Improving Aqueous Solubility of Natural Antioxidant Mangiferin through Glycosylation by Maltogenic Amylase from Parageobacillus galactosidasius DSM 18751

Mangiferin is a natural antioxidant C-glucosidic xanthone originally isolated from the Mangifera indica (mango) plant. Mangiferin exhibits a wide range of pharmaceutical activities. However, mangiferin’s poor solubility limits its applications. To resolve this limitation of mangiferin, enzymatic glycosylation of mangiferin to produce more soluble mangiferin glucosides was evaluated. Herein, the recombinant maltogenic amylase (MA; E.C. 3.2.1.133) from a thermophile Parageobacillus galactosidasius DSM 18751T (PgMA) was cloned into Escherichia coli BL21 (DE3) via the expression plasmid pET-Duet-1. The recombinant PgMA was purified via Ni2+ affinity chromatography. To evaluate its transglycosylation activity, 17 molecules, including mangiferin (as sugar acceptors), belonging to triterpenoids, saponins, flavonoids, and polyphenol glycosides, were assayed with β-CD (as the sugar donor). The results showed that puerarin and mangiferin are suitable sugar acceptors in the transglycosylation reaction. The glycosylation products from mangiferin by PgMA were isolated using preparative high-performance liquid chromatography. Their chemical structures were glucosyl-α-(1→6)-mangiferin and maltosyl-α-(1→6)-mangiferin, determined by mass and nucleic magnetic resonance spectral analysis. The newly identified maltosyl-α-(1→6)-mangiferin showed 5500-fold higher aqueous solubility than that of mangiferin, and both mangiferin glucosides exhibited similar 1,1-diphenyl-2-picrylhydrazyl free radical scavenging activities compared to mangiferin. PgMA is the first MA with glycosylation activity toward mangiferin, meaning mangiferin glucosides have potential future applications.


Introduction
Mangiferin is a natural C-glucosidic xanthone originally isolated from the Mangifera indica (mango) plant. Mangiferin has been reported to possess diverse health-promoting activities, such as antioxidant [1,2], anticancer [3,4], anti-inflammatory [5], and anti-osteoarthritis pain activities [6], allowing it to prevent memory impairment [7], neurodegeneration [8], and organ fibrosis [9]. Furthermore, it offers protection from the deleterious effects of heavy metals [10]. However, the pharmacological use of mangiferin is restricted owing to its poor solubility and low bioavailability [11,12]. As the glycosylates of small molecules have been proven to have better aqueous solubility and bioavailability than the original molecules [13,14], the glycosylation of mangiferin should be further improved for better usage.

Assay of Hydrolysis Activity
The standard reaction was performed with 1% (w/v) β-CD, 5.6 µg/mL PgMA, and 50 mM of PB at pH 6 and 60 • C for 30 min. After the reaction was stopped by boiling, the amount of reducing sugars produced from each reaction was estimated using the dinitrosalicylic acid method [40]. One unit of MA activity was defined as the amount of the enzyme that released 1 µmol of reducing sugar as maltose per min under the assay condition described earlier. For optimal conditions, the reaction was further performed at different temperatures and pH values, including pH 5 (acetate buffer), pH 6-7 (PB), and pH 8-10 (glycine buffer). Accordingly, the substrate specificity was measured with 1% (w/v) of the studied sugars, including α-CD, β-CD, γ-CD, soluble starch, and pullulan, performed at pH 7 and 65 • C. To realize the effects of metal ions and DMSO on the hydrolysis activity of PgMA, 10 mM of tested metal ion or 5-20% (v/v) of DMSO was added into the reaction mixture.

Assay of Transglycosylation Activity
To determine the transglycosylation activity of the PgMA, β-CD was used as a sugar donor, and the molecules, which belonged to triterpenoids, saponins, flavonoids, or polyphenol glycosides, were tested as sugar acceptors. The reaction mixture containing 5% (w/v) β-CD, 5.6 µg/mL PgMA, and 1 mg/mL tested molecules, dissolved in DMSO with 50 mM of PB (pH 7), was incubated at 65 • C for 24 h. The reaction mixture was then mixed with an equal volume of methanol and analyzed with high-performance liquid chromatography (HPLC).

HPLC Analysis
HPLC was performed with the Agilent 1100 series HPLC system (Santa Clara, CA, USA) equipped with a gradient pump (Waters 600, Waters, Milford, MA, USA). The stationary phase was a C18 column (5 µm, 4.6 i.d. × 250 mm; Sharpsil H-C18, Sharpsil, Bei-jing, China), and the mobile phase was 1% acetic acid in water (A) and methanol (B). The elution condition was a linear gradient from 0 min with 40% B to 20 min with 70% B, an isocratic elution from 20 to 25 min with 70% B, a linear gradient from 25 min with 70% B to 28 min with 40% B, and an isocratic elution from 28 to 35 min with 40% B. All eluants were eluted at a flow rate of 1 mL/min. The sample volume was 10 µL. The detection condition was set at 254 nm.

Purification of Mangiferin Glycosides
The purification process was a previously described method [41]. A 100 mL reaction mixture containing 50% (w/v) maltodextrin, 1 mg/mL mangiferin, 5.6 µg/mL PgMA, and 50 mM PB (pH 7) was incubated at 65 • C for 24 h. After the large-scale reaction, an equal volume of methanol was added to stop the transglycosylation. The mixture was then filtrated through a 0.2 µm nylon membrane, and the filtrate was injected in a preparative YoungLin HPLC system (YL9100, YL Instrument, Gyeonggi-do, South Korea) equipped with a preparative C18 reversed-phase column (10 µm, 20.0 i.d. × 250 mm, ODS 3; Inertsil, GL Sciences, Eindhoven, The Netherlands) for the purification of biotransformation products. The operational conditions for the preparative HPLC analysis were the same as those in the HPLC analysis. The elution corresponding to the peak of the metabolite in the HPLC analysis was collected, condensed under a vacuum, and then crystallized by freeze drying. Finally, 20.1 mg of compound (1) and 9.3 mg of compound (2) were obtained, and the structures of the compounds were confirmed with nucleic magnetic resonance (NMR) and mass spectral analyses. The mass analysis was performed using the Finnigan LCQ Duo mass spectrometer (ThermoQuest Corp., San Jose, CA, USA) with electrospray ionization (ESI). 1 H-and 13 C-NMR, distortionless enhancement by polarization transfer (DEPT), heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond connectivity (HMBC), correlation spectroscopy (COSY), and nuclear Overhauser effect spectroscopy (NOESY) spectra were recorded on a Bruker AV-700 NMR spectrometer at ambient temperature. Standard pulse sequences and parameters were used for the NMR experiments, and all chemical shifts were reported in parts per million (ppm, δ).  The composition of compound (2)

Determination of Aqueous Solubility
The aqueous solubility of mangiferin and its glucoside derivative were examined as follows: each compound was vortexed in double-deionized H 2 O for 1 h at 25 • C. The mixture was centrifuged at 10,000× g for 30 min at 25 • C. The supernatant was filtrated with 0.2 µM of nylon membrane and analyzed with HPLC. Based on their peak areas, the concentrations of the tested compounds were determined by using calibration curves prepared with HPLC analyses of authentic samples.

Determination of Antiradical Activity Using a DPPH Assay
The assay was performed as previously described [42] with minor modifications. The tested sample (dissolved in DMSO) was added to the DPPH solution (1 mM in methanol) to a final volume of 0.1 mL. After 30 min of reaction, the absorbance of the reaction mixture was measured at 517 nm with a microplate reader (Sunrise, Tecan, Männedorf, Switzerland). Ascorbic acid (dissolved in DMSO) was used as a positive antioxidant standard. The DPPH free radical scavenging activity was calculated as follows: DPPH free radical scavenging activity = (OD 517 of the control reaction − OD 517 of the reaction)/(OD 517 of the control reaction).

Selection of Candidate MAs via Online Genome Sequences
Maltogenic amylase from Bacillus stearothermophilus (BsMA, GenBank accession number: AAC46346) is a well-studied MA for the glycosylation of bioactive molecules [31,33,34,36,37]. Thus, the amino acid sequence of BsMA was selected to search for new MAs from the NCBI GenBank. The highest but distinct MA sequences from bacterial whole genomes were further identified as our study's candidates. The available bacterial strains with known genome sequences in BCRC (Hsinchu, Taiwan) were also included for comparison. Accordingly, an α-glycosidase gene (GenBank accession number: OXB94089.1) from the genome data of P. galactosidasius DSM 18751T (GenBank accession number: PRJNA383662) showed the highest homology (79.1%) with BsMA ( Figure 1) and the top five candidates with the best-hit of BsMA from NCBI GenBank in Table S1. Therefore, the α-glycosidase gene from the DSM 18,751 strain was identified as a suitable candidate in the present study. Figure 1 shows the α-glycosidase gene from P. galactosidasius DSM 18751, which was classified as a maltogenic amylase gene, and the gene product was named PgMA in this study.

Production of Recombinant PgMA in E. coli
To produce the recombinant PgMA, an expression plasmid pETDuet-PgMA was constructed (Figure 2a), and the recombinant PgMA was produced in recombinant E. coli BL21 (DE3) and purified as a major band shown by SDS-PAGE with a Ni 2+ affinity chromatography ( Figure 2b). The purified PgMA showed an estimated 68 kD molecular weight in the SDS-PAGE. The production yield was 18.73 mg/L, and the specific activity of the purified enzyme was determined to be 91.46 U/mg by using β-CD as a substrate at pH 7 and 60 °C.

Determination of Hydrolysis Activity by Recombinant PgMA
To determine the optimal pH and temperature, β-CD was used as a substrate, and the reaction was performed at different pH levels and temperatures. The results showed that the optimal pH and temperature for the PgMA catalytic reaction were pH 7 (Figure 3a)

Production of Recombinant PgMA in E. coli
To produce the recombinant PgMA, an expression plasmid pETDuet-PgMA was constructed (Figure 2a), and the recombinant PgMA was produced in recombinant E. coli BL21 (DE3) and purified as a major band shown by SDS-PAGE with a Ni 2+ affinity chromatography ( Figure 2b). The purified PgMA showed an estimated 68 kD molecular weight in the SDS-PAGE. The production yield was 18.73 mg/L, and the specific activity of the purified enzyme was determined to be 91.46 U/mg by using β-CD as a substrate at pH 7 and 60 • C.

Production of Recombinant PgMA in E. coli
To produce the recombinant PgMA, an expression plasmid pETDuet-PgMA was constructed (Figure 2a), and the recombinant PgMA was produced in recombinant E. coli BL21 (DE3) and purified as a major band shown by SDS-PAGE with a Ni 2+ affinity chromatography (Figure 2b). The purified PgMA showed an estimated 68 kD molecular weight in the SDS-PAGE. The production yield was 18.73 mg/L, and the specific activity of the purified enzyme was determined to be 91.46 U/mg by using β-CD as a substrate at pH 7 and 60 °C.

Determination of Hydrolysis Activity by Recombinant PgMA
To determine the optimal pH and temperature, β-CD was used as a substrate, and the reaction was performed at different pH levels and temperatures. The results showed that the optimal pH and temperature for the PgMA catalytic reaction were pH 7 (Figure 3a)

Determination of Hydrolysis Activity by Recombinant PgMA
To determine the optimal pH and temperature, β-CD was used as a substrate, and the reaction was performed at different pH levels and temperatures. The results showed that the optimal pH and temperature for the PgMA catalytic reaction were pH 7 (Figure 3a) at 65 • C (Figure 3b). Moreover, the addition of Mg 2+ , K + , or ethylenediaminetetraacetic acid did not significantly affect the activity of PgMA; by contrast, DMSO decreased the activity of PgMA (Figure 3c). One of the specific features of MA is that the enzyme prefers CD as its substrate over other polysaccharides, such as starch or pullulan. To characterize the PgMA hydrolysis activity, different polysaccharides were used as a substrate for the PgMA catalytic reaction. The results showed that PgMA exhibited almost equally high specific activities toward α-CD, γ-CD, and β-CD. The specific activities of PgMA toward CD were 65-and 650-fold higher than those toward pullulan and starch, respectively (Figure 4). This CD preference was consistent with other known MAs [20][21][22][23][24][25][26][27]. The 130 residues at the Nterminal of the MA are key for the enzymes to form dimers and largely increase hydrolysis activities toward CD [18,19]. In addition, some recombinant MAs have been purified using N-terminal His-tag fusion [18][19][20][21][22][23][24][25][26][27]. The N-terminal His-tag fusion did not seem to affect its dimerization; herein, the recombinant PgMA also remained as the CD preference. The reaction was performed with 1% (w/v) β-cyclodextrin (CD), 5.6 µg/mL of PgMA, and 50 mM of a different buffer at the tested temperature in the absence or presence of the tested metal ion or DMSO for 30 min. After the reaction was stopped by boiling, the hydrolytic activity of PgMA was determined by measuring the reducing sugars produced from the reaction as described in Section 2.
One of the specific features of MA is that the enzyme prefers CD as its substrate over other polysaccharides, such as starch or pullulan. To characterize the PgMA hydrolysis activity, different polysaccharides were used as a substrate for the PgMA catalytic reaction. The results showed that PgMA exhibited almost equally high specific activities toward α-CD, γ-CD, and β-CD. The specific activities of PgMA toward CD were 65-and 650-fold higher than those toward pullulan and starch, respectively (Figure 4). This CD preference was consistent with other known MAs [20][21][22][23][24][25][26][27]. The 130 residues at the N-terminal of the MA are key for the enzymes to form dimers and largely increase hydrolysis activities toward CD [18,19]. In addition, some recombinant MAs have been purified using Nterminal His-tag fusion [18][19][20][21][22][23][24][25][26][27]. The N-terminal His-tag fusion did not seem to affect its dimerization; herein, the recombinant PgMA also remained as the CD preference.

Determination of Transglycosylation Activity by Recombinant PgMA
Transglycosylation activity is an important property of MAs for biotechnology applications. To clarify the transglycosylation activity of recombinant PgMA, 17 different molecules, including mangiferin (Table S2), belonging to triterpenoids, saponins, flavonoids, flavonoid glycosides, or xanthone glycoside, were used as sugar acceptors with 1% (w/v) β-CD (as the sugar donor) for activity. The reaction mixture was then analyzed with HPLC. The results showed that only puerarin ( Figure 5a) and mangiferin (Figure 5b

Determination of Transglycosylation Activity by Recombinant PgMA
Transglycosylation activity is an important property of MAs for biotechnology applications. To clarify the transglycosylation activity of recombinant PgMA, 17 different molecules, including mangiferin (Table S2), belonging to triterpenoids, saponins, flavonoids, flavonoid glycosides, or xanthone glycoside, were used as sugar acceptors with 1% (w/v) β-CD (as the sugar donor) for activity. The reaction mixture was then analyzed with HPLC. The results showed that only puerarin ( Figure 5a) and mangiferin (Figure 5b  Except mangiferin and puerarin, the other four tested triterpenoids, two triterpenoids saponins, nine flavonoid aglycones, and glycosides could not act as the sugar acceptors in the transglycosylation of PgMA. PgMA could transglycosylate puerarin, which has the isoflavone-8-C-glucosdie structure. However, PgMA could not transglycosylate isoflavone-7-O-glucoside (8-hydroxydaidzein-7-α-O-glucoside) or flavone-8-C-glucoside (vi- Figure 5. High-performance liquid chromatography (HPLC) analysis of the biotransformation products of puerarin (a) and mangiferin (b) by PgMA. The reaction was performed with 1% (w/v) β-CD, 5.6 µg/mL of PgMA, and 1 mg/mL of puerarin or mangiferin at 50 mM of PB (pH 7) and 65 • C for 24 h. After the reaction, the reaction mixture was analyzed with HPLC. The conditions for HPLC are described in Section 2.
The results revealed that PgMA glycosylates mangiferin to produce low amounts of the M1 compound with a yield of 2.3% (Figure 5b). This is the first study to report that MA could glycosylate mangiferin, of which mangiferin glycoside may have better aqueous solubility for different applications. Therefore, we mainly focused on the unknown mangiferin glycoside by PgMA in the following assays.

Optimization of Biotransformation of Mangiferin by PgMA
As the yield of the M1 compound from the biotransformation by PgMA is too low to be easily purified, the glycosylation condition must be optimized. The glycosylation reaction was optimized with different sugar donors, concentrations of sugar donors, and reaction times. First, although the yield of the M1 using α-CD showed the best output (Figure 6a), maltodextrin was selected as the sugar donor for experiments due to its highly aqueous solubility at a much lower price.
Second, the GH enzymes contained both hydrolysis and transglycosylation activities. The transglycosylation activity of GH has been reported to increase under low water concentrations [43]. Thus, a solution with a higher sugar concentration and lower water concentration would increase its transglycosylation activity. The transglycosylation activity of PgMA was indeed increased in 50% maltodextrin, the highest soluble concentration. When the maltodextrin concentration was increased from 1% to 50% (w/v), three compounds (M1, M2, and M3 in Figure 6b) were formed with higher yields (Figure 6c). The maximal yields of M1 and M2 reached 10% and 21%, respectively. The M3 compound was not completely separated with mangiferin, which is similar to the situation of the T3 compound with puerarin by BsMA [34]. Therefore, only M1 and M2 were further studied.
Antioxidants 2021, 10, x FOR PEER REVIEW 9 of 16 texin). The results imply that PgMA has a narrow and/or specific substrate range. Nevertheless, the main finding is that PgMA can glycosylate mangiferin, which will expand the biotechnological applications of MAs in the future. MAs have been proven to glycosylate some small molecules, such as hydroquinone [29], caffeic acid [30], ascorbic acid [31], puerarin [32][33][34], genistin [35], neohesperidin [36], and naringin [37]. Our results also showed that PgMA glycosylated puerarin to three major products, P1, P2, and P3 ( Figure 5a). These three major products were not identified in advance because the glycosylation of puerarin has been studied based only on known MAs [32,34]. Li et al. (2004) reported that BsMA glycosylated puerarin to three products (T1, T2, and T3), two of which were identified as maltosyl-α-(1→6)-puerarin (T1) and glucosyl-α-(1→6)-puerarin (T2), while T3 was not identified [34]. Li et al. (2011) further reported that a maltogenic amylase (TfMA) from the archaeon T. pendens glycosylated puerarin to a series of products containing glucosyl puerarin and maltosyl puerarin, although they did not identify the exact chemical structures of the products [32]. From the results of the two studies, the P1-P3 products might contain glucosyl and maltosyl puerarin. The results revealed that PgMA glycosylates mangiferin to produce low amounts of the M1 compound with a yield of 2.3% (Figure 5b). This is the first study to report that MA could glycosylate mangiferin, of which mangiferin glycoside may have better aqueous solubility for different applications. Therefore, we mainly focused on the unknown mangiferin glycoside by PgMA in the following assays.

Optimization of Biotransformation of Mangiferin by PgMA
As the yield of the M1 compound from the biotransformation by PgMA is too low to be easily purified, the glycosylation condition must be optimized. The glycosylation reaction was optimized with different sugar donors, concentrations of sugar donors, and reaction times. First, although the yield of the M1 using α-CD showed the best output (Figure 6a), maltodextrin was selected as the sugar donor for experiments due to its highly aqueous solubility at a much lower price.
Second, the GH enzymes contained both hydrolysis and transglycosylation activities. The transglycosylation activity of GH has been reported to increase under low water concentrations [43]. Thus, a solution with a higher sugar concentration and lower water concentration would increase its transglycosylation activity. The transglycosylation activity of PgMA was indeed increased in 50% maltodextrin, the highest soluble concentration. When the maltodextrin concentration was increased from 1% to 50% (w/v), three compounds (M1, M2, and M3 in Figure 6b) were formed with higher yields (Figure 6c). The maximal yields of M1 and M2 reached 10% and 21%, respectively. The M3 compound was not completely separated with mangiferin, which is similar to the situation of the T3 compound with puerarin by BsMA [34]. Therefore, only M1 and M2 were further studied. Third, the yields of M1 and M2 in 50% maltodextrin by PgMA we mined under different time courses (Figure 7). The results showed that t of M1 and M2 reached 13.2% at 168 h and 33.8% at 72 h, respectively.

Isolation and Identification of Mangiferin Glycosides by PgMA
The glycosylation of mangiferin by PgMA was scaled up to 100 mL. and M2 were purified using preparative HPLC. From the 100 mL reac compound (1) (M1) and 9.3 mg of compound (2) (M2) were isolated The reaction was performed with 5.6 µg/mL PgMA, 1 mg/mL mangiferin, and the tested sugar donors at 50 mM of PB (pH 7) and 65 • C for 24 h. After the reaction, the reaction mixture was analyzed with HPLC. The conditions for HPLC are described in Section 2. The yield of the product was calculated by dividing the area of the product by that of mangiferin without an enzyme reaction (control reaction).
Third, the yields of M1 and M2 in 50% maltodextrin by PgMA were further determined under different time courses (Figure 7). The results showed that the yields plateau of M1 and M2 reached 13.2% at 168 h and 33.8% at 72 h, respectively.
(c) The reaction was performed with 5.6 μg/mL PgMA, 1 mg/mL mangiferin, and the tested sugar donors at 50 mM of PB (pH 7) and 65 °C for 24 h. After the reaction, the reaction mixture was analyzed with HPLC. The conditions for HPLC are described in Section 2. The yield of the product was calculated by dividing the area of the product by that of mangiferin without an enzyme reaction (control reaction).
Third, the yields of M1 and M2 in 50% maltodextrin by PgMA were further deter mined under different time courses (Figure 7). The results showed that the yields plateau of M1 and M2 reached 13.2% at 168 h and 33.8% at 72 h, respectively.

Isolation and Identification of Mangiferin Glycosides by PgMA
The glycosylation of mangiferin by PgMA was scaled up to 100 mL. The products M1 and M2 were purified using preparative HPLC. From the 100 mL reaction, 20.1 mg o compound (1) (M1) and 9.3 mg of compound (2) (M2) were isolated. The molecula

Isolation and Identification of Mangiferin Glycosides by PgMA
The glycosylation of mangiferin by PgMA was scaled up to 100 mL. The products M1 and M2 were purified using preparative HPLC. From the 100 mL reaction, 20.1 mg of compound (1) (M1) and 9.3 mg of compound (2) (M2) were isolated. The molecular weights of the purified products were then determined with mass spectrometry. The mass spectrometry of compound (1) revealed an [M-H] − ion peak at m/z: 583.4 in the electrospray ionization mass spectrum (ESI-MS) corresponding to the molecular formula C 25 H 28 O 16 ( Figure S1). The mass data imply that M1 contains one glucosyl moiety attached to the mangiferin structure. In the mass data of M2, an [M-H] − ion peak at m/z: 745.3 in the ESI-MS corresponded to the molecular formula C 31 H 38 O 21 ( Figure S2), which implies that compound (2) contains two glucosyl moieties attached to the mangiferin structure. To identify the structures in advance, the structures of both compounds were determined using NMR spectroscopy. 1 H and 13 C NMR, including the DEPT, HSQC, HMBC, COSY, and NOESY spectra, were obtained.
The characteristic 1 H and 13 C NMR sugar signals in compound (1) were assigned to C-glucosyl and O-glucosyl moieties by one-dimensional (1-D) and 2-D NMR experiments. The 1 H spectrum of compound (1) in DMSO-d 6 showed three singlets at 6.36, 6.86, and 7.37 ppm and a complex 10-spin system between 3.0 and 5.0 ppm. Analysis of this secondorder system revealed coupling constants typical of two glucose moieties. The compound (1) glucosidic linkage of the C-glucosyl moiety on the xanthone C-2 was revealed by the presence of HMBC correlations between C-2/H-1 (107.5/4.58 ppm), and the anomeric proton H-1 at 4.58 (d, J = 9.1 Hz) indicated a C-βconfiguration of mangiferin that was confirmed by the data reported in the literature [44]. The mangiferin O-glucosyl moiety was a doublet signal at H-1 (4.73 ppm, d, J = 4.2 Hz) with the corresponding carbon atom at C-1 (98.7 ppm) assigned to the anomeric proton and indicating an O-α-configuration by HSQC, which is in the O-α-configuration. The H-1 (δ = 4.73 ppm) of mangiferin and the HMBC cross signaled H-1 /C-6 (4.73/66.9 ppm) and H-6 a, 6 b/C-1 (3.46, 3.52/98.7 ppm). The significant downfield shift of the 13 C signal of C-6 indicated the connection of the second glucosyl moiety, which confirmed the α-(1→6) linkage of the second glucosyl moiety. The NMR signals were identified as shown in Table S3. The compound (1) was thus confirmed as glucosyl-α-(1→6)-mangiferin ( Figures S3-S9).
The 1 H spectrum of compound (2) in the same compound (1) solvent also showed three singlets at 6.36, 6.86, and 7.37 ppm and a complex 11-spin system between 3.0 and 5.0 ppm. Analysis of this second-order system revealed coupling constants typical of three glycose moieties, which included the chemical shifts listed in Table S3. The glucosyl moiety chemical shifts of C-2 at 107.4 ppm and H-1 at 4.58 ppm (d, J = 9.8 Hz) according to the corresponding HMBC indicated a C-C bond between the sugar and the aglycone of mangiferin (C-glucosyl-xanthone) and were confirmed by the data reported in the literature [44]. The O-maltosyl moiety connected to mangiferin was confirmed by HMBC from the anomeric carbon C-6 (66.9 ppm), and the corresponding anomeric proton H-  /H-4 (100.9/3.34 ppm) confirmed the α-(1→4) between the two-glucosyl moiety. Our experimental 1 H and 13 C chemical shifts listed in Table  S2 confirmed compound (2) as maltosyl-α-(1→6)-mangiferin (Figures S10-S16). Figure 8 summarizes the biotransformation process of mangiferin by PgMA.

Compound
Aqueous Solubility (mg/L) 1 Fold 2 Mangiferin 92.2 ± 4.60 1.0 Maltosyl-α-(1→6)-mangiferin (2) 5.11 × 10 5 ± 2.64 × 10 3 5.5 × 10 3 1 The mean (n = 2) is shown, and the standard deviations are represented by error bars. 2 The folds of the aqueous solubilities of the mangiferin glucoside derivatives are expressed as relative to that  Mangiferin exhibits a wide pharmacological profile, and its antioxidant property is well known from previous studies. Furthermore, it has been associated with the redox aromatic system of the xanthone nucleus [3][4][5][6][7][8][9][10]12]. Thus, the antioxidative activities of the two mangiferin glucosides were determined using DPPH free radical scavenging assay. The assay showed that the antioxidant activity levels of mangiferin and its two glucosides were all higher than those of ascorbic acid (Figure 9). In other words, the antioxidant activities of the two mangiferin glucosides are comparable with those of mangiferin. The ortho-dihydroxyl groups on the benzene ring of the mangiferin structure have been reported to play a key role in exerting its antioxidant activity [1,2]. Both mangiferin glucosides remained the key functional groups after glycosylation; therefore, most of the antioxidant activity remained in the mangiferin derivatives. These glycoside derivatives (glucoside and fructoside) might possess different pharmacological properties. A futher study will focus on the bioactivities and bioavailability of these mangiferin derivatives ported to play a key role in exerting its antioxidant activity [1,2]. Both mangiferin glucosides remained the key functional groups after glycosylation; therefore, most of the antioxidant activity remained in the mangiferin derivatives. These glycoside derivatives (glucoside and fructoside) might possess different pharmacological properties. A futher study will focus on the bioactivities and bioavailability of these mangiferin derivatives Figure 9. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity of mangiferin, mangiferin glucosides, and ascorbic acid. The DPPH scavenging activity was determined as described in Section 2. The IC50 values represent the concentrations required for 50% DPPH free radical scavenging activity. The mean (n = 3) is shown, and the standard deviations are represented by error bars. M1 and M2 are glucosyl-α-(1→6)-mangiferin and maltosyl-α-(1→6)-mangiferin, respectively.

Conclusions
The recombinant PgMA from P. galactosidasius DSM 18751 T was confirmed to exhibit the bifunctions of hydrolysis and transglycosylation activities. The recombinant PgMA can glycosylate mangiferin and produce glucosyl-α-(1→6)-mangiferin and maltosyl-α-(1→6)-mangiferin with a high maltodextrin concentration. The novel maltosyl-α-(1→6)mangiferin showed much higher aqueous solubility than that of mangiferin. The two mangiferin glucosides exhibited similar DPPH antioxidative activity compared to mangiferin. To our knowledge, PgMA is the first MA identified with glycosylation activity toward mangiferin. With higher water solubility and compatible antioxidant activity, the two mangiferin glucoside derivatives have better pharmaceutical applicability.

Data Availability Statement:
The data presented in this study are available in the article or supplementary material.

Conflicts of Interest:
The authors declare no conflict of interest.