Phosphorylation of Isoflavone by Bacillus subtilis BCRC 80517 May

Article Cite This: J. Agric. Food Chem. 2018, 66, 127−137

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Phosphorylation of Isoflavones by Bacillus subtilis BCRC 80517 May Represent Xenobiotic Metabolism Chen Hsu,† Bo-Yuan Wu,† Yu-Chuan Chang,† Chi-Fon Chang,‡ Tai-Ying Chiou,§ and Nan-Wei Su*,† †

Laboratory of Food Chemistry, Department of Agricultural Chemistry, National Taiwan University, Taipei 10617, Taiwan Genomics Research Center, Academia Sinica, Taipei 11529, Taiwan § Laboratory of Food Science and Technology, Department of Biotechnology and Environmental Chemistry, Kitami Institute of Technology, Kitami 090-8507, Japan ‡

S Supporting Information *

ABSTRACT: The soy isoflavones daidzein (DAI) and genistein (GEN) have beneficial effects on human health. However, their oral bioavailability is hampered by their low aqueous solubility. Our previous study revealed two water-soluble phosphorylated conjugates of isoflavones, daidzein 7-O-phosphate and genistein 7-O-phosphate, generated via biotransformation by Bacillus subtilis BCRC80517 cultivated with isoflavones. In this study, two novel derivatives of isoflavones, daidzein 4′-O-phosphate and genistein 4′-O-phosphate, were identified by HPLC-ESI−MS/MS and 1H, 13C, and 31P NMR, and their biotransformation roadmaps were proposed. Primarily, isoflavone glucosides were deglycosylated and then phosphorylated predominantly into 7-O-phosphate conjugates with traces of 4′-O-phosphate conjugates. Inevitably, trace quantities of glucosides were converted into 6″-O-succinyl glucosides. GEN was more efficiently phosphorylated than DAI. Nevertheless, the presence of GEN prolonged the time until the exponential phase of cell growth, whereas the other isoflavones showed little effect on cell growth. Our findings provide new insights into the novel microbial phosphorylation of isoflavones involved in xenobiotic metabolism. KEYWORDS: isoflavone, biotransformation, phosphorylation, Bacillus subtilis, xenobiotic metabolism

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cytochrome P450 (CYP)16−18 or glycosyltransferase19,20 to biosynthesize regio- and stereospecific hydroxylated or glycosylated isoflavones, which showed higher antioxidant activities and solubilities than their parent forms. Despite the many studies on the biotransformation of isoflavones by diverse enzymes and microbial factors, to the best of our knowledge, the biological phosphorylation of isoflavones has rarely been explored.21 In our previous study, we generated two highly water-soluble isoflavones, daidzein 7-O-phosphate (DAI-7P) and genistein 7-O-phosphate (GEN-7P), by cultivating B. subtilis BCRC 80517 with aglycones and glucosides of soy isoflavones. This bacterial strain could convert the isoflavone glucosides, via the aglycones, into the corresponding 7-O-phosphate conjugates.22 Furthermore, we found that GEN-7P improved water solubility and enhanced intestinal permeability in vitro and in situ as well as greatly increasing plasma exposure to GEN after oral administration in rats.23 The major metabolites of isoflavones, namely, 7-O-phosphate conjugates, produced by B. subtilis BCRC 80517 have been investigated, but some minor metabolites and their related metabolisms remain unclear and uncharacterized. In this study, we isolated and identified new metabolites of isoflavones, daidzein 4′-O-phosphate (DAI-4′P) and genistein 4′-O-phosphate (GEN-4′P), from cultures of B. subtilis BCRC 80517 and then characterized each metabolite during the biotransformations.

INTRODUCTION The relationship between soy isoflavones and Bacillus subtilis var. natto has been extensively studied in the past two decades. Natto, cheonggukjang, and doenjang, which are made from soybeans fermented with B. subtilis, are popular traditional foods that have been consumed in East Asia for thousands of years.1 Soybean and soy-based food products contain high amounts of isoflavones, including the aglycones daidzein (DAI) and genistein (GEN), the glycosides daidzin (DAI-G) and genistin (GEN-G), and the malonyl glycosides malonyl daidzin (DAI-GM) and malonyl genistin (GEN-GM).2,3 Much evidence has indicated that soy isoflavones possess phytoestrogenic activity and physiological activities, including lowering the risks of breast cancer and prostate cancer,4,5 decreasing cholesterol levels and preventing cardiovascular diseases,6 increasing bone mass density to prevent osteoporosis and reducing menopause symptoms,7−9 preventing obesity and diabetes,10,11 and improving cognitive function,12 all with high efficacy, low toxicity, and minimal side effects. However, according to the Merck Index, both DAI and GEN are practically water-insoluble and belong to Biopharmaceutical Classification System (BCS) class IV, so they are not physiologically absorbed easily because of their low aqueous solubilities, low gastrointestinal permeabilities, and therefore low bioavailabilities.13,14 In the past decade, enzyme or microbial biocatalysis has been promoted as a new strategy and powerful tool to modify the chemical structures of isoflavones.15 Several recent studies have used a microbial-biotransformation method focused on hydroxylation and glycosylation to generate new water-soluble isoflavones. Many studies have cloned and expressed genes encoding © 2017 American Chemical Society

Received: Revised: Accepted: Published: 127

October 8, 2017 December 9, 2017 December 12, 2017 December 12, 2017 DOI: 10.1021/acs.jafc.7b04647 J. Agric. Food Chem. 2018, 66, 127−137

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Journal of Agricultural and Food Chemistry

identified according to LC-MS-spectra data and NMR analyses. LC-ESI-MS/MS involved the use of a Thermo Finnigan LXQ Mass Spectrometer System (San Jose, CA) equipped with an ESI source. The experiments were carried out in the positive-ion mode. The source parameters were a spray voltage of 3.73 kV, cone voltage of 33.0 V, collision voltage of 35 V, sheath gas of 20.0 arbitrary units, auxiliary gas of 20.0 arbitrary units, sweep gas of 2.0 arbitrary units, and capillary temperature of 200.5 °C. Nitrogen was used as the nebulizing gas at a pressure of 50 psi and at 350.0 °C. Separation involved use of a Finnigan Surveyor LC Pump (San Jose, CA) and a flow rate of 1.0 mL/min with a postcolumn-split-volume ratio of 1/5 to the mass detector at room temperature. The column and chromatographic conditions as for the HPLC-DAD analyses were the same as those mentioned previously. Full scan spectra were acquired in the 100 to 1000 m/z range. Helium was used as the damping gas. The NMR spectra were recorded on a Bruker Avanve-500 spectrometer. The chemical shifts (δ) and coupling constants (J) were expressed in ppm and Hz, respectively. The 1H and 13C chemical shifts were calibrated at 2.58 and 39.9, respectively, with DMSO-d6 used as the internal standard. For the phosphorus-31 NMR spectra, triphenylphosphate (TPP) in acetone-d6 was used as an external standard with −17.5 ppm for the chemical-shift calibration of the 31P resonance. Effects of Isoflavones and the Corresponding 7-O-Phosphate Conjugates on the Growth of B. subtilis BCRC 80517. Seed-culture broth (1 mL, OD600 = 1.0, 108 cfu/mL) was inoculated into a 500 mL Hinton’s flask containing 100 mL of NB medium, then an individual isoflavone predissolved in DMSO was added to the culture broth to give a final substrate concentration of 100 μM, and the bacteria were incubated at 37 °C on a rotary shaker (150 rpm) for 24 h. DMSO was added without an isoflavone substrate as a control. Aliquots of 1 mL of culture broth were withdrawn at intervals of 2 h to monitor the cell growth by measuring the absorbance at 600 nm. Data Analysis. The isoflavone profiles from the HPLC were analyzed by using a SISC Chromatography Data Station v3.2 (SISC Inc., Taipei, Taiwan). The LC-ESI-MS/MS data were analyzed by using Xcalibur v2.0 (Thermo Finnigan, San Jose, CA). All NMR spectra were analyzed by using a TopSpin 3.5 (Bruker BioSpin, Billerica, MA), and the chemical shifts were referenced to those of the solvent signals. The data are the means ± SD calculated by using Microsoft Excel 2013. All experiments were performed in triplicate.

We further examined the bioconversion rates of the individual isoflavones in B. subtilis BCRC 80517 cultures and their inhibition of cell growth. We proposed that phosphorylation modifications of isoflavones may play a role in xenobiotic metabolism in B. subtilis.

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MATERIALS AND METHODS

Chemicals and Biomaterials. The isoflavone standards DAI, GEN, DAI-G, and GEN-G were obtained from Shanghai Aladdin Bio-Chem Technology Company (Shanghai, China). High-performance liquid chromatography (HPLC)-grade acetonitrile and methanol were from Merck (Darmstadt, Germany). Analytical-grade acetic acid, benzoic acid, ethanol, and formic acid were from Sigma-Aldrich (St. Louis, MO). B. subtilis BCRC 80517 was from the Bioresource Collection and Research Centre (Hsinchu, Taiwan). The strain was maintained on nutrient agar, stored at 4 °C, and subcultured periodically. Nutrient broth (NB) and agar were from Difco (Detroit, MI). Culture Conditions of B. subtilis BCRC 80517 with the Individual Isoflavones. A colony of B. subtilis BCRC 80517 from an agar plate was placed in a 30 mL glass tube containing 5 mL of sterilized NB medium (pH 7.0) and cultivated at 37 °C and 150 rpm until OD600 = 1.0 (108 cfu/mL); the culture broth was used as the seed culture for further incubation. For the microbial biotransformation, a 500 mL Hinton’s flask containing 85 mL of medium, 10 mL of the individual isoflavones at 15 mg/mL, and a 5 mL inoculum from the seed culture was incubated at 37 °C and 150 rpm for 48 h. Cell growth was determined by colony-forming units (cfu) on an NB-agar plate. The experimental blanks and controls were incubated under the same culture conditions without the addition of isoflavones and inoculum, respectively. Analysis of Isoflavones and Their Derivatives by HPLC-DAD. Culture broth (500 μL) was mixed with 500 μL of methanol containing 10 000 mg/L benzoic acid as an internal standard (IS) at various intervals, then the mixture was centrifuged to remove the insoluble matter, and the supernatant underwent HPLC. An analytical Shimadzu LC-20 AD HPLC system equipped with a YMC-Pack ODS-AM C18 column (250 × 4.6 mm, 5 μm) and a Thermo Scientific UV6000 photodiode array detector (DAD) was used. The mobile phase involved the use of 0.1% acetic acid in H2O (solvent A) and 0.1% acetic acid in acetonitrile (solvent B) under a linear combination gradient. After an injection of 20 μL of the sample, solvent B was increased from 15 to 20% over 20 min and to 24% within the next 10 min, kept isocratic at 24% for 6 min, then increased to 35% within the next 8 min, kept isocratic at 35% for 6 min, then decreased to 15% within 5 min, and held there for the next 15 min. The flow rate was set at 1 mL/min. The eluted components were detected at 254 nm. The bioconversion rate was calculated by dividing the amount of generated isoflavone 7-O-phosphate in the culture broth by that of the isoflavone in the initial culture broth and expressed as a percentage of molar ratios. Isolation and Identification of DAI-4′P and GEN-4′P. The purification of the isoflavone derivatives was as previously described in detail.22 Briefly, the harvested broth was centrifuged to remove the cells, and then the supernatant was adjusted to pH 1.0 with concentrated HCl and extracted three times with an equal volume of ethyl acetate. The organic layers were combined and then evaporated under reduced pressure to remove the solvent. The residue was suspended in a small amount of distilled water, placed on a prepacked DIAION HP-20 resin column, and eluted with distilled water. The nonadsorbed fraction was collected and lyophilized. Further separation of DAI-4′P and GEN-4′P involved a semipreparative HPLC system equipped with a Hypersil ODS C18 HPLC column (250 × 10 mm, 10 μm). The mobile phase consisted of 0.2% formic acid in H2O (solvent A) and 0.2% formic acid in methanol (solvent B) with a gradient elution. After an injection of 100 μL of the sample, solvent B was increased from 20 to 35% over 20 min and then to 80% within the next 5 min, decreased to 20% within 5 min, and held there for the next 10 min. The flow rate was 5.0 mL/min. The eluate was collected and monitored at 254 nm. The fractions containing the desired compounds were further concentrated and resuspended with hot alcohol to obtain a pale, amorphous crystal for identifying the chemical structure. DAI-4′P and GEN-4′P were

Figure 1. High-performance liquid chromatograms of the culture broth of the biotransformation of GEN and DAI by B. subtilis BCRC 80517 at (a) 0 h and (b) 48 h after incubation. The culture broth was incubated at 37 °C and 150 rpm. Peak 1, DAI-7P; 2, DAI-4′P; 3, GEN-7P; 4, GEN-4′P; 5, DAI-GS; 6, GEN-GS; 7, DAI; 8, GEN. Benzoic acid was used as the IS. 128

DOI: 10.1021/acs.jafc.7b04647 J. Agric. Food Chem. 2018, 66, 127−137

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RESULTS

biotransformation process, peaks 5 and 6 showed slight accumulations after 24 h and were identified as 6′′-O-succinyl daidzin (DAI-GS, peak 5, Rt: 27.3 min, [M+H]+ m/z 517.4, MS2 m/z 255.2; Figure S1) and 6′′-O-succinyl genistin (GEN-GS, peak 6, Rt: 37.4 min, [M+H]+ m/z 533.1, MS2 m/z 271.2; Figure S2), which agreed with the literature for their UV absorbances and ESI-MS/MS spectra.24 With their polar functional groups, the above minor isoflavone derivatives were considered more watersoluble than their parent aglycones. Characterization of all the newly generated peaks elucidated by HPLC-ESI-MS/MS is in Table 1. Identification of Novel Derivatives of Isoflavones: Daidzein 4′-O-Phosphate (DAI-4′P) and Genistein 4′-OPhosphate (GEN-4′P). Peaks 2 and 4 showed the same spectra

HPLC Profiles of Isoflavones and Their Derivatives during Biotransformation with B. subtilis BCRC 80517. To investigate the change in composition of isoflavones during biotransformation, we used HPLC to analyze the isoflavone profiles at different times (Figure 1). As described previously, two major peaks, representing DAI-7P and GEN-7P (Figure 1b, peaks 1 and 3, respectively) were the predominant products of the biotransformation. In the meanwhile, we observed two new minor peaks (peaks 2 and 4), representing DAI-4′P and GEN-4′P, which eluted after DAI-7P and GEN-7P. The next section describes the identification of these two novel compounds. Moreover, during the

Table 1. Chemical Characteristics of the Isoflavone Derivatives Generated from the Biotransformation Process of B. subtilis BCRC 80517 Cultivated with DAI and GENa peak no.

compound name

abbreviation

formula

Rt (min)a

λmax (nm)

[M+H]+ (m/z)

MS2 fragment ions (m/z)

1 2 3 4 5 6 7 8

daidzein 7-O-phosphate daidzein 4′-O-phosphate genistein 7-O-phosphate genistein 4′-O-phosphate 6′′-O-succinyl daidzin 6′′-O-succinyl genistin daidzein genistein

DAI-7P DAI-4′P GEN-7P GEN-4′P DAI-GS GEN-GS DAI GEN

C15H11O8P C15H11O8P C15H11O8P C15H11O8P C25H24O12 C25H24O13 C15H10O4 C15H10O5

8.8 11.9 14.2 19.2 27.3 37.4 39.8 51.2

251 251 259 259 258 260 249 262

335.2 335.2 351.2 351.2 516.4 532.1 255.0 271.0

317.2, 255.1 317.0, 255.2 333.1, 271.1 333.1, 271.1 499.0, 255.2 515.0, 271.2 227.2, 199.2, 137.1 243.2, 215.2, 153.0

HPLC conditions: column, YMC ODS-AM (250 × 4.6 mm, 5 μm); mobile phase, 0.1% acetic acid in H2O (solvent A) and 0.1% acetic acid in acetonitrile (solvent B); gradient, 15%−20% B in 20 min, 20%−24% B in 10 min, 24% B for 6 min, 24%−35% B in 8 min, 35% B for 6 min; flow rate, 1.0 mL/min; temperature, ambient. More details are described in the Materials and Methods. a

Figure 2. Mass and NMR spectra for DAI-4′P. (a) ESI-MS of the protonated parent ion (m/z 335), (b,c) ESI-MS/MS of the protonated fragment ions (m/z 255, 137, and 119), (d) 2D HMQC 1H−31P (TPP in acetone-d6, 202 MHz for the 31P NMR), and (e) chemical structure of DAI-4′P. 129


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were 1H NMR (DMSO-d6, 500 MHz) δ: 6.28 (d, J = 1.8 Hz, H-6), 6.44 (d, J = 1.8 Hz, H-8), 7.26 (2H; d; J = 8.5 Hz; H-3′, -5′), 7.45 (2H; d; J = 8.5 Hz; H-2′, -6′), and 8.37 (s, H-2); 13C NMR (DMSO-d6, 125 MHz) δ: 94.2 (C-8), 99.6 (C-6), 120.0 (C-3′, -5′), 122.6 (C-4), 123.9 (C-1′), 129.7 (C-2′, -6′), 154.5 (C-2, -4′), 162.3 (C-5), 165.4 (C-7), and 180.5 (C-4); and 31P NMR (TPP in acetone-d6, 202 MHz) δ: −5.45 (s) (Figure S4). The phosphate’s presence at the 4′-O position of GEN but not at the 5-O position was established by measuring the 2D HMQC 1 H−31P NMR spectra, which showed correlations of the phosphorus at δ −5.45 with the protons of peak 4 at δ 7.26 (H-3′, -5′) and δ 7.45 (H-2′, -6′) (Figure 3d). These data together confirmed that the structure of peak 4 was genistein 4′-O-phosphate (GEN-4′P) (Figure 3e). Biotransformation of Individual DAI and GEN by B. subtilis BCRC 80517. A time-course study was used to investigate the biotransformation of DAI and GEN individually in cultures with B. subtilis BCRC 80517 (Figures 4 and 5). At the beginning of the biotransformation, both DAI and GEN contents decreased slowly in the lag phase of growth. After 12 h of cultivation, both contents decreased rapidly during the exponential phase. The GEN in the culture broth was almost consumed after 48 h of incubation, whereas approximately 22% of the original content of DAI remained. DAI-7P and GEN-7P started to accumulate at 6 h, and then GEN-7P content peaked at 5.4 mM in the culture broth at 48 h (Figure 5a). In contrast, DAI-7P content was relatively low, around 4.3 mM (Figure 4a).

of UV absorption and peak tailing profiles as those of the isoflavone 7-O-phosphate (Figure 1b). The tailing peaks of ionic compounds are often caused by a silanol effect, so these two peaks should be ionic compounds. The ESI-MS/MS spectra for peak 2 exhibited [M+H]+ at m/z 335.2 and an MS2 peak at m/z 255.2 [M−H2PO3+H]+ (Figure 2a,b). The fragment-ion m/z 80.0 might suggest the presence of a phosphate moiety. MS3 analysis of the m/z 255.2 ion brought out the DAI spectrum (Figure 2c). The mass fragmentation profiles of peak 2 were the same as for DAI-7P, so the two compounds are structural isomers. The NMR spectral data for peak 2 were 1H NMR (DMSO-d6, 500 MHz) δ: 6.93 (d, J = 2.0 Hz, H-8), 7.00 (dd, J = 8.8, 2.0 Hz, H-6), 7.27 (2H; d; J = 8.3 Hz; H-3′, -5′), 7.48 (2H; d; J = 8.3 Hz; H-2′, -6′), 8.00 (d, J = 8.8 Hz, H-5), and 8.35 (s, H-2); 13C NMR (DMSO-d6, 125 MHz) δ: 102.5 (C-8), 115.7 (C-6), 120.0 (C-3′, -5′), 123.7 (C-3), 125.8 (C-1′), 127.5 (C-5), 129.8 (C-2′, -6′), 153.5 (C-2, -4′), 163.4 (C-7), and 175.0 (C-4); and 31P NMR (TPP in acetone-d6, 202 MHz) δ: −5.46 (s) (Figure S3). The 2D HMQC 1H−31P NMR spectra show the presence of correlation cross peaks between the main phosphorus resonance at δ −5.46 with the hydrogens of peak 2 at δ 7.27 (H-3′, -5′) and δ 7.48 (H-2′, -6′) (Figure 2d). Therefore, the monophosphate was located at the 4′-O position. From all this evidence, peak 2 was characterized as daidzein 4′-O-phosphate (DAI-4′P) (Figure 2e). The ESI-MS/MS spectra for peak 4 showed [M+H]+ at m/z 351.2 and an MS2 peak at m/z 271.1 [M−H2PO3+H]+ (Figure 3a,b). MS3 analysis of the m/z 271.1 ion revealed the GEN spectrum (Figure 3c). The NMR spectral data for peak 4

Figure 3. Mass and NMR spectra for GEN-4′P. (a) ESI-MS of the protonated parent ion (m/z 351), (b,c) ESI-MS/MS of the protonated fragment ions (m/z 271 and 153), (d) 2D HMQC 1H−31P (TPP in acetone-d6, 202 MHz for the 31P NMR), and (e) chemical structure of GEN-4′P. 130


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Figure 4. (a) Time course analysis of the biotransformation and (b) the determination of cell growth and the bioconversion rate during the incubation of B. subtilis BCRC 80517 with DAI. (c) HPLC elution profiles of the biotransformation of DAI by B. subtilis BCRC 80517 at (c1) 0 h, (c2) 24 h, and (c3) 48 h of incubation. The culture broth was incubated at 37 °C and 150 rpm. The control experiments involved the use of NB-medium culture without DAI. The data are the means ± SD from three experiments. Benzoic acid was used as the IS.

The contents of both DAI-G and GEN-G remained at their original levels during the first 12 h and then decreased rapidly in the exponential phase of growth, with increases in the contents of predominantly DAI-7P and GEN-7P after 12 h of incubation. Small amounts of DAI-GS and GEN-GS were also detected in the 6 h culture broths and increased only slightly in the culture broths. These phenomena indicated that the enzymatic interconversion of isoflavone glucosides and the corresponding 6″-O-succinylated derivatives occurred in the culture broths during the biotransformation process. At the end of the 48 h incubation, the content of GEN-7P reached 5.0 mM, for about a 90% conversion rate (Figure 7a,b). However, only 3.8 mM DAI-7P remained at 48 h, for a 68% bioconversion rate (Figure 6a,b). Both DAI-G and GEN-G seemed not to influence the growth of bacteria, because the cell-growth curves were almost identical to those of the control (Figures 6b and 7b). The formation of the phosphate conjugates was positively related to cell growth, so the biosynthesis of phosphorylated isoflavones could be attributed to cell growth. In addition, DAI-4′P and GEN-4′P were detected, and the levels of these two new compounds showed increasing trends during biotransformation (Figures 6c and 7c). Kuo et al. reported that glucoside conjugates could be effectively hydrolyzed into their aglycones from soybean fermented with B. subtilis natto.26 However, in the current study, the DAI and GEN contents were observed at 6 h of incubation and then were maintained at

At the end of the 48 h incubation, the bioconversion rate of GEN-7P was 96%, but it was only 78% for DAI-7P. Although GEN could be phosphorylated more efficiently than DAI by B. subtilis BCRC 80517, it retarded the growth of the bacteria and prolonged the time before the exponential phase of cell growth, whereas DAI had only minor effects (Figures 4b and 5b). Moreover, according to the bioconversion rate and cell growth results, the generation of phosphate derivatives seems to be highly related to cell growth. Additionally, two new minor peaks were generated and accumulated in the separate culture broths with increasing biotransformation time; these peaks were further identified as DAI-4′P and GEN-4′P. Furthermore, a small amount of DAI-GS and GEN-GS remained after 12 h of culturing, and the contents of these two compounds increased slightly with incubation (Figures 4c and 5c). Park et al. indicated that isoflavone glucosides were the only substrates for the formation of succinyl derivatives by B. subtilis natto.25 However, we found that B. subtilis BCRC 80517 could generate 6″-O-succinyl conjugations from the corresponding aglycones, and we did not observe any peaks representing DAI-G or GEN-G in the culture broth during the biotransformation process. Biotransformation of Individual DAI-G and GEN-G by B. subtilis BCRC 80517. We examined separately the bioconversions of DAI-G and GEN-G by B. subtilis BCRC 80517 by sampling the culture broths at different incubation times to detect changes in the isoflavone contents (Figures 6 and 7). 131


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Figure 5. (a) Time course analysis of the biotransformation and (b) the determination of cell growth and the bioconversion rate during the incubation of B. subtilis BCRC 80517 with GEN. (c) HPLC elution profiles of the biotransformation of GEN by B. subtilis BCRC 80517 at (c1) 0 h, (c2) 24 h, and (c3) 48 h of incubation. The culture broth was incubated at 37 °C and 150 rpm. The control experiments involved the use of NB-medium culture without GEN. The data are the means ± SD from three experiments. Benzoic acid was used as the IS.

Effect of Isoflavones and Their 7-O-Phosphate Derivatives on the Growth of B. subtilis BCRC 80517. We found that GEN had a higher phosphorylation efficiency with B. subtilis BCRC 80517 as the biotransformation substrate than did DAI. Nevertheless, the amount of cfu was severely inhibited by having GEN in the culture broth, whereas DAI did not affect the growth of the bacteria. According to these production rates of isoflavone phosphate conjugates and growth patterns with GEN, B. subtilis BCRC 80517 may be equipped with an isoflavone-phosphorylation system for xenobiotic metabolism to avoid harmful effects. We performed initial growth-inhibition experiments with DAI and GEN at 100 μM. The concentration was chosen based on the solubility of aglycones in NB medium containing 1% DMSO for measuring bacterial growth by detecting optical density (Figure 9). GEN maximally prohibited the growth of B. subtilis BCRC 80517 within 12 h of incubation. After 16 h of incubation, the effect of GEN on cell viability declined because of the phosphorylation of GEN. We investigated whether the phosphorylated metabolites had less growth inhibition than the aglycones by applying them at equimolar inhibitory concentrations as their corresponding compounds. Indeed, as expected, GEN suppressed growth at the concentration used, whereas 100 and 500 μM GEN-7P had only minor effects on bacterial growth. In contrast, cell growth was not affected by the addition of 100 μM DAI or 100 or 500 μM DAI-7P.

trace levels to the end of the biotransformation by B. subtilis BCRC 80517. Proposed Metabolic Route of Isoflavones in B. subtilis BCRC 80517 Culture. According to the changes in the contents of the isoflavones and isoflavone derivatives in the culture broths at different incubation times, we propose a potent metabolic route for isoflavone biotransformation by B. subtilis BCRC 80517 in Figure 8. During the bioconversion, aglycones are predominantly phosphorylated at the 7-O position, and a small amount of 4′-O-phosphate conjugates is generated. In addition, the aglycones are glycosylated into glycosides and further converted into a small amount of 6″-O-succinyl glucosides. The glucosides are mainly deglycosylated into aglycones, followed by rapid 7-O-phosphorylation, along with which a small amount of isoflavone 4′-O-phosphates are produced. Moreover, glucosides are converted into a small amount of 6″-O-succinyl glucosides. Furthermore, 6″-O-succinyl glucosides may be desuccinylated by esterase and then deglycosylated by β-glucosidase to form aglycones, which can be further converted into isoflavone phosphate conjugates. During the biotransformation, various isoflavone derivatives in the metabolites pool could be converted mutually by particular enzymes produced from B. subtilis BCRC 80517. Further intensive study is needed to characterize specific enzymes, especially isoflavone-phosphorylation enzymes, to explore the detailed molecular mechanism of the biotransformation of isoflavone by B. subtilis BCRC 80517. 132


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Figure 6. (a) Time course analysis of the biotransformation and (b) the determination of cell growth and the bioconversion rate during the incubation of B. subtilis BCRC 80517 with DAI-G. (c) HPLC elution profiles of the biotransformation of DAI-G by B. subtilis BCRC 80517 at (c1) 0 h, (c2) 24 h, and (c3) 48 h of incubation. The culture broth was incubated at 37 °C and 150 rpm. The control experiments involved the use of NB-medium culture without DAI-G. The data are the means ± SD from three experiments. Benzoic acid was used as the IS.

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DISCUSSION In this study, we propose a metabolism roadmap for the biotransformation of isoflavones by B. subtilis BCRC 80517 and identify two novel isoflavone derivatives, DAI-4′P and GEN-4′P, from culture broths. The beneficial effects of isoflavones on human health has received growing attention over the past few decades. However, since both DAI and GEN have poor solubility and low permeability, they are categorized into BCS class IV, representing the characteristic of low oral bioavailability.13 To enhance their practical applications, many researchers have concentrated on increasing the solubility of isoflavones by means of microbial biotransformation, which offers an alternative method for modifying chemical structures, with the advantages of ease of cultivation, rapid growth, mild conditions, stereoselectivity, and high production with few byproducts as compared with organic synthesis.27 To date, hydroxylated and glycosylated isoflavones have been the major water-soluble derivatives produced by microbial biotransformation. Isoflavones are commonly hydroxylated at the C3′ position of the B ring by microorganisms. Streptomyces avermitilis MA-4680 hydroxylated DAI at the C3′ and C6 or C8 positions via the cytochrome P450 (CYP) enzymes CYP105D7 and CYP105D6, respectively.17,18 CYP107H1, known as fatty acid hydroxylase from B. subtilis 168, exhibited ortho-specific hydroxylation activity toward DAI.16 Moreover, Esaki et al.

revealed the formation mechanism of hydroxylated isoflavones during soybean fermentation: isoflavone glycosides were gradually hydrolyzed into their corresponding aglycones by β-glucosidase and further hydroxylated to form dihydroisoflavones by CYP monooxygenases from Aspergillus saitoi.28 Regarding glycosylation, Li et al. used maltosyltransferase from Thermotoga maritima to obtain DAI conjugation with multiglucoside19 and used cyclodextrin glucanotransferase from Bacillus sp. I-5 to create a cycloamylose GEN complex,20 all of which showed about 104 times higher solubility than their aglycones. Succinylated isoflavones were first isolated and identified in 1999. In the present study, we found a small amount of DAI-GS and GEN-GS in culture broths during the bioconversion of isoflavones. Toda et al. found DAI-GS and GEN-GS in soybean foods (natto and cheonggukjang) fermented with B. subtilis.24 These two compounds showed biological activities in preventing bone loss and bone-strength reduction without causing uterine atrophy or bodyweight gain in an ovarian-hormone-deficient-rat model. Very recently, a high yield of 6″-O-succinyl isoflavones (>90%) from aglycones was achieved by using the resting cells of a solvent-tolerant strain, Bacillus licheniformis ZSP0, in an aqueous miscible organic medium (10% (v/v) DMSO);29 the conventional aqueous-phase biocatalysis of B. subtilis was incompetent for the accumulation of succinyl-glucosides because of the low substrate solubility in the aqueous phase. 133


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Figure 7. (a) Time course analysis of the biotransformation and (b) the determination of cell growth and the bioconversion rate during the incubation of B. subtilis BCRC 80517 with GEN-G. (c) HPLC elution profiles of the biotransformation of GEN-G by B. subtilis BCRC 80517 at (c1) 0 h, (c2) 24 h, and (c3) 48 h of incubation. The culture broth was incubated at 37 °C and 150 rpm. The control experiments involved the use of NB-medium without GEN-G. The data are the means ± SD from three experiments. Benzoic acid was used as the IS.

Figure 8. A proposed roadmap of soy-isoflavone biotransformation by B. subtilis BCRC 80517. 134


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Figure 9. Effects of individual isoflavones and their derivatives on the growth of B. subtilis BCRC 80517. Effects of (a) GEN (100 μM) and GEN-7P (100 and 500 μM) and (b) DAI (100 μM) and DAI-7P (100 and 500 μM). Growth was monitored by measuring the OD600 values, and 1% DMSO was added to the control culture broth. The culture broths were incubated at 37 °C and 150 rpm for 24 h. The data are the means ± SD from three experiments.

against topoisomerase I but inhibits topoisomerase II only weakly, thus having only weak antibacterial activity against B. subtilis 168.34 Furthermore, we found that GEN was phosphorylated more efficiently by fewer cells during biotransformation than DAI. GEN-7P applied at equimolar concentrations as GEN did not inhibit bacterial growth. The main biological function of the phosphorylation of isoflavones by B. subtilis BCRC 80517 may be a mechanism to reduce the growth inhibition of xenobiotic molecules by the bacteria strain. This viewpoint is similar to that of antibiotic resistance via phosphorylation in global microbiota.36 Antibiotic kinases, which include aminoglycoside and macrolide O-phosphotransferase, are widely distributed among bacterial pathogens and pose a serious threat to the currently used antimicrobial therapies.37,38 Also, the phosphorylation of the hydroxyl groups in positions 21 and 23 of the ansa chain of rifampin has been reported in pathogenic bacteria of the genera Nocardia and Rhodococcus; these phosphorylated products did not show antimicrobial activity.39 Additionally, S. avermitilis MA4680 expresses chloramphenicol 3′-O-phosphotransferase to inactivate the corresponding antibiotic molecules,40 and Bacillus pumilus can form a phosphate ester with the 8′-O position of amicoumacin.41 These microbial agents gain resistance against antibiotics by phosphorylation. Xenobiotic metabolism plays an essential role in the bacterial defense against xenobiotic molecules, which might have harmful effects on biological systems.42 In the current study, we demonstrated that phosphorylation modifications change the physicochemical properties of GEN, which alters its biological and physiological effects. However, the enzymes that specifically catalyze the phosphorylation of isoflavones have not been revealed. Further research is under way to reveal the mysteries of the isoflavone-phosphorylation enzyme and catalysis mechanism.

Although broad biological methods have been used to discover the formation of various water-soluble isoflavone derivatives, the microbial ability to form phosphate conjugates is limited to a few bacterial strains. Several fungal strains, such as Circinella muscae, Absidia cylindrospora, and Absidia glauca were found to phosphorylate compactin (ML-236B) and monacolin K at the C5′ hydroxyl group; these water-soluble derivatives also showed high hypocholesterolemic activity in animal experiments.30 Lack et al. elucidated that the formation of phenylphosphate is the first step in phenol degradation under anaerobic conditions in the denitrifying bacteria Pseudomonas sp. and Thauera aromatica.31 The incubation of puerarin, the 8-C-glycoside of DAI, with Bacillus cereus NT02 generated a 6″-O-phosphate conjugate, but the phosphate moiety was attached via a hydroxyl group in the 6″-O position on the glucose moiety and not directly to a phenolic hydroxyl group.32 Recently, Zühlke et al. isolated Bacillus amyloliquefaciens from sewage sludge and found that it could convert bisphenols to their phosphate conjugates; these derivatives were less toxic and less estrogenically active than their parent bisphenols.33 In this work, we purified and characterized DAI-4′P and GEN-4′P, two novel metabolites of isoflavones, in aglyconecontaining broths cultivated with B. subtilis BCRC 80517. Moreover, we examined the effects of each isoflavone on growth of B. subtilis BCRC 80517 to determine the relationship between cell growth and the bioconversion rates of isoflavone 7-O-phosphates. Although the chemical structure of DAI is similar to that of GEN and only lacks the hydroxyl group at the 5-O position, GEN had an inhibitory effect on B. subtilis BCRC 80517 growth relative to DAI. In contrast, DAI-G and GEN-G, with similar chemical structures, had the same minor effect on cell growth. Ulanowska et al. indicated that GEN inhibited the growth of B. subtilis type-culture-strain 168 at concentrations in the culture broth as low as 100 μM as compared with other flavonoids.34 GEN negatively affected the global synthesis of DNA, RNA, and protein by hampering the function of DNA topoisomerases type I and type II, so it might influence DNA topology significantly and have strong negative effects on DNA replication.35 Contrary to GEN, DAI has inhibitory activity

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b04647. ESI-MS/MS spectra of DAI-GS and GEN-GS and NMR spectra of DAI-4′P and GEN-4′P (PDF) 135


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postmenopausal osteoporosis: a consensus document by the Belgian Bone Club. Osteoporosis Int. 2010, 21 (10), 1657−1680. (9) Levis, S.; Strickman-Stein, N.; Ganjei-Azar, P.; Xu, P.; Doerge, D. R.; Krischer, J. Soy isoflavones in the prevention of menopausal bone loss and menopausal symptoms: a randomized, double-blind trial. Arch. Intern. Med. 2011, 171 (15), 1363−1369. (10) Zimmermann, C.; Cederroth, C. R.; Bourgoin, L.; Foti, M.; Nef, S. Prevention of diabetes in db/db mice by dietary soy is independent of isoflavone levels. Endocrinology 2012, 153 (11), 5200−5211. (11) Most, J.; Goossens, G.; Jocken, J.; Blaak, E. Short-term supplementation with a specific combination of dietary polyphenols increases energy expenditure and alters substrate metabolism in overweight subjects. Int. J. Obes. 2014, 38 (5), 698. (12) Wrenn, C. C. Dietary isoflavones and learning and memory. In Isoflavones: Chemistry, Analysis, Function and Effects; Preedy, V. R., Ed.; Royal Society of Chemistry: London, 2013; pp 451−464. (13) Waldmann, S.; Almukainzi, M.; Bou-Chacra, N. A.; Amidon, G. L.; Lee, B.-J.; Feng, J.; Kanfer, I.; Zuo, J. Z.; Wei, H.; Bolger, M. B.; Löbenberg, R. Provisional biopharmaceutical classification of some common herbs used in Western medicine. Mol. Pharmaceutics 2012, 9 (4), 815−822. (14) McClements, D. J.; Li, F.; Xiao, H. The nutraceutical bioavailability classification scheme: classifying nutraceuticals according to factors limiting their oral bioavailability. Annu. Rev. Food Sci. Technol. 2015, 6, 299−327. (15) Wang, A.; Zhang, F.; Huang, L.; Yin, X.; Li, H.; Wang, Q.; Zeng, Z.; Xie, T. New progress in biocatalysis and biotransformation of flavonoids. J. Med. Plants Res. 2010, 4 (10), 847−856. (16) Roh, C.; Choi, K.-Y.; Pandey, B. P.; Kim, B.-G. Hydroxylation of daidzein by CYP107H1 from Bacillus subtilis 168. J. Mol. Catal. B: Enzym. 2009, 59 (4), 248. (17) Roh, C.; Seo, S. H.; Choi, K. Y.; Cha, M.; Pandey, B. P.; Kim, J. H.; Park, J. S.; Kim, D. H.; Chang, I. S.; Kim, B. G. Regioselective hydroxylation of isoflavones by Streptomyces avermitilis MA-4680. J. Biosci. Bioeng. 2009, 108 (1), 41−6. (18) Roh, C. Biotransformation for multiple regio-selective hydroxylation of isoflavonoid. Biocatal. Agric. Biotechnol. 2013, 2 (4), 403−408. (19) Li, D.; Park, J.-H.; Park, J.-T.; Park, C. S.; Park, K.-H. Biotechnological production of highly soluble daidzein glycosides using Thermotoga maritima maltosyltransferase. J. Agric. Food Chem. 2004, 52 (9), 2561−2567. (20) Li, D.; Roh, S.-A.; Shim, J.-H.; Mikami, B.; Baik, M.-Y.; Park, C.-S.; Park, K.-H. Glycosylation of genistin into soluble inclusion complex form of cyclic glucans by enzymatic modification. J. Agric. Food Chem. 2005, 53 (16), 6516−6524. (21) Mitchell, S. C. Xenobiotic conjugation with phosphate−a metabolic rarity. Xenobiotica 2016, 46 (8), 743−756. (22) Hsu, C.; Ho, H.-W.; Chang, C.-F.; Wang, S.-T.; Fang, T.-F.; Lee, M.-H.; Su, N.-W. Soy isoflavone-phosphate conjugates derived by cultivating Bacillus subtilis var. natto BCRC 80517 with isoflavone. Food Res. Int. 2013, 53 (1), 487−495. (23) Wang, S.-T.; Fang, T.-F.; Hsu, C.; Chen, C.-H.; Lin, C.-J.; Su, N.W. Biotransformed product, genistein 7-O-phosphate, enhances the oral bioavailability of genistein. J. Funct. Foods 2015, 13, 323−335. (24) Toda, T.; Uesugi, T.; Hirai, K.; Nukaya, H.; Tsuji, K.; Ishida, H. New 6-O-acyl isoflavone glycosides from soybeans fermented with Bacillus subtilis (natto). I. 6-O-succinylated isoflavone glycosides and their preventive effects on bone loss in ovariectomized rats fed a calcium-deficient diet. Biol. Pharm. Bull. 1999, 22 (11), 1193−1201. (25) Park, C. U.; Jeong, M. K.; Park, M. H.; Yeu, J.; Park, M. S.; Kim, M. J.; Ahn, S. M.; Chang, P. S.; Lee, J. Formation of succinyl genistin and succinyl daidzin by Bacillus species. J. Food Sci. 2010, 75 (1), C128− C133. (26) Hyung Ko, J.; Gyu Kim, B.; Joong-Hoon, A. Glycosylation of flavonoids with a glycosyltransferase from Bacillus cereus. FEMS Microbiol. Lett. 2006, 258 (2), 263−268. (27) Cao, H.; Chen, X.; Jassbi, A. R.; Xiao, J. Microbial biotransformation of bioactive flavonoids. Biotechnol. Adv. 2015, 33 (1), 214−223.

AUTHOR INFORMATION

Corresponding Author

*Tel.: +886-2-33664806, Fax: +886-2-23632714, E-mail: snw@ ntu.edu.tw. ORCID

Nan-Wei Su: 0000-0002-1402-7349 Funding

This work was part of a research project supported by the Ministry of Science and Technology, Executive Yuan, Taiwan (Grant Number MOST 104-2320-B-002-027-MY3). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The MS spectra were obtained at the Joint Center for Instruments and Research, College of Bioresources and Agriculture, National Taiwan University. The NMR spectra were obtained at the Core Facility for Protein Structural Analysis supported by the National Core Facility Program for Biotechnology at Academia Sinica, Taiwan. We thank Laura Smales (BioMedEditing, Toronto, Canada) for her Englishlanguage editing.

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ABBREVIATIONS USED DAI, daidzein; GEN, genistein; DAI-G, daidzin; GEN-G, genistin; DAI-GS, 6″-O-succinyl daidzin; GEN-GS, 6″-Osuccinyl genistin; DAI-7P, daidzein 7-O-phosphate; GEN-7P, genistein 7-O-phosphate; DAI-4′P, daidzein 4′-O-phosphate; GEN-4′P, genistein 4′-O-phosphate; IS, internal standard; DMSO, dimethyl sulfoxide; HPLC, high-performance liquid chromatography; LC-MS, liquid-chromatography mass spectrometry; SD, standard deviation; UV, ultraviolet

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REFERENCES

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Phosphorylation of Isoflavone by Bacillus subtilis BCRC 80517 May

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