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).
Preparation of recombinant PBOR_28850.
pbor_28850 was amplified from the genomic DNA of P. borealis DSM 13188 (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) using the primers (5′-ATGGCTGGCTTCGGCAATTTC-3′ and 5′-CTACATATCCACCTCTACAGTAT-3′) and KOD FX DNA polymerase (Toyobo, Osaka, Japan). The amplified DNA fragment was used as a template for subsequent PCR using the primers 5′-AAGAAGGAGATATACATATGGCTGGCTTCGGCAATTTC-3′ and 5′-CAGTGGTGGTGGTGGTGGTGCATATCCACCTCTACAGTAT-3′. The resulting DNA fragment was inserted into pET-23a (Novagene, Darmstadt, Germany) using the In-Fusion HD Cloning Kit (Takara Bio, Kusatsu, Japan). The inserted DNA and flanking regions were sequenced using Applied Biosystems 3130 Genetic Analyzer (Life Technologies, Carlsbad, CA, USA).
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 A600 reached 0.5. Production of the recombinant protein was induced by adding 0.1 mM isopropyl b-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 cell-free 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.
Screening of acceptor substrate.
The acceptor substrate of PBOR_28850 for reverse phosphorolysis was screened based on the rate of release of inorganic phosphate from α-Glc1P and various sugars. d-Allose, d-galactose, d-glucose, d-mannose, d-glucosamine, N-acetyl-d-glucosamine, d-gluconic acid, d-glucuronic acid, d-galacturonic acid, d-xylose, cellobiose, lactose, and β-(1→4)-mannobiose were tested as acceptors. A reaction mixture (50 µL) containing 72.4–3620 nM PBOR_28850, 20 mM α-Glc1P, 20 mM acceptor, and 0.2 M 2-(N-morpholino)ethanesulfonic acid (MES)-NaOH buffer (pH 6.5) was incubated at 30°C for 10 min. The reaction was terminated by heating the sample at 80°C for 3 min. The liberated Inorganic phosphate was measured as described by Lowry and Lopez [30].
Time course of reverse phosphorolysis with α-Glc1 P and d-galactose
A reaction mixture 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. After 60 h, 66 µL of 18.1 µM PBOR_28850 (the same amount of enzyme present at the start of the reaction) was added and the reaction mixture was further incubated at 30°C. To monitor reaction progression, 100 µL of the reaction mixture was taken at the indicated time and incubated at 80°C for 3 min to stop the reaction. Inorganic phosphate concentration was determined as described above.
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 D2O (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 in this study. 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, 9.05 µM PBOR_28850 was incubated in 250 mM buffer (pH 4.6–9.4) at 4°C for 24 h. Sodium acetate buffer (pH 4.6–5.6), 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 1–16 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).
Eq. 1: v = kcat[E0][A][B]/([A][B] + KmB[A] + KmA[B] + KiAKmB)
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.
HPLC analysis of sugar content.
The sugar content of the samples (10 mg/mL) was analyzed via HPLC under the following conditions: sample injection volume, 10 µL; column, Hilicpak VG-50 4E (Shodex, Tokyo, Japan; 4.6 mm i.d. × 250 mm); column temperature, 40°C; eluent, acetonitrile/methanol/water = 75/20/5 (v/v/v); flow rate, 1 mL/min; detection, refractive index.