Discovery of solabiose phosphorylase and its application for enzymatic synthesis of solabiose from sucrose and lactose

Glycoside phosphorylases (GPs), which catalyze the reversible phosphorolysis of glycosides, are promising enzymes for the efficient production of glycosides. Various GPs with new catalytic activities are discovered from uncharacterized proteins phylogenetically distant from known enzymes in the past decade. In this study, we characterized Paenibacillus borealis PBOR_28850 protein, belonging to glycoside hydrolase family 94. Screening of acceptor substrates for reverse phosphorolysis, in which α-d-glucose 1-phosphate was used as the donor substrate, revealed that the recombinant PBOR_28850 produced in Escherichia coli specifically utilized d-galactose as an acceptor and produced solabiose (β-d-Glcp-(1 → 3)-d-Gal). This indicates that PBOR_28850 is a new GP, solabiose phosphorylase. PBOR_28850 catalyzed the phosphorolysis and synthesis of solabiose through a sequential bi-bi mechanism involving the formation of a ternary complex. The production of solabiose from lactose and sucrose has been established. Lactose was hydrolyzed to d-galactose and d-glucose by β-galactosidase. Phosphorolysis of sucrose and synthesis of solabiose were then coupled by adding sucrose, sucrose phosphorylase, and PBOR_28850 to the reaction mixture. Using 210 mmol lactose and 280 mmol sucrose, 207 mmol of solabiose was produced. Yeast treatment degraded the remaining monosaccharides and sucrose without reducing solabiose. Solabiose with a purity of 93.7% was obtained without any chromatographic procedures.

www.nature.com/scientificreports/ the glycosidic oxygen of the substrate, and the inorganic phosphate attacks anomeric carbon to produce sugar phosphate in an inverted anomeric configuration. Rapidly increasing information on genome sequences has made it possible to screen novel enzymes based on the phylogenetic analysis of putative proteins. In GH94, CBAP and 12BOGP were discovered using this approach 11,12 . A putative GH94 protein PBOR_28850 (GenBank accession number, AIQ60507.1) from Paenibacillus borealis DSM 13188 is phylogenetically distant from any characterized enzymes of GH94, and its sequence identity with the characterized GH94 enzymes is 11.5-29.2% (Fig. 1). In this study, we characterized the enzymatic functions of PBOR_28850 and found that it can phosphorolyze solabiose (β-d-Glcp-(1 → 3)-d-Gal), which was originally identified in the sugar part of solanine from Solanum tuberosum 13 .

Results
Screening of substrates with PBOR_28850. Recombinant PBOR_28850 with a C-terminal His-tag (6 His residues were attached to Met768) was produced in an Escherichia coli transformant and purified via Ni-affinity and gel filtration column chromatography. From E. coli cells harvested from 1 L of the culture broth, 38.9 mg of purified protein was obtained. The purified PBOR_28850 showed a single band of 82 kDa on an SDS-polyacrylamide gel (Fig. 2). This molecular mass matched well with the value estimated from the amino acid sequence (85.9 kDa).
In the synthetic reactions with α-d-glucose 1-phosphate (α-Glc1P) and various sugars, PBOR_28850 liberated inorganic phosphate only in the presence of d-galactose. The rate of the reaction with d-galactose and α-Glc1P was 3.23 ± 0.23 s −1 at 30 °C and pH 6.5, while that for the other acceptor sugars tested was < 0.1 s −1 .
Isolation and structural analysis of the reaction product using PBOR_28850. The reaction of PBOR_28850 with α-Glc1P and d-galactose was monitored by measuring the amount of inorganic phosphate liberated at the same molar concentration as the sugar product (Fig. 3a). The inorganic phosphate concentration reached 65 mM after 60 h. Supplementation of the enzyme at this point slightly increased the inorganic phosphate concentration to 70 mM after further incubation for 8 h (68 h from the reaction start). The inorganic phosphate concentration remained stable during the reaction period from reaction 68 h to 87 h (71 mM at 87 h).
The reaction product from α-Glc1P and d-galactose was produced at an 11 mL scale (1.1 mmol of α-Glc1P and d-galactose). The concentration of the liberated inorganic phosphate was 60 mM after a 24 h reaction. From this  www.nature.com/scientificreports/ reaction mixture, 200 mg of the product was purified via gel-filtration column chromatography. The purity of the product was 99.4%, which was estimated from the relative peak area in the HPLC analysis (Fig. 3b). The signal at 365.11 m/z [M + Na] + was obtained by electrospray ionization mass spectrometry analysis, and the product was determined to be a disaccharide. In the heteronuclear multiple bond correlation spectroscopy analysis of the product, correlation signals between H-1 of the d-glucosyl residue and C-3 of the d-galactose residue, and between C-1 of the d-glucosyl residue and H-3 of the β-d-galactose residue were detected (Fig. 3c). This indicates that the glycosidic linkage was formed between C-1 of the d-glucosyl residue and C-3 of the d-galactose residue.
As the J value of H-1 of the d-glucosyl residue was 7.55 Hz, the d-glucosyl residue was linked to d-galactose through a β-linkage. Thus, the reaction product was determined to be solabiose (β-d-Glcp-(1 → 3)-d-Gal). The molar ratio of the α-and β-anomers of solabiose, estimated from the signal intensity of H-1 of the reducing-end d-galactose residue in 1 H-NMR, was 1:3. This molar ratio is consistent with that of d-galactose 14  Enzymatic characteristics of PBOR_28850. The optimum pH of PBOR_28850 for the phosphorolysis and synthesis of solabiose was determined based on the reaction velocities at various pH values. PBOR_28850 showed the highest activity at pH 7.6 and pH 7.8 in the phosphorolysis and synthesis of solabiose, respectively (Fig. 4). This enzyme retained more than 85% of its original activity in a pH range of 6.5-7.4 (at 4 °C for 24 h) and below 25 °C (at pH 8.0 for 15 min). As PBOR_28850 produced inorganic phosphate linearly during the 10 min reaction at 30 °C with 1 mM α-Glc1P and d-galactose (the lowest substrate concentration used in this study), kinetic analysis was conducted at 30 °C. The reaction velocity for the phosphorolysis and synthesis of solabiose was measured at various substrate concentrations (Fig. 5). The reaction rates for both directions of the reaction obeyed the rate equation of the sequential bi-bi mechanism. Kinetic parameters for the phosphorolysis of solabiose were calculated to be: k cat , 9.14 ± 0.11 s -1 ;  www.nature.com/scientificreports/ PBOR_28850 were coupled (Fig. 3d). After a reaction time for 120 h, almost all the d-galactose was consumed, and the concentration of solabiose reached 259 mM (207 mmol), indicating that the yield of solabiose was 98.6% (Fig. 3e). Yeast treatment was also performed, which showed that yeast consumed d-fructose, d-glucose, and sucrose in the reaction mixture without decreasing the solabiose content. The purity of solabiose in the reaction mixture increased from 44.2% (after the reaction for 120 h) to 93.7% (Fig. 3b).
Prediction of substrate binding mechanism of PBOR_28850. To predict the substrate-binding mechanism, a model structure of PBOR_28850 was constructed using AlphaFold 15 . This model structure was superimposed onto Saccharophagus degradans CBAP in complex with 3-O-β-d-glucopyranosyl-α-dglucopyranuronic acid (PDB entry, 4ZLI) 16 (Fig. 6). The residues of PBOR_28850, responsible for the catalysis (Asp456) and substrate binding in subsite − 1 (Arg342, Asp343, Trp454, Tyr609, His611, and Thr676) are predicted to be spatially similar to the corresponding residues of CBAP. These residues are well conserved in GH94 enzymes (Asp343 is substituted by Asn350 in the CBAP, but most GH94 enzymes have Asp at this position) (Fig. 7). Consistent with the diversity of substrate specificity of GH94 enzymes, amino acid residues forming subsite + 1 are less conserved. To predict the binding mode of d-galactose in subsite + 1 of PBOR_28850, β-d-galactose in ideal form (PDB entory, GAL) was superimposed onto the α-d-glucopyranuronic acid residue bound to subsite + 1. Trp603 and Phe672 are predicted to be suitably located to interact with C-6 of the d-galactose residue of solabiose in subsite + 1. The axial hydroxy group at the C-4 position of d-galactose points to Thr337. These residues are conserved in the homologous proteins of PBOR_28850 from Paenibacillus species (Fig. 7). Open symbols indicate activity of reverse phosphorolysis. Circles, triangles, squares, and diamond show the reaction in sodium acetate buffer, MES-NaOH buffer, HEPES-NaOH buffer, and glycine-NaOH buffer, respectively. Closed triangles show residual activity after the pH treatment at pH 4.

Discussion
GPs are promising biocatalysts for the efficient production of oligosaccharides. However, the number of known GPs is small; therefore, the discovery of new GPs is desirable. Owing to the rapid increase in genome information, GPs with new catalytic activity have recently been discovered from uncharacterized proteins that are phylogenetically distant from known enzymes 7,11,12,[17][18][19][20][21][22][23][24] . In this study, we conducted biochemical characterization of an uncharacterized GH94 protein, PBOR_28850, from P. borealis, and identified this protein as a new GP, solabiose phosphorylase. Solabiose was first isolated from the partial acid hydrolysate of Solanum tuberosum 13 . This disaccharide was also prepared from the extracellular polysaccharide of phytopathogenic Xanthomonas spp. 25 and lipid-bound sugars from Rhizobium meliloti 26   Substrate binding mechanism of PBOR_28850 was predicted based on the structural comparison of the model structure of PBOR_28850 and the crystal structure of the complex of S. degradans CBAP and 3-O-β-dglucopyranosyl-α-d-glucopyranuronic acid. The residues of PBOR_28850, involved in the catalysis, phosphate binding, and sugar binding in subsite − 1, are conserved well as in GH94 enzymes, and predicted to be arranged similarly to those of the compared enzyme. This indicates that PBOR_28850 obeys the common catalytic mechanism of GH94 enzymes. In the prediction of d-galactose binding in subsite + 1 of PBOR_28850, Thr337 and two aromatic residues Trp603 and Phe672 are found as candidate binding residues. Considering that PBOR_28850 is very specific to d-galactose as acceptor substrate of the reverse phosphorolysis (this enzyme does not use d-glucose as acceptor), PBOR_28850 strictly recognizes axial 4-OH of d-galactose in subsite + 1 or equatorial 4-OH of d-glucose causes steric hindrance upon binding to subsite + 1. Since no amino acid residue, which causes steric hindrance against 4-OH of d-glucose, is not found in the model structure of PBOR_28850, Thr337, situated close to 4-OH of d-galactose, presumably has an essential interaction with d-galactose in subsite + 1.
In this study, we established an efficient enzymatic synthesis of solabiose from sucrose and lactose using PBOR_28850. As PBOR_28850 had very high acceptor specificity to d-galactose, solabiose was specifically produced from α-Glc1P and d-galactose, even in the presence of d-glucose and d-fructose as byproducts. As solabiose was not utilized as a carbon source for yeast, high purity solabiose was successfully obtained without any column chromatographic procedures. The yield of solabiose by this enzymatic synthesis is much higher than that of organic synthesis, as established previously 28 . Benzyl 2,6-di-O-acetyl-3-O-(2,3,4,6-tetra-O-acetylβ-d-glucopyranosyl)-β-d-galactopyranoside was synthesized at a yield of 34% from benzyl 2,6-di-O-acetyl-βd-galactopyranoside and tetra-O-acetyl-α-d-glucopyranosyl bromide, and solabiose was obtained from this compound through the removal of acetyl and benzyl groups. Furthermore, enzymatic synthesis is much simpler than organic synthesis and does not require any harmful reagents. The enzymatic synthesis of solabiose can easily be scaled up, making it possible to provide solabiose for physiological analysis in animals, plants, and microorganisms. The beneficial physiological functions of solabiose can be identified through such biological analyses.

Methods
Materials. d-Allose, d-glucose, α-Glc1P, d-gluconic acid, lactose, d-mannose, and d-xylose were purchased from Fujifilm Wako Pure Chemical (Osaka, Japan); N-acetyl-d-glucosamine, d-galactose, and sucrose were purchased from Nacalai Tesque (Kyoto, Japan); d-glucosamine was purchased from Tokyo Chemical Industry (Tokyo, Japan); cellobiose, d-galacturonic acid, and d-glucuronic acid were purchased from Sigma (St. Louis, MO, USA). β-(1 → 4)-Mannobiose was prepared as previously described 29 . Lactoless L3 (β-galactosidase) was provided by Daiwa Kasei (Shiga, Japan). SP from Bifidobacterium longum was prepared according to the method described by Nishimoto and Kitaoka 5 . One unit of SP was defined as the amount of enzyme required to phosphorolyze 1 μmol of sucrose in 1 min. SP activity was measured as follows: a reaction mixture (50 μL) containing appropriate concentration of enzyme, 20 mM sucrose, 40 mM sodium phosphate, 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-NaOH buffer (pH 7.0), and 0.2 mg/mL bovine serum albumin (BSA, Nacalai Tesque) was incubated at 37 °C for 10 min. The enzymatic reaction was terminated by incubating the sample at 80 °C for 3 min, and the liberated d-fructose was measured using a d-Fructose/d-Glucose Assay Kit (Megazyme, Brey, Ireland). Transformants of E. coli BL21 (DE3) harboring the expression plasmid were cultured in 1 L of LB medium, containing 100 μg/mL ampicillin, at 37 °C until A 600 reached 0.5. Production of the recombinant protein was induced by adding 0.1 mM isopropyl β-thiogalactoside, followed by incubation with vigorous shaking at 18 °C for 24 h. Bacterial cells, harvested via centrifugation (9600 × g, 4 °C, 10 min), were disrupted by sonication, and cellfree extracts were obtained by centrifugation (9600 × g, 4 °C, 10 min). Recombinant PBOR_28850 was purified from the extract via Ni affinity column chromatography using Chelating Sepharose Fast Flow (GE Healthcare, Uppsala, Sweden; 2.6 cm i.d. × 2.0 cm) equilibrated with 20 mM imidazole-HCl buffer (pH 7.0) containing 0.5 M NaCl. The adsorbed protein was eluted using a linear gradient of 20-500 mM imidazole (total elution volume, 200 mL) after elution of the non-adsorbed protein with the equilibration buffer. The collected sample was concentrated to 7.7 mg/mL by ultrafiltration using a Vivaspin YM-30 concentrator (Sartorius, Göttingen, Germany) and separated via gel filtration column chromatography using a Toyopearl HW-55S column (Tosoh, Tokyo, Japan; 2.6 cm i.d. × 100 cm) equilibrated with 10 mM HEPES-NaOH buffer (pH 7.0). Concentration of the purified enzyme was determined via amino acid analysis. Preparation and structural analysis of oligosaccharide products. A reaction mixture (11 mL) containing 120 nM PBOR_28850, 0.1 M α-Glc1P, 0.1 M d-galactose, and 50 mM HEPES-NaOH buffer (pH 8.0) was incubated at 30 °C for 24 h. The oligosaccharide product was purified via gel filtration column chromatography using a Toyopearl HW-40S column (5.0 cm i.d. × 100 cm). Water was used as the mobile phase. The pooled fractions were desalted with Amberlite MB-4 (Organo, Tokyo, Japan) and lyophilized. The molecular masses of the products were measured via electrospray ionization mass spectrometry using an Exactive mass spectrometer (Thermo Scientific, San Jose, CA, USA). The sample was applied to a mass spectrometer by flow injection, using methanol as the mobile phase solvent. The positive ion was detected under following conditions: spray voltage, 3.00 kV; capillary temperature, 300 °C. NMR spectra were recorded in D 2 O (Sigma) at 27 °C using a Bruker AMX500 spectrometer (500 MHz; Bruker, Billerica, MA, USA). A series of two-dimensional homo-and heteronuclear correlated spectra (correlated spectroscopy, heteronuclear single quantum correlation spectroscopy, heteronuclear single quantum correlation total correlation spectroscopy, heteronuclear 2-bond correlation spectroscopy, and heteronuclear multiple bond correlation spectroscopy) were acquired to determine chemical structures of the reaction products.
Standard enzyme assay. A reaction mixture (50 μL) containing an appropriate concentration of enzyme, 20 mM α-Glc1P, 20 mM d-galactose, and 0.2 M HEPES-NaOH buffer (pH 8.0) was incubated at 30 °C for 10 min. The reaction was terminated by incubating the sample at 80 °C for 3 min. The amount of liberated inorganic phosphate was measured as described above. One U of solabiose phosphorylase was defined as the amount of enzyme required to produce 1 μmol of solabiose in 1 min. The specific activity of purified PBOR_28850 was 8.90 U/mg. Optimum pH. The optimum pH of PBOR_28850 for the phosphorolysis and synthesis of solabiose was evaluated from the rate of the reaction at various pH values. The reaction rate for the phosphorolysis of solabiose was measured as follows: a reaction mixture (50 μL) containing 36.2 nM PBOR_28850, 20 mM solabiose, and 80 mM Britton-Robinson buffer (mixture of sodium acetate buffer, sodium phosphate buffer, and glycine-NaOH buffer; pH 5.0-8.4) was incubated at 30 °C for 10 min, and the generated d-galactose was measured using an l-Arabinose/d-Galactose Assay Kit (Megazyme) after stopping the reaction by heating at 80 °C for 3 min. Solabiose, synthesized from α-Glc1P and d-galactose, was used. The reaction rate for the synthesis of solabiose was measured as described above, except that sodium acetate buffer (pH 4.4-5.4), MES-NaOH buffer (pH 5.8-6.8), HEPES-NaOH buffer (pH 6.8-8.2), and glycine-NaOH buffer (pH 8.6) were used as reaction buffers.
Stability range of pH and temperature. The stability range of pH and temperature was determined from the residual activity after the pH and temperature treatments, respectively. For the pH treatment, 9. www.nature.com/scientificreports/ MES-NaOH buffer (pH 6.5), HEPES-NaOH buffer (pH 7.4-8.3), and glycine-NaOH buffer (pH 9.4) were used. For the temperature treatment, 201 nM PBOR_28850 was incubated in 336 mM HEPES-NaOH buffer (pH 8.0) containing 0.33 mg/mL BSA at 15-40 °C for 15 min. Residual activity was measured using the standard enzyme assay. The stability range was defined as the range in which the enzyme retained more than 85% of its original activity.
Kinetic parameters for the phosphorolysis and synthesis of solabiose. For the phosphorolysis of solabiose, 50 μL of a reaction mixture containing 72.4 nM PBOR_28850, 1-16 mM solabiose, and 1-16 mM sodium phosphate, 0.2 M HEPES-NaOH buffer (pH 7.5), and 0.2 mg/mL BSA was incubated at 30 °C for 10 min, and the generated d-galactose was measured as described above. For the synthesis of solabiose, the rate of release of inorganic phosphate from 1 to 16 mM α-Glc1P and 1-16 mM d-galactose was measured using the standard enzyme assay. The reaction equation for the sequential bi-bi mechanism (Eq. 1) was fitted to the reaction rates at various substate concentrations using Grafit version 7.0.2 (Erithacus Software, East Grinstead, UK).

Synthesis of solabiose from lactose and sucrose.
A reaction mixture (700 mL) containing 300 mM lactose, 20 mM sodium phosphate buffer (pH 7.0), and 2% (v/v) Lactoless L3 was incubated at 30 °C for 21 h, and the reaction was stopped by heating the sample up to 85 °C using microwaves. After cooling the sample, 95.8 g of sucrose, 7 mL of 153 U/mL SP, and 20 mL of 44.8 U/mL PBOR_28850 were added to the mixture (increasing the volume of the reaction mixture to 800 mL) and the reaction mixture was incubated at 30 °C for 120 h. To consume the remaining sucrose and monosaccharides in the reaction mixture, 4.6 g of dry yeast (Nisshin Foods, Tokyo, Japan) was added, and the sample was incubated at 30 °C for 24 h with gentle shaking. The supernatant was obtained by centrifugation (9600 × g, 4 °C, 10 min) and decolored by incubation at 60 °C for 2 h in the presence of 3 g/L of active carbon (Nacalai Tesque). The sample was filtered with Celite No. 545 (Fujifilm Wako Pure Chemical) and deionized using Amberlite MB-4. The sample was filtrated through a 0.45 μm membrane filter (Advantec, Tokyo, Japan) and concentrated to 200 mL in vacuo.

Data availability
The datasets generated and/or analyzed in this study are available from the corresponding author upon reasonable request.