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Advances in Chemical Engi neering and Science , 2011, 1, 90-95 doi:10.4236/aces.2011.13015 Published Online July 2011 (http://www.SciRP.org/journal/aces) Copyright © 2011 SciRes. ACES Bioremediation of Bisphenol A by Glycosylation with Immobilized Marine Microalga Amphidinium crassum —Bioremediation of Bisphenol A by Immobilized Cells Kei Shimoda1*, Ryohei Yamamoto2, Hiroki Hamada2* 1Department of Chemistry, Faculty of Medicine, Oita University, Oita, Japan 2Department of Life Science, Faculty of Science , Okayama Uni vers i t y of Sc ience, Okayama, Japan E-mail: *shimoda@med.oita-u.ac.jp, *hamada@dls.ous.ac.jp Received April 14, 2011; revised May 17, 2011; accepted May 26, 2011 Abstract Glycosylation of bisphenol A, which is an endocrine disrupting chemical, was investigated using immobi- lized marine microalga and plant cells from the viewpoint of bioremediation of bisphenol A. Immobilized marine microalga of Amphidinium crassum glucosylated bisphenol A to the corresponding glucoside. On the other hand, bisphenol A was glycosylated to its glucoside, diglycoside, gentiobioside, and gentiobiosylgluco- side, which was a new compound, by immobilized plant cells of Catharanthus roseus. Keywords: Glycosylation, Biosphenol A, Amphidinium crassum, Catharanthus roseus, Immobilized Cells 1. Introduction Bisphenol A is widely used as the starting material for the production of polyacrylates, ether resins, phenol resins, photostabilizers, insecticides, fragrance ingredients, ag- ricultural chemicals, pharmaceuticals, and coatings, and are released as pollutants and toxic compounds into rivers and seas [1]. Recently, bisphenol A has attracted consid- erable attention as it exhibited estrogenic activity in bio- assays [2] and has been listed among “chemicals sus- pected of having endocrine disrupting effects” by the World Wildlife Fund, the National Institute of Environ- mental Health Sciences in the USA and the Japanese En- vironment Agency. From the viewpoint of pollution con- trol, many studies on the biological metabolites of aro- matic compounds have been reported, e.g., the benzene rings of aromatic compounds are degraded through the gentisic acid intermediate by some soil bacteria [3-8]. However, little attention has been paid to the biological degradation of endocrine disrupting chemicals. On the other hand, the metabolic pathway of aromatic compounds in plant cells is quite different from that in microorganisms; plant cells glycosylate phenols and accumulate them as glycosides in the cells [9-15]. Recently, the biotransformation of exogenous sub- strates by cultured marine microalga and plant cells has been reported [16,17]. These cells have the abilities of hydroxylation, glycosylation, oxido-reduction, hydrogena- tion, and hydrolysis for various organic compounds. Par- ticularly, glycosylation seems to be an efficient procedure for the bioremediation of environmental pollution, be- cause the estrogenicity of endocrine disrupting compound, i.e., bisphenol A, was eliminated by formation of its glycosides [18]. This paper describes the glycosylation of bisphenol A by the immobilized marine microalga of Am- phidinium crassum and immobilized plant cells of Ca- tharanthus roseus. 2. Experimental 2.1. General Bisphenol A was purchased from Aldrich Chemical Co. The 1H and 13C NMR, H-H COSY, C-H COSY, and HMBC spectra were recorded in CD3OD using a Varian XL-400 spectrometer (Varian Inc.). The chemical shifts were expressed in δ (ppm) referring to tetramethylsilane. The FABMS spectra were measured using a JEOL MStation JMS-700 spectrometer (JEOL Ltd.). HPLC was carried out on a YMC-Pack R&D ODS column (150 × 30 mm) at 25˚C [solvent: methanol-water (9:11, v/v); detection: UV (280 nm); flow rate: 1.0 ml/min]. 2.2. Cell Line and Culture Conditions A. crassum, a gift from Ehime Prefectural Fisheries Ex- K. SHIMODA ET AL. Copyright © 2011 SciRes. ACES 91 perimental Station, Japan, cells were cultivated in a syn- thetic seawater (500 ml) for 2 weeks at 20˚C with con- stant aeration by air (1 l/min) in 1 l flasks under illumi- nation (1000 lx). The synthetic seawater contained 20.747 g NaCl, 0.8 g MnCl2·4H2O, 9.474 g MgCl2·6H2O, 1.326 g CaCl2·6H2O, 3.505 g Na2SO4, 597 mg KCl, 171 mg NaHCO3, 85mg KBr, 34mg Na2B4O7·10H2O, 12 mg SrCl2, 3 mg NaF, 1 mg LiCl, 0.07 mg KI, 0.2g CoCl2·6H2O, 8 g AlCl3·6H2O, 5 g FeCl3·6H2O, 0.2 g Na2WO4·2H2O, 0.02 mg (NH4)6Mo7O24, 0.0045% Na2SiO 3 and 1.07 ml of NM solution per 1 l of distilled water. The NM solution (1 l) is a kind of vitamin solutions and composed of NaNO3 (150 g), Na2HO4 (10 g), EDTA-2Na (0.9 g), Vi- tamin B12 (1.5 mg), thiamine·HCl (75 mg), biotin (1 mg), EDTA-Fe (2.5 g), and H2NC(CH3OH)3 (5 g) in distilled water. The cultured plant cells of C. roseus have been culti- vated over 20 years in our laboratory and subcultured in 300 ml conical flasks containing Schenk and Hildebrand (SH) medium (100 ml, pH 5.7) on a rotary shaker (120 rpm) at 25˚C in the dark for every 3 - 5 weeks. Part of the callus tissues (fresh weight 30 g) was transplanted to freshly prepared SH medium (100 ml in a 500 ml conical flask, pH 5.7) containing 3% sucrose and was incubated for 3 weeks prior to use for this work. 2.3. Glycosylation of Bisphenol A by A. crassum and C. roseus Cultured A. crassum cells were harvested by centrifuga- tion at 3000 rpm for 15 min and washed twice by adding 100 ml of synthetic seawater followed by centrifugation (3000 rpm for 15 min). To the 500 ml flask containing 9 g of cultured A. crassum cells and 300 ml of a synthetic seawater was added 0.2 mmol of bisphenol A. The cul- tures were incubated at 20˚C on a rotary shaker (120 rpm) for five days under illumination (1000 lx). After the incubation period, the cells and synthetic seawater were separated by centrifugation at 1000 g for 15 min. The syn- thetic seawater was extracted with ethylacetate and then n-butanol. The cells were extracted (three times) by ho- mogenization with methanol, and the methanol fraction was concentrated and partitioned between water and ethylacetate. The ethylacetate fractions were analyzed by HPLC, combined, and concentrated. The water and n- butanol fractions were analyzed by HPLC, combined, evaporated, and re-dissolved in water. This water fraction was applied to a Diaion HP-20 column and the column was washed with water followed by elution with metha- nol. The methanol eluate was subjected to preparative HPLC [column: CAPCELLPAK R&D C18 column (250 × 30 mm); solvent: MeOH: H2O (9:11, v/v); detection: UV (340 nm); flow rate: 1.0 ml/min] to give glycosyla- tion products. Bisphenol A (0.2 mmol) was administered to the 500 ml flask containing 300 ml of SH medium and 70 g of the suspension cultured cells of C. roseus, and the cul- tures were incubated at 25˚C for five days on a rotary shaker (120 rpm) under illumination (1000 lx). After the incubation, the cells and medium were separated by fil- tration with suction. The filtered medium was extracted with EtOAc. The medium was further extracted with n- BuOH. The cells were extracted (x3) by homogenization with MeOH. The MeOH fraction was concentrated and partitioned between H2O and EtOAc. The EtOAc fractions were combined and concentrated. The H2O fraction was applied to a Dianion HP-20 column and the column was washed with H2O followed by elution with MeOH. The MeOH eluate was subjected to HPLC to give products. Spectral data of a new compound, 2-(4--gentiobiosy- loxphenyl)-2-(4--D-glucopyranosyloxyphenyl)propane (5): FAB MS: m/z 737 [M + Na]+; 1H NMR (400 MHz, CD3OD, in ppm): 1.60 (s, 6H, H-1, 3), 3.25-3.90 (m, 18H, H-2', 3', 4', 5', 6', 2'', 3'', 4'', 5'', 6'', 2''', 3''', 4''', 5''', 6'''), 4.50 (d, 1H, J=7.6 Hz, H-1'''), 4.86 (d, 1H, J=8.0 Hz, H-1'), 4.87 (d, 2H, J = 8.0 Hz, H-1''), 6.95 (d, 2H, J = 8.5 Hz, H-12, 14), 7.00 (d, 2H, J = 8.5 Hz, H-6, 8), 7.12 (d, 2H, J = 8.5 Hz, H-11, 15), 7.15 (d, 2H, J = 8.5 Hz, H-5, 9); 13C NMR (CD3OD): 31.5 (C-1, C-3), 42.9 (C-2), 62.3, 62.5 (C-6', C-6'''), 68.8 (C-6''), 71.2, 71.5 (C-4', C-4'', C-4'''), 75.0, 75.1 (C-2', C-2'', C-2'''), 77.7, 78.0, 78.1 (C-3', C-3'', C-3'''), 78.2, 78.3, 78.8 (C-5', C-5'', C-5'''), 102.5 (C-1', C-1''), 105.1 (C-1'''), 117.0 (C-12, C-14), 117.2 (C-6, C-8), 128.1 (C-11, C-15), 128.7 (C-5, C-9), 146.1 (C-10), 146.8 (C-4), 157.0 (C-13), 157.6 (C-7). 2.4. Preparation of Immobilized A. crassum and C. roseus in Sodium Alginate Gel Sodium alginate (2%) was suspended in water (500 ml), which was autoclaved at 120˚C for 30 min. The cultured cells in the stationary growth phase have been used for experiments. Cultured cells of A. crassum (9 g) and C. roseus (70 g) were individually added to this solution and the mixture was stirred for 2 h until it became ho- mogeneous. The suspension was added dropwise from a dropping funnel with a glass tube into a 5% CaCl2 solu- tion (1 l) with stirring to form pieces of spherical sodium alginate gel with 5 mm diameter immediately. Washing with water gave each immobilized cells of A. crassum and C. roseus which were used for biotransformation of bisphenol A. 2.5. Time Course Experiments Time course experiments to examine the biotransforma- K. SHIMODA ET AL. Copyright © 2011 SciRes. ACES 92 tion of bisphenol A by A. crassum were carried out using eight flasks containing cultured cells (9 g) or immobi- lized cells, which included 9 g cells. In the case of the biotransformation by the cultured and immobilized C. roseus cells, cultured cells (70 g) or immobilized cells, which included 70 g cells, were partitionated to each flask. Substrate (0.2 mmol) was administered to each of flasks and the mixtures were incubated on a rotary shaker at 25˚C. At a day interval, one of the flasks was taken out from the rotary shaker, and the cells (or immobilized cells) and medium were separated by filtration. The ex- traction and analysis procedures were same as described above. The yield of the products was determined on the basis of the peak area from HPLC and expressed as a relative percentage to the total amount of the whole reac- tion products extracted. 3. Results and Discussions The biotransformation product was isolated from A. crassum cell cultures, which had been incubated with bisphenol A (1) for five days, by a combination of Diaion HP-20 column chromatography and preparative HPLC in 4% yield. The glycosylation product 2 was detected by HPLC. No additional conversion products were observed in spite of careful analyses by HPLC. Incubation of the substrate in medium without cells gave no transformation products. The structure of the product 2 was determined as 2-(4--D-glucopyranosyloxyphenyl)-2-hydroxypheny- lpropane (bisphenol A glucoside) by FABMS, 1H and 13C NMR analyses (Figure 1). To investigate the biotrans- formation pathway, the time course in the conversion of 1 was followed. Figure 2 showed that the amount of pro- duct 2 increased with time during the reaction with cul- tured A. crassum cells. Next, A. crassum cells were immobilized with sodium alginate at concentrations of 2%. The immobilized A. crassum cells were incubated with bisphenol A (1) for five days. The product 2 was obtained in 6% yield. The time course of the conversion of bisphenol A (1) with immobilized A. crassum cells was investigated. As shown in Figure 3, the glycosylation activity for bisphenol A (1) was increased and the compound 2 was produced in higher yield in comparison with the case of the biotrans- formation using normal cells. On the other hand, four biotransformation products 2-5 were isolated by a combination of Diaion HP-20 column chromatography and preparative HPLC after five days incubation of cultured plant cells of C. roseus with bisphenol A (1). The yields of 2-5 were 5, 12, 8, and 2%. The structures of the products 3-6 were identifined as 2,2-bis(4--D-glucopyranosyloxyphenyl)propane (di- glucoside, 3), 2-(4--genbiobiosyloxyphenyl)-2-(4- hydroxyphenyl) propane (gentiobioside, 4), and 2-(4-- gentiobiosylophenyl)-2-(4--D-glucopyranosyloxyph- enyl) propane (gentiobiosylglucoside, 5) (Figure 4). The CHO OH CH3 CH3 COOH CH3 CH3 O OH HO HO OH Bisphenol A (1)2 1 2 3 4 56 7 89 10 11 12 13 1415 A. crassum Figure 1. Glycosylation of bisphenol A (1) by cultured cells of A. crassum. 50 100 48 Time/day Figure 2. Time course of the glycosylation of bisphenol A (1) by the cultured cells of A. crassum. Yields of 1 (■) and 2 (●) are plotted. K. SHIMODA ET AL. Copyright © 2011 SciRes. ACES 93 50 100 48 Time/day Figure 3. Time course of the glycosylation of bisphenol A (1) by the immobilized cells of A. crassum. Yields of 1 (■) and 2 (●) are plotted. product 5 was a new compound. To investigate the bio- transformation pathway, the time course in the conver- sion of 1 by cultured C. roseus cells was examined. Products 2, 3, and 4 were produced at an early stage of incubation. On the other hand, 5 was accumulated after 3 days of incubation (Figure 5). These findings indicated that 1 was first converted to 2-4 and further glycosylation gave 5 as shown in Figure 4. Immobilized C. roseusm cells were tested for their ability to convert bisphenol A (1). The substrate, bisphe- nol A (1), was converted into products 2-5 in 7, 17, 11, and 4% yields by five days incubation. The time course of the conversion of bisphenol A (1) with immobilized A. crassum cells was investigated. As shown in Figure 6, the products 2-5 were obtained in higher yields in com- parison with the case of the biotransformation using nor- CHO OH CH3 CH3 COOH CH3 CH3 O OH HO HO OH COO CH3 CH3 O OH HO HO OH COOH CH3 CH3 O OH HO HO O O HO OH OH HO O OH HO HO OH COO CH3 CH3 O OH HO HO O O HO OH OH HO O OH HO HO OH 1 2 C. roseus 3 4 5 Figure 4. Glycosylation of bisphenol A (1) by cultured cells of C. roseus. 50 100 48 Time/day Figure 5. Time course of the glycosylation of bisphenol A (1) by the cultured cells of C. roseus. Yields of 1 (■), 2 (●), 3 (♦), 4 (▲), and 5 (○) are plotted. K. SHIMODA ET AL. Copyright © 2011 SciRes. ACES 94 50 100 48 Time/day Figure 6. Time course of the glycosylation of bisphenol A (1) by the immobilized cells of C. rose us. Yields of 1 (■), 2 (●), 3 (♦), 4 (▲), and 5 (○) are plotted. mal cells. The results of this experiment demonstrate that cul- tured marine microalga of A. crassum converted bisphe- nol A into its glucoside and that cultured plant cells of C. roseus glycosylate bisphenol A to its glucoside, digluco- side, gentiobioside, gentiobiosylglucoside.The use of immobilized cells of both A. crassum and C. roseus in sodium alginate gel much improved the yield of the products. Recently, it has been reported that freshwater micro- alga of Pseudokirchneriella subcapitata, Scenedes- musacutus, and Coelastrum reticulatum converted bisphenol A into its glucoside [19]. Also, bisphenol A was shown to be transformed to its glucoside, digluco- side, gentiobioside, and trisaccharide, i.e., O--D- glucopysyl-(1→4)-[-D-glucopyranosyl-(1→6)]-D- glucopyranoside, by cultured plant cells of Nicotiana tabacum [20]. 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