Plant Cell Reports
Plant Cell Reports (1987) 6:275-278
© Springer-Verlag 1987
Potential of Datura innoxia cell suspension cultures for glucosylating hydroquinone T o s h i y u k i S u z u k i 1, T o s h i h i r o Y o s h i o k a 1, M a m o r u T a b a t a 2, and Y a s u h i r o Fujita 1 Bioscience Research Center, Mitsui Petrochemical Industries, Ltd., Waki-cho, Kuga-gum Yamaguchi-ken 740, Japan 2 Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606, Japan Received February 5, 1987 / Revised version received May 7, 1987 - Communicated by F. Constabel
ABSTRACT
The efficiency of g l u c o s y l a t i Q n of h y d r o q u i n o n e by Datura innoxia cell suspension cultures was investigated. The yield of arbutin was 2.4 g/l m e d i u m when I0 m M of h y d r o q u i n o n e was added to a suspension culture that was then incubated for 24 h, but the yield d e c r e a s e d at a higher concentration. This decrease, which could not be overcome by changing the growth phase or i n c r e a s i n g the cell density used, could be avoided by the r e p e a t e d addition of a low c o n c e n t r a t i o n of h y d r o q u i n o n e over 3 days. This increased the yield of arbutin to 4.2 g/l at the usual cell d e n s i t y and to 7.1 g/l at a high density. The kinetics of this r e a c t i o n were explained by the M i c h a e l i s - M e n t e n formula. The theoretical m a x i m u m v e l o c i t y of the a r b u t i n - f o r m i n g reaction was estim a t e d as 0.77 mg/h/g. The v e l o c i t y increased linearly up to a cell density of 300 g/l under standard aeration, the theoretical m a x i m u m yield of arbutin b e i n g c a l c u l a t e d to be 5.5 g/I/day. INTRODUCTION
Several plant cell cultures have been shown to be capable of c o n v e r t i n g exogenous compounds such as phenolics (Tabata et al 1976, 1984), steroids (Furuya 1978), and c a r d e n o l i d e s (D~ller et al 1977, Furuya 1978, R e i n h a r d and Alfermann 1980) to the corresponding glucosides. Results suggest that largescale p r o d u c t i o n of useful glucosides may be possible by b i o t r a n s f o r m a t i o n if the g l u c o s y l a t i n g ability of cultured cells can be improved. Tabata et al (1976) r e p o r t e d that h y d r o q u i n o n e added to Datura innoxia cell suspension cultures was readily c o n v e r t e d to the c o r r e s p o n d i n g ~ - D - m o n o glucoside, arbutin and that this reaction was catalyzed by a g l u c o s y l t r a n s f e r a s e that require uridine d i p h o s p h a t e glucose as its high energy donor of glucose. The yield of arbutin in this e x p e r i m e n t was a p p r o x i m a t e l y 800 mg/l. To study the potential of Datura innoxia cells for the industrial p r o d u c t i o n of arbutin from hydroquinone, we investigated the effects of substrate concentration, its r e p e a t e d a d d i t i o n and the d e n s i t y of cell culture on the rate of g l u c o s y l a tion. Our results w h i c h include kinetic studies of arbutin formation are discussed. MATERIALS
Cell suspension culture
Offprint requests to: T. Suzuki
AND METHODS
Datura innoxia cell suspension cultures established by Tabata et al (1976) were used. Cultured cells (0.5 g fr. wt) were inoculated to a flask (I00 ml) containing L i n s m a i e r - S k o o g m e d i u m (20 ml) containing 10 -6 M 2 , 4 - d i c h l o r o p h e n o x y a c e t i c acid. The cultures w e r e agitated on a rotary shaker at I00 rpm for 2 or 3 weeks at 25°C in the dark. Under these culture conditions the periods of the exponential growth phase were found to be at days 10-15; the linear growth phase, at days 15-16 and the stationary phase, at days 16-18. A n a l y s i s of arbutin and h y d r o q u i n o n e H y d r o q u i n o n e (5-30 mM), as powder or aqueous solution, was added to the culture 5 to 14 days after inoculation. After further incubation for 24 h, cells were h a r v e s t e d by filtration then d r i e d at 45°C overnight. Arbutin and h y d r o q u i n o n e were extracted by sonication of the cells in methanol. In some cases, they also were p r e s e n t in the filtered medium. The arbutin and h y d r o q u i n o n e o b t a i n e d were a n a l y z e d by HPLC (column: TSK gel ODS 120A, Toyo Soda Co. Ltd.; solvent: 10% aqueous methanol; pH: a d j u s t e d to 2.5 by H3P04; flow rate: 1.5 ml/min; detection: UV at 292 nm). In this system, the retention time of arbutin was a p p r o x i m a t e l y 5.7 min and that of h y d r o q u i n o n e 8.2 min. The structure of the hydroquinone reaction product from our cell suspension cultures was confirmed to be that of arbutin by IH-NMR and FD-MS after p u r i f i c a t i o n by HPLC. The quantitative accuracy of the HPLC analysis was confirmed by IH-NMR, in w h i c h the intensities of the signals of the p u r i f i e d arbutin were compared with signals of an added internal standard (~-acetotoluidine). The amount of arbutin m e a s u r e d by IH-NMR was nearly the same as that m e a s u r e d by HPLC. R e p e a t e d additon of h y d r o q u i n o n e Equal amounts of h y d r o q u i n o n e (5-15 raM) were added to the m e d i u m on days I0, ii and 12 of culture at 24 h intervals, and cells were then h a r v e s t e d on day 13. Experiments were done both with cultures of normal i n o c u l u m size (25 g fr. wt/l) and with those of large inoculum size (i00 g fr. wt/l). M e a s u r e m e n t of the v e l o c i t y of g l u c o s y l a t i o n Various concentrations (i-i0 mM) of h y d r o q u i n o n e were added to 100-ml flasks containing 1 0 - d a y - o l d cells in the e x p o n e n t i a l ' g r o w t h stage (2.5 g fr. wt) in fresh m e d i u m (17.5 ml), the reaction taking place
276 at 25°C on a rotary shaker at I00 rpm. Cells were h a r v e s t e d at intervals of 1 or 2 h up to 8 h after inoculation. The amounts of arbutin formed were m e a s u r e d as described above. When the amount of arbutin formed per g fr. wt of cells was plotted against the r e a c t i o n time, a slight curve was obtained. The slope of the line tangent to the curve at reaction time zero was regarded as the initial v e l o c i t y of the a r b u t i n - f o r m i n g reaction. D e p e n d e n c e of the reaction v e l o c i t y on cell density Various amounts of cultured Datura innoxia cells (2-12 g fr. wt, 10-day-old) were t r a n s f e r r e d to fresh m e d i u m in 100-ml flasks, to make the total volume of the cells and m e d i u m 20 ml. After the hydroquinone (I0 mM) was added to the flasks, the v e l o c i t y of the reaction was m e a s u r e d as described above. RESULTS
AND
i
3000 4O
30
Plant Cell Reports (1987) 6:275-278
© Springer-Verlag 1987
Potential of Datura innoxia cell suspension cultures for glucosylating hydroquinone T o s h i y u k i S u z u k i 1, T o s h i h i r o Y o s h i o k a 1, M a m o r u T a b a t a 2, and Y a s u h i r o Fujita 1 Bioscience Research Center, Mitsui Petrochemical Industries, Ltd., Waki-cho, Kuga-gum Yamaguchi-ken 740, Japan 2 Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606, Japan Received February 5, 1987 / Revised version received May 7, 1987 - Communicated by F. Constabel
ABSTRACT
The efficiency of g l u c o s y l a t i Q n of h y d r o q u i n o n e by Datura innoxia cell suspension cultures was investigated. The yield of arbutin was 2.4 g/l m e d i u m when I0 m M of h y d r o q u i n o n e was added to a suspension culture that was then incubated for 24 h, but the yield d e c r e a s e d at a higher concentration. This decrease, which could not be overcome by changing the growth phase or i n c r e a s i n g the cell density used, could be avoided by the r e p e a t e d addition of a low c o n c e n t r a t i o n of h y d r o q u i n o n e over 3 days. This increased the yield of arbutin to 4.2 g/l at the usual cell d e n s i t y and to 7.1 g/l at a high density. The kinetics of this r e a c t i o n were explained by the M i c h a e l i s - M e n t e n formula. The theoretical m a x i m u m v e l o c i t y of the a r b u t i n - f o r m i n g reaction was estim a t e d as 0.77 mg/h/g. The v e l o c i t y increased linearly up to a cell density of 300 g/l under standard aeration, the theoretical m a x i m u m yield of arbutin b e i n g c a l c u l a t e d to be 5.5 g/I/day. INTRODUCTION
Several plant cell cultures have been shown to be capable of c o n v e r t i n g exogenous compounds such as phenolics (Tabata et al 1976, 1984), steroids (Furuya 1978), and c a r d e n o l i d e s (D~ller et al 1977, Furuya 1978, R e i n h a r d and Alfermann 1980) to the corresponding glucosides. Results suggest that largescale p r o d u c t i o n of useful glucosides may be possible by b i o t r a n s f o r m a t i o n if the g l u c o s y l a t i n g ability of cultured cells can be improved. Tabata et al (1976) r e p o r t e d that h y d r o q u i n o n e added to Datura innoxia cell suspension cultures was readily c o n v e r t e d to the c o r r e s p o n d i n g ~ - D - m o n o glucoside, arbutin and that this reaction was catalyzed by a g l u c o s y l t r a n s f e r a s e that require uridine d i p h o s p h a t e glucose as its high energy donor of glucose. The yield of arbutin in this e x p e r i m e n t was a p p r o x i m a t e l y 800 mg/l. To study the potential of Datura innoxia cells for the industrial p r o d u c t i o n of arbutin from hydroquinone, we investigated the effects of substrate concentration, its r e p e a t e d a d d i t i o n and the d e n s i t y of cell culture on the rate of g l u c o s y l a tion. Our results w h i c h include kinetic studies of arbutin formation are discussed. MATERIALS
Cell suspension culture
Offprint requests to: T. Suzuki
AND METHODS
Datura innoxia cell suspension cultures established by Tabata et al (1976) were used. Cultured cells (0.5 g fr. wt) were inoculated to a flask (I00 ml) containing L i n s m a i e r - S k o o g m e d i u m (20 ml) containing 10 -6 M 2 , 4 - d i c h l o r o p h e n o x y a c e t i c acid. The cultures w e r e agitated on a rotary shaker at I00 rpm for 2 or 3 weeks at 25°C in the dark. Under these culture conditions the periods of the exponential growth phase were found to be at days 10-15; the linear growth phase, at days 15-16 and the stationary phase, at days 16-18. A n a l y s i s of arbutin and h y d r o q u i n o n e H y d r o q u i n o n e (5-30 mM), as powder or aqueous solution, was added to the culture 5 to 14 days after inoculation. After further incubation for 24 h, cells were h a r v e s t e d by filtration then d r i e d at 45°C overnight. Arbutin and h y d r o q u i n o n e were extracted by sonication of the cells in methanol. In some cases, they also were p r e s e n t in the filtered medium. The arbutin and h y d r o q u i n o n e o b t a i n e d were a n a l y z e d by HPLC (column: TSK gel ODS 120A, Toyo Soda Co. Ltd.; solvent: 10% aqueous methanol; pH: a d j u s t e d to 2.5 by H3P04; flow rate: 1.5 ml/min; detection: UV at 292 nm). In this system, the retention time of arbutin was a p p r o x i m a t e l y 5.7 min and that of h y d r o q u i n o n e 8.2 min. The structure of the hydroquinone reaction product from our cell suspension cultures was confirmed to be that of arbutin by IH-NMR and FD-MS after p u r i f i c a t i o n by HPLC. The quantitative accuracy of the HPLC analysis was confirmed by IH-NMR, in w h i c h the intensities of the signals of the p u r i f i e d arbutin were compared with signals of an added internal standard (~-acetotoluidine). The amount of arbutin m e a s u r e d by IH-NMR was nearly the same as that m e a s u r e d by HPLC. R e p e a t e d additon of h y d r o q u i n o n e Equal amounts of h y d r o q u i n o n e (5-15 raM) were added to the m e d i u m on days I0, ii and 12 of culture at 24 h intervals, and cells were then h a r v e s t e d on day 13. Experiments were done both with cultures of normal i n o c u l u m size (25 g fr. wt/l) and with those of large inoculum size (i00 g fr. wt/l). M e a s u r e m e n t of the v e l o c i t y of g l u c o s y l a t i o n Various concentrations (i-i0 mM) of h y d r o q u i n o n e were added to 100-ml flasks containing 1 0 - d a y - o l d cells in the e x p o n e n t i a l ' g r o w t h stage (2.5 g fr. wt) in fresh m e d i u m (17.5 ml), the reaction taking place
276 at 25°C on a rotary shaker at I00 rpm. Cells were h a r v e s t e d at intervals of 1 or 2 h up to 8 h after inoculation. The amounts of arbutin formed were m e a s u r e d as described above. When the amount of arbutin formed per g fr. wt of cells was plotted against the r e a c t i o n time, a slight curve was obtained. The slope of the line tangent to the curve at reaction time zero was regarded as the initial v e l o c i t y of the a r b u t i n - f o r m i n g reaction. D e p e n d e n c e of the reaction v e l o c i t y on cell density Various amounts of cultured Datura innoxia cells (2-12 g fr. wt, 10-day-old) were t r a n s f e r r e d to fresh m e d i u m in 100-ml flasks, to make the total volume of the cells and m e d i u m 20 ml. After the hydroquinone (I0 mM) was added to the flasks, the v e l o c i t y of the reaction was m e a s u r e d as described above. RESULTS
AND
i
3000 4O
30
