addition into the reaction. Reactions were incubated up to 24 hours. IP = inorganic phosphate.

Pro_7066-catalysed reactions of Gal1P with cognate acceptors 3 and 4
The reactions were performed using Gal1P as donor and Glc-β-1-3-Glc-β-1-4-Glc (3) or laminaribiose (4) as acceptors in a ratio ranging from 16:1 to 1:2, with Pro_7066 at 0.125 mg/ml in 25 mM HEPES buffer (pH 7) (all concentrations are final concentrations). Reaction mixtures were incubated at 37 °C overnight with shaking at 300 rpm leading to the formation of a number of oligosaccharide products as judged by TLC analyses. To decrease a proportion of high-molecular weight oligosaccharides formed both reactions were performed using a 1:1 donor to acceptor ratio, incubated at 37 °C overnight and stopped by heating at 95 °C for 5 min. Reaction mixtures were initially purified by ion exchange chromatography for removal of residual Gal1P/Glc1P, and then freeze dried. The crude mixture (25 mg) of products was then incubated with β-1-3-glucanase (25 U/ml, Megazyme) in 50 mM sodium acetate buffer (1 ml, pH 5). Progress of these reactions was monitored by TLC and MALDI, as shown in Fig. S3. Finally, the crude mixtures were purified by gel filtration chromatography as described in Section 1.

CDP and Pro_7066 reactions with non-cognate acceptors and donors
This general protocol describes reactions between Gal1P, ClcN1P and Man1P and a number of oligosaccharide acceptors as shown in Table 1 (main text). All reactions were performed using sugar 1-phosphate (25 mM), an acceptor (8 mM) and CDP (0.1 mg/ml) in 50 mM sodium citrate buffer (pH 5-6) or Pro_7066 (0.1 mg/ml) in 50 mM HEPES buffer (pH 7). Reactions were incubated at 37 °C with shaking at 300 rpm for 3 days keeping pH constant and adding extra amount of enzyme (10 µg) after 24 and 48 h. Reactions were stopped upon completion by heating at 95 °C for 5 min. Then, the reaction mixtures were first purified by ion exchange chromatography to remove residual sugar 1-phosphates, followed by gel filtration chromatography to isolate the final products as described in Section 1.

Influence of acceptor chain length on the enzymatic galactosylation of glucans
The impact of increasing degree of polymerization on efficiency of enzymatic galactosylation of glucooligosaccharide acceptors was evaluated in two series of experiments which aimed at measuring the released inorganic phosphate. In the first series, Gal1P (25 mM) and laminarioligosaccharide acceptors 4 -6 (8 mM) were incubated with CDP (0.1 mg/ml) in 50 mM sodium acetate buffer (pH 5). In the second series of experiments Gal1P (25 mM) was incubated with cellooligosaccharides acceptors 1, 3 and cellotetraose (8 mM

Kinetic analysis of donor specificities in reactions catalysed by CDP and Pro_7066
Kinetic analyses were performed for Pro_7066 and CDP with the 4 different donors, natural Glc1P, and unnatural Gal1P, GlcN1P and Man1P, in combination with the corresponding cognate and non-cognate acceptors, laminaribiose and cellobiose, respectively. All kinetic analyses were performed in the synthetic direction, measuring release of inorganic phosphate from sugar-1-P donor. The concentration of released Pi was measured colorimetrically, as described in Section 3.