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[54] Biotransformation of hydrocortisone into prednisolone
154l
PREDNISOLONE
559
[54] B i o t r a n s f o r r n a t i o n of H y d r o c o r t i s o n e into P r e d n i s o l o n e By RAJNI KAtJL and Bo MATTIASSON
Introduction Biotransformations of organic compounds involve processes that are often characterized by dilute product streams owing to the low solubility of the substrate and/or product in the reaction medium. Several methods to improve these processes have been presented. ~-3 Aqueous two-phase systems offer a useful environment for biotransformations as evidenced by a model study on hydrocortisone transformation using Arthrobacter simplex. 4 Aqueous two-phase systems are biocompatible and even permit cells to grow.5'6 These systems may also possess hydrophobic components as chains or side chains linked to phase-forming polymers and thereby enhance the solubility of the reactants. These properties make two-phase systems a favorable setting for bioconversions. Transformation in Aqueous Two-Phase Systems Two-Phase Systems
Most of the studies have been carried out with two-phase systems having fractionated dextran (Dx) as the bottom phase polymer. However, the use of Dx on a large scale is not economical. Hence, some relatively inexpensive polymers are being studied as substitutes for the Dx. Here, Reppal PES, a hydroxypropyl starch is used as such a replacement. All the phase systems are prepared in 50 mM Tris-HCl solution, pH 7.0. The polymers forming the two-phase system are weighed and mixed in the buffer at the desired concentrations (w/w). Complete phase separation takes place within 30 min at room temperature. The volumes of the two phases are noted. The phase systems used with Dx T40 and Dx T70 (Pharmacia, Uppsala, Sweden) as the bottom phase polymer and different top phase polymers, namely, poly(ethylene glycol) (PEG) 8000 (Union Carbide, New York, i G. 2 E. 3 B. 4 R. 5 M.
Carrea, TIBTECH. 2, 102 (1984). K o n d o and E. M a s u o , J. Gen. Appl. Microbiol. 7, 113 (1961). M a t t i a s s o n a n d P. Adlercreutz, Trends Biotechnol. 9, 394 (1991). K a u l and B. Mattiasson, Appl. Microbiol. Biotechnol. 24, 259 (1986). L a r s s o n , V. A r a s a r a t n a m , and B. Mattiasson, J. Microb. Biotechnol. 3, 22 (1988).
6M. Larsson, V. Arasaratnam,and B. Mattiasson,Biotechnol. Bioeng. 3, 58 (1989). METHODS IN ENZYMOLOGY, VOL. 228
Copyright © 1994by AcademicPress, Inc. All rights of reproduction in any form reserved.
560
BIOTECHNOLOGY
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TABLE I EFFECTS OF VARIATIONS IN PHASE COMPOSITION ON PARTITIONING BEHAVIOR OF HYDROCORTISONE AND PREDNISOLONE a
Partition coefficient (K) Phase system [% (w/w) : % (w/w)]
Volume ratio (top to bottom phase)
Hydrocortisone
12.3 : 1 15.7 : I 19.0 : 1 8.5:1
2.6 2.8 2.8 3.3
6.4 6.6 6.8 7.1
7.0 : 1 4.3 : 1
2.6 2.7
2.9 3.2
0.5:1
11.5
15.6
24.0 : 1
2.0
2.2
8.1:1 27.6 : 1
1.7 1.5
5.0 4.3
P E G 8 0 0 0 - d e x t r a n T40 15 : 3 20 : 3 25 : 3 25:6 Pluronic F68-dextran T40 14 : 4 14 : 6 PPG 2 0 2 5 - d e x t r a n T40 25:3 M - P E G 5 5 0 - d e x t r a n T40 25 : 3 M - P E G 5 0 0 0 - d e x t r a n T70 9.7:4 10.7 : 2
Prednisolone
a F r o m R. Kaul and B. Mattiasson, Appl. Microbiol. Biotechnol. 24, 259 (1986), with permission.
NY), methoxypoly(ethylene glycol) (M-PEG) 550 and 5000 (Polysciences, Northampton, UK), poly(propylene glycol) (PPG) 2025 (BDH, Poole, UK), or Pluronic F-68 (Montoil AB, Stockholm, Sweden) are listed in Table I. Two-phase systems with Reppal PES 200 (Reppe Glykos, Viixj6, Sweden) and PEG 8000 as phase polymers are also prepared in the same manner.
Estimation of Steroid Content A steroid sample (50 /~1) is shaken with chloroform (0.2 ml). 7 The chloroform phase is subjected to analysis by high-performance liquid chromatography (HPLC) of hydrocortisone and prednisolone (Serva, Heidelberg, Germany) using an LDC chromatograph (Tillquist, Stockholm, Sweden). The steroid separation is carried out on a Spherisorb column (250 × 4.6 mm) with 5/zm particle size and eluting with a mobile phase of 98% (v/v) chloroform and 2% (v/v) methanol at a flow rate of 1.5 ml/min. Detection is by absorbance at 254 nm. Under these conditions the retention times for hydrocortisone and prednisolone are 6.1 and 7.7 rain, respectively. 7 R. Kaul, P. Adlercreutz, and B. Mattiasson, Biotechnol. Bioeng. 28, 1432 (1986).
[54]
PREDNISOLONE
561
T A B L E II SOLUBILITY OF HYDROCORTISONE IN INDIVIDUAL PHASES OF Two-PHASE SYSTEMSa
System 25% PEG 8000-6% dextran T40 12% PEG 8000-3% Reppal PES 200
Phase
Solubility (mg/ml)b
PEG-rich Dextran-rich PEG-rich Reppal-rich
1.0 0.1 0.69 0.74
From R. Kauland B. Mattiasson,Appl. Microbiol.Biotechnol.24, 259 (1986), withpermission. b Solubilityin pure water under these conditionswas 0.2 mg/ml.
Determination of Partition Coefficients, K, of Steroids The K values of hydrocortisone and prednisolone are determined by first stirring the respective steroids into a two-phase system for a few hours at 20° . The phase separation is carded out by allowing the systems to stand for 30 min or by centrifuging them at 1625 g for 5 min at 20°. Aliquots are removed from both phases and their steroid concentration determined. K is calculated as the ratio between the concentrations in top and bottom phases. Table I shows the partitioning of both hydrocortisone and prednisolone in the two-phase systems using Dx and different top phase polymers. The steroids partition more toward the top phase. PPG was particularly effective in producing high K values. The steroid crystals which did not go into solution settled onto the interface.
Determination of Hydrocortisone Solubility in Polymer Solutions The respective phases of the two-phase systems, 25% PEG 8000-Dx T40 and 12% PEG 8000-3% Reppal PES 200, are separated. Hydrocortisone (4 mg/ml) is added to the respective phases and stirred for about 24 hr at 20°. The undissolved steroid is sedimented by high-speed centrifugation (12,000 g for 20 rain) at 4°, and the amount dissolved is measured in the supernatant solution. The solubility in water is determined under identical conditions. The solubility of hydrocortisone in the PEG solution was I0 times greater than in Dx (Table II). Reppal solubilized even a little more steroid than PEG. Reppal contains hydroxypropyl groups, which imparts to the polymer a weak hydrophobic character.
562
BIOTECHNOLOGY
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Organism and Culture Conditions Arthrobacter simplex ATCC 6946 (DSM, No. 20130, Braunschweig, Germany) is used for the transformation of hydrocortisone to prednisolone. 7The bacteria are grown at 30° with shaking in a medium (pH 7.2-7.4) containing 1% (w/v) casein peptone tryptic digest (Difco, Detroit, MI), 0.5% yeast extract (Difco), 0.5% (w/v) NaCI, and 0.5% (w/v) glucose. After 24 hr of growth, the cells are induced for the enzyme, steroid A~dehydrogenase, by addition of hydrocortisone (1 mg/ml) to the medium and continued incubation for another 6 hr. The bacteria are collected by centrifugation at 12,000 gmax (4°) for 10 min, washed with 50 mM TrisHC1 solution, pH 7.0, and stored at -20 ° until use. Transformation Reaction To 20 ml of the phase system, in a 100-ml beaker, is added hydrocortisone powder, and the mixture is stirred for a few minutes. The reaction is started by addition of bacterial cells. Samples are withdrawn periodically to follow the course of transformation. At the end of the reaction the phases are allowed to separate, and concentrations of the product in the phases are determined. At a substrate concentration of 1 mg/ml and 22 mg (dry weight) bacterial cells, more than 99% of hydrocortisone could be transformed in the two-phase systems (Table III). Because phase volume ratios (top/bottom) of these systems are high (except for those of PPG-Dx) the major amount of product formed is in the top phase. The bottom phase contains a small percentage of product and some untransformed substrate (Table III). The reaction efficiency in the PEG-Reppal system was found to be similar to that in the PEG-Dx system. However, with PEG-Reppal the bottom phase contained more product, namely, 12% in the 12% PEG-3% Reppal system and 20% in the 10% PEG-5% Reppal system, corresponding well to the steroid solubility. Studies in a 25% PEG-6% Dx system with a constant substrate to cell ratio reveal that nearly complete transformation occurs only up to a substrate concentration of 2 mg/ml. At 5 mg/ml the conversion attained is 95% (Fig. 1). Incomplete hydrocortisone transformation at higher concentrations has been reported. 2,~ Many situations demand the complete conversion of a substrate, since ultimately the separation of product from unreacted substrate becomes critical.
S. Ohlson, P.-O. Larsson, and K. Mosbach, Fur. J. Appl. Microbiol. Biotechnol. 7, 103 (1979).
[54]
PREDNISOLONE
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TABLE III HYDROCORTISONE TRANSFORMATION IN AQUEOUS Two-PHASE SYSTEMSa Hydrocortisone conversion (%) in Phase system [% (w/w) : % (w/w)] PEG 8000-dextran T40 15 : 3 20 : 3 25 : 3 25 : 6 Pluronic F68-dextran T40 14 : 4 14 : 6 PPG 2025-dextran T40 25 : 3 M-PEG 550-dextran T40 25 : 3 M-PEG 5000-dextran T70 9.7 : 4 10.7 : 2
Amount of prednisolone in bottom phase (% of total)
Reaction time (hr)
Total system
Top phase b
2.5 2.5 2.5 2.5
99.5 99.8 99.6 100
99.8 100 99.6 100
76 62 65 65
4. I 4.0 1.0 1.5
2.5 1.75
100 100
100 100
94 85
6.8 8.6
Bottom phase b
3.0
99.5
99.3
93
20
6.5
99.4
99.4
99
2.1
4.0 8.0
99.4 98.5
99.8 98.4
99.2 91
8.5 1.2
From R. Kaul and B. Mattiasson, Appl, Microbiol. Biotechnol. 24, 259 (1986), with permission. b After completion of the reaction, the phases were separated, and the degree of conversion was determined in the respective phases.
Increasing Oxygen Supply One limiting factor during conversion of hydrocortisone to predniso[one is the lack of oxygen. By improving the oxygen supply to the reaction mixture it is possible to increase the rate of transformation, especially at high substrate concentrations. To increase the oxygen supply, the reaction can be carried out in the presence of perfluorochemicals, which are good solvents for gases. 9 Preparation of Emulsion of Perfluorochemical. The emulsion (30% perfluorochemical, v/v) is prepared as described earlier.l° The buffer solution (water phase), the perfluorochemical FC-72 (3M, St. Paul, MN) (organic phase), and Pluronic F-68 (emulsifier) are mixed in appropriate proportions. The amount of Pluronic F-68 is 0.21 g/ml FC-72. The mixture (50 ml) is placed in a 400-ml beaker and cooled in crushed ice. It is 9 B. Mattiasson and P. Adlercreutz, Trends Biotechnol. 5, 250 (1987). l0 p. Adlercreutz and B. Mattiasson, Eur. J. Appl. Microbiol. Biotechnol. 16, 165 (1982).
564
BIOTECHNOLOGY
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FIo. 1. Time course for hydrocortisonetransformationby Arthrobactersimplex cells in a 25% (w/w)PEG 8000-6% (w/w)dextranT40 systemat 20°, Hydrocortisoneconcentrations used were 1 (x), 2 (O), 3 (E2), 4 (A), and 5 mg (~7) per milliliterof reaction medium. Experimental details are described in the text. [From R. Kaul and B. Mattiasson, Appl. Microbiol. Biotechnol. 24, 259 (1986), with permission.] then sonicated using MSE Soniprep 150 Ultrasonic disintegrator (MSE Scientific Instruments, Sussex, England) for 5 min until a white emulsion forms. Use o f Perfluorochemical Emulsion During Bioconversion. The emulsion (4 ml) is added to 20 ml of the two-phase system containing hydrocortisone (either 1 or 3 mg/ml), and the cells (22 or 66 mg dry weight, respectively) are then added. In the presence of the perfluorochemical emulsion, the transformation rate was found to increase, and the conversion of higher concentrations of hydrocortisone nearly reached 100% (Fig. 2). On completion of the reaction, as the phase system was allowed to stand, the emulsion formed a separate phase below that of Dx and could easily be recovered. Perfluorochemicals are very poor solvents for organic chemicals and the product was, hence, recovered in the polymer phases.
Product Extraction from Top Phase o f Aqueous Two-Phase System Amberlite XAD-4 (BDH) is packed into a column (17 x 0.6 cm) and rinsed with 50 mM Tris-HCl solution, pH 7.0. The PEG-rich top phase from the 25% (w/w) PEG 8000-6% (w/w) Dx T40 system is separated after completion of hydrocortisone transformation, then passed through the Amberlite column at a flow rate of 0.5 ml/min. The column is washed with buffer and flushed with methanol for the desorption of prednisolone. The column is then regenerated by washing with buffer. The PEG eluate is generally devoid of steroid product, and the latter
[54]
PREDNISOLONE
565
1.0
0.8 0.6 0.4 o
~o.2 3 ~o
I
(h~
C O
.81.o, @
.~0.8 "0.6 0.4 0.2
o
~
Time (h)
,~
FIG. 2. Transformation of hydrocortisone to prednisolone by Arthrobaeter simplex cells in 25% (w/w) PEG 8000-6% (w/w) dextran T40 system at 20° in the presence (A) and absence (O) of an emulsion of a perfluorochemical (F-72). The concentrations of hydrocortisone used were (a) 1 and (b) 3 mg/ml of the phase system. [From R. Kaul and B. Mattiasson, Appl. Microbiol. Biotechnol. 24, 259 (1986), with permission.]
is totally desorbed by methanol. The solvent is evaporated so as to obtain prednisolone crystals. Sometimes prednisolone, up to 5% of the amount loaded, could be detected in the eluate. This is extracted by reloading the eluate onto the column. Adsorption to the Amberlite resin is a mild process and permits the phase polymer to be recycled. Amberlite could be used repeatedly after regeneration with buffer.
Recycling of Cells and Phase Polymers The r e c o v e r e d P E G phase is supplemented with substrate and returned to the bioreactor, which contains the Dx-rich phase and the cells. P E G
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lost during product extraction is replaced. Alternatively, a fresh PEGrich phase can be used. The transformation rate improved by 25-75% in a number of runs. 4 This was attributed to further induction of enzyme activity in the cells by the substrate. A similar observation had been reported earlier. 7 A decline in enzyme activity occurred, however, after about 7 runs, which resulted in slightly longer times for complete conversion. Enhancement of A~-dehydrogenaseactivity of immobilized A. simplex is possible when the cells are incubated in nutrient media. 8 This activation is due to an increase in the number of cells that are simultaneously being induced for dehydrogenase activity by the substrate hydrocortisone. Application of a similar approach here, that is, adding the nutrients to the aqueous two-phase system, helped maintain the catalytic activity of the cells. 4 It is important, however, to maintain sterile conditions in longterm operations. Remarks Table IV compares aqueous two-phase systems with some of the methods previously used for hydrocortisone transformation. It is evident that the reaction rate achieved in the two-phase system is among the highest reported. Immobilized cells have earlier been employed to protect the biocatalyst from denaturation. 1~Conversion of hydrocortisone to prednisolone is also subject to product inhibition. 12Aqueous two-phase systems offer suitable reaction conditions even when the viability of the microbial cells is to be maintained. The top phase serves as a steroid reservoir, whereas the cells are confined to the bottom phase. As the reaction proceeds the product is extracted simultaneously to the top phase and away from the biocatalyst. By manipulation of the phase volume ratios, it is possible to obtain most of the product in the top phase. Acknowledgments The authors are grateful to The National Swedish Board for Technical and Industrial Development (NUTEK) for financial support.
ii K. Sonomoto, A. Tanaka, T. Omata, T. Yamane, and S. Fukui, Fur. J. Appl. MicrobioL BiotechnoL 6, 325 (1979). ~2j. Kloosterman IV and M. D. Lilly, Enzyme Microb. Technol. 6, 113 (1984).
PREDNISOLONE
559
[54] B i o t r a n s f o r r n a t i o n of H y d r o c o r t i s o n e into P r e d n i s o l o n e By RAJNI KAtJL and Bo MATTIASSON
Introduction Biotransformations of organic compounds involve processes that are often characterized by dilute product streams owing to the low solubility of the substrate and/or product in the reaction medium. Several methods to improve these processes have been presented. ~-3 Aqueous two-phase systems offer a useful environment for biotransformations as evidenced by a model study on hydrocortisone transformation using Arthrobacter simplex. 4 Aqueous two-phase systems are biocompatible and even permit cells to grow.5'6 These systems may also possess hydrophobic components as chains or side chains linked to phase-forming polymers and thereby enhance the solubility of the reactants. These properties make two-phase systems a favorable setting for bioconversions. Transformation in Aqueous Two-Phase Systems Two-Phase Systems
Most of the studies have been carried out with two-phase systems having fractionated dextran (Dx) as the bottom phase polymer. However, the use of Dx on a large scale is not economical. Hence, some relatively inexpensive polymers are being studied as substitutes for the Dx. Here, Reppal PES, a hydroxypropyl starch is used as such a replacement. All the phase systems are prepared in 50 mM Tris-HCl solution, pH 7.0. The polymers forming the two-phase system are weighed and mixed in the buffer at the desired concentrations (w/w). Complete phase separation takes place within 30 min at room temperature. The volumes of the two phases are noted. The phase systems used with Dx T40 and Dx T70 (Pharmacia, Uppsala, Sweden) as the bottom phase polymer and different top phase polymers, namely, poly(ethylene glycol) (PEG) 8000 (Union Carbide, New York, i G. 2 E. 3 B. 4 R. 5 M.
Carrea, TIBTECH. 2, 102 (1984). K o n d o and E. M a s u o , J. Gen. Appl. Microbiol. 7, 113 (1961). M a t t i a s s o n a n d P. Adlercreutz, Trends Biotechnol. 9, 394 (1991). K a u l and B. Mattiasson, Appl. Microbiol. Biotechnol. 24, 259 (1986). L a r s s o n , V. A r a s a r a t n a m , and B. Mattiasson, J. Microb. Biotechnol. 3, 22 (1988).
6M. Larsson, V. Arasaratnam,and B. Mattiasson,Biotechnol. Bioeng. 3, 58 (1989). METHODS IN ENZYMOLOGY, VOL. 228
Copyright © 1994by AcademicPress, Inc. All rights of reproduction in any form reserved.
560
BIOTECHNOLOGY
[54]
TABLE I EFFECTS OF VARIATIONS IN PHASE COMPOSITION ON PARTITIONING BEHAVIOR OF HYDROCORTISONE AND PREDNISOLONE a
Partition coefficient (K) Phase system [% (w/w) : % (w/w)]
Volume ratio (top to bottom phase)
Hydrocortisone
12.3 : 1 15.7 : I 19.0 : 1 8.5:1
2.6 2.8 2.8 3.3
6.4 6.6 6.8 7.1
7.0 : 1 4.3 : 1
2.6 2.7
2.9 3.2
0.5:1
11.5
15.6
24.0 : 1
2.0
2.2
8.1:1 27.6 : 1
1.7 1.5
5.0 4.3
P E G 8 0 0 0 - d e x t r a n T40 15 : 3 20 : 3 25 : 3 25:6 Pluronic F68-dextran T40 14 : 4 14 : 6 PPG 2 0 2 5 - d e x t r a n T40 25:3 M - P E G 5 5 0 - d e x t r a n T40 25 : 3 M - P E G 5 0 0 0 - d e x t r a n T70 9.7:4 10.7 : 2
Prednisolone
a F r o m R. Kaul and B. Mattiasson, Appl. Microbiol. Biotechnol. 24, 259 (1986), with permission.
NY), methoxypoly(ethylene glycol) (M-PEG) 550 and 5000 (Polysciences, Northampton, UK), poly(propylene glycol) (PPG) 2025 (BDH, Poole, UK), or Pluronic F-68 (Montoil AB, Stockholm, Sweden) are listed in Table I. Two-phase systems with Reppal PES 200 (Reppe Glykos, Viixj6, Sweden) and PEG 8000 as phase polymers are also prepared in the same manner.
Estimation of Steroid Content A steroid sample (50 /~1) is shaken with chloroform (0.2 ml). 7 The chloroform phase is subjected to analysis by high-performance liquid chromatography (HPLC) of hydrocortisone and prednisolone (Serva, Heidelberg, Germany) using an LDC chromatograph (Tillquist, Stockholm, Sweden). The steroid separation is carried out on a Spherisorb column (250 × 4.6 mm) with 5/zm particle size and eluting with a mobile phase of 98% (v/v) chloroform and 2% (v/v) methanol at a flow rate of 1.5 ml/min. Detection is by absorbance at 254 nm. Under these conditions the retention times for hydrocortisone and prednisolone are 6.1 and 7.7 rain, respectively. 7 R. Kaul, P. Adlercreutz, and B. Mattiasson, Biotechnol. Bioeng. 28, 1432 (1986).
[54]
PREDNISOLONE
561
T A B L E II SOLUBILITY OF HYDROCORTISONE IN INDIVIDUAL PHASES OF Two-PHASE SYSTEMSa
System 25% PEG 8000-6% dextran T40 12% PEG 8000-3% Reppal PES 200
Phase
Solubility (mg/ml)b
PEG-rich Dextran-rich PEG-rich Reppal-rich
1.0 0.1 0.69 0.74
From R. Kauland B. Mattiasson,Appl. Microbiol.Biotechnol.24, 259 (1986), withpermission. b Solubilityin pure water under these conditionswas 0.2 mg/ml.
Determination of Partition Coefficients, K, of Steroids The K values of hydrocortisone and prednisolone are determined by first stirring the respective steroids into a two-phase system for a few hours at 20° . The phase separation is carded out by allowing the systems to stand for 30 min or by centrifuging them at 1625 g for 5 min at 20°. Aliquots are removed from both phases and their steroid concentration determined. K is calculated as the ratio between the concentrations in top and bottom phases. Table I shows the partitioning of both hydrocortisone and prednisolone in the two-phase systems using Dx and different top phase polymers. The steroids partition more toward the top phase. PPG was particularly effective in producing high K values. The steroid crystals which did not go into solution settled onto the interface.
Determination of Hydrocortisone Solubility in Polymer Solutions The respective phases of the two-phase systems, 25% PEG 8000-Dx T40 and 12% PEG 8000-3% Reppal PES 200, are separated. Hydrocortisone (4 mg/ml) is added to the respective phases and stirred for about 24 hr at 20°. The undissolved steroid is sedimented by high-speed centrifugation (12,000 g for 20 rain) at 4°, and the amount dissolved is measured in the supernatant solution. The solubility in water is determined under identical conditions. The solubility of hydrocortisone in the PEG solution was I0 times greater than in Dx (Table II). Reppal solubilized even a little more steroid than PEG. Reppal contains hydroxypropyl groups, which imparts to the polymer a weak hydrophobic character.
562
BIOTECHNOLOGY
[54]
Organism and Culture Conditions Arthrobacter simplex ATCC 6946 (DSM, No. 20130, Braunschweig, Germany) is used for the transformation of hydrocortisone to prednisolone. 7The bacteria are grown at 30° with shaking in a medium (pH 7.2-7.4) containing 1% (w/v) casein peptone tryptic digest (Difco, Detroit, MI), 0.5% yeast extract (Difco), 0.5% (w/v) NaCI, and 0.5% (w/v) glucose. After 24 hr of growth, the cells are induced for the enzyme, steroid A~dehydrogenase, by addition of hydrocortisone (1 mg/ml) to the medium and continued incubation for another 6 hr. The bacteria are collected by centrifugation at 12,000 gmax (4°) for 10 min, washed with 50 mM TrisHC1 solution, pH 7.0, and stored at -20 ° until use. Transformation Reaction To 20 ml of the phase system, in a 100-ml beaker, is added hydrocortisone powder, and the mixture is stirred for a few minutes. The reaction is started by addition of bacterial cells. Samples are withdrawn periodically to follow the course of transformation. At the end of the reaction the phases are allowed to separate, and concentrations of the product in the phases are determined. At a substrate concentration of 1 mg/ml and 22 mg (dry weight) bacterial cells, more than 99% of hydrocortisone could be transformed in the two-phase systems (Table III). Because phase volume ratios (top/bottom) of these systems are high (except for those of PPG-Dx) the major amount of product formed is in the top phase. The bottom phase contains a small percentage of product and some untransformed substrate (Table III). The reaction efficiency in the PEG-Reppal system was found to be similar to that in the PEG-Dx system. However, with PEG-Reppal the bottom phase contained more product, namely, 12% in the 12% PEG-3% Reppal system and 20% in the 10% PEG-5% Reppal system, corresponding well to the steroid solubility. Studies in a 25% PEG-6% Dx system with a constant substrate to cell ratio reveal that nearly complete transformation occurs only up to a substrate concentration of 2 mg/ml. At 5 mg/ml the conversion attained is 95% (Fig. 1). Incomplete hydrocortisone transformation at higher concentrations has been reported. 2,~ Many situations demand the complete conversion of a substrate, since ultimately the separation of product from unreacted substrate becomes critical.
S. Ohlson, P.-O. Larsson, and K. Mosbach, Fur. J. Appl. Microbiol. Biotechnol. 7, 103 (1979).
[54]
PREDNISOLONE
563
TABLE III HYDROCORTISONE TRANSFORMATION IN AQUEOUS Two-PHASE SYSTEMSa Hydrocortisone conversion (%) in Phase system [% (w/w) : % (w/w)] PEG 8000-dextran T40 15 : 3 20 : 3 25 : 3 25 : 6 Pluronic F68-dextran T40 14 : 4 14 : 6 PPG 2025-dextran T40 25 : 3 M-PEG 550-dextran T40 25 : 3 M-PEG 5000-dextran T70 9.7 : 4 10.7 : 2
Amount of prednisolone in bottom phase (% of total)
Reaction time (hr)
Total system
Top phase b
2.5 2.5 2.5 2.5
99.5 99.8 99.6 100
99.8 100 99.6 100
76 62 65 65
4. I 4.0 1.0 1.5
2.5 1.75
100 100
100 100
94 85
6.8 8.6
Bottom phase b
3.0
99.5
99.3
93
20
6.5
99.4
99.4
99
2.1
4.0 8.0
99.4 98.5
99.8 98.4
99.2 91
8.5 1.2
From R. Kaul and B. Mattiasson, Appl, Microbiol. Biotechnol. 24, 259 (1986), with permission. b After completion of the reaction, the phases were separated, and the degree of conversion was determined in the respective phases.
Increasing Oxygen Supply One limiting factor during conversion of hydrocortisone to predniso[one is the lack of oxygen. By improving the oxygen supply to the reaction mixture it is possible to increase the rate of transformation, especially at high substrate concentrations. To increase the oxygen supply, the reaction can be carried out in the presence of perfluorochemicals, which are good solvents for gases. 9 Preparation of Emulsion of Perfluorochemical. The emulsion (30% perfluorochemical, v/v) is prepared as described earlier.l° The buffer solution (water phase), the perfluorochemical FC-72 (3M, St. Paul, MN) (organic phase), and Pluronic F-68 (emulsifier) are mixed in appropriate proportions. The amount of Pluronic F-68 is 0.21 g/ml FC-72. The mixture (50 ml) is placed in a 400-ml beaker and cooled in crushed ice. It is 9 B. Mattiasson and P. Adlercreutz, Trends Biotechnol. 5, 250 (1987). l0 p. Adlercreutz and B. Mattiasson, Eur. J. Appl. Microbiol. Biotechnol. 16, 165 (1982).
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FIo. 1. Time course for hydrocortisonetransformationby Arthrobactersimplex cells in a 25% (w/w)PEG 8000-6% (w/w)dextranT40 systemat 20°, Hydrocortisoneconcentrations used were 1 (x), 2 (O), 3 (E2), 4 (A), and 5 mg (~7) per milliliterof reaction medium. Experimental details are described in the text. [From R. Kaul and B. Mattiasson, Appl. Microbiol. Biotechnol. 24, 259 (1986), with permission.] then sonicated using MSE Soniprep 150 Ultrasonic disintegrator (MSE Scientific Instruments, Sussex, England) for 5 min until a white emulsion forms. Use o f Perfluorochemical Emulsion During Bioconversion. The emulsion (4 ml) is added to 20 ml of the two-phase system containing hydrocortisone (either 1 or 3 mg/ml), and the cells (22 or 66 mg dry weight, respectively) are then added. In the presence of the perfluorochemical emulsion, the transformation rate was found to increase, and the conversion of higher concentrations of hydrocortisone nearly reached 100% (Fig. 2). On completion of the reaction, as the phase system was allowed to stand, the emulsion formed a separate phase below that of Dx and could easily be recovered. Perfluorochemicals are very poor solvents for organic chemicals and the product was, hence, recovered in the polymer phases.
Product Extraction from Top Phase o f Aqueous Two-Phase System Amberlite XAD-4 (BDH) is packed into a column (17 x 0.6 cm) and rinsed with 50 mM Tris-HCl solution, pH 7.0. The PEG-rich top phase from the 25% (w/w) PEG 8000-6% (w/w) Dx T40 system is separated after completion of hydrocortisone transformation, then passed through the Amberlite column at a flow rate of 0.5 ml/min. The column is washed with buffer and flushed with methanol for the desorption of prednisolone. The column is then regenerated by washing with buffer. The PEG eluate is generally devoid of steroid product, and the latter
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is totally desorbed by methanol. The solvent is evaporated so as to obtain prednisolone crystals. Sometimes prednisolone, up to 5% of the amount loaded, could be detected in the eluate. This is extracted by reloading the eluate onto the column. Adsorption to the Amberlite resin is a mild process and permits the phase polymer to be recycled. Amberlite could be used repeatedly after regeneration with buffer.
Recycling of Cells and Phase Polymers The r e c o v e r e d P E G phase is supplemented with substrate and returned to the bioreactor, which contains the Dx-rich phase and the cells. P E G
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lost during product extraction is replaced. Alternatively, a fresh PEGrich phase can be used. The transformation rate improved by 25-75% in a number of runs. 4 This was attributed to further induction of enzyme activity in the cells by the substrate. A similar observation had been reported earlier. 7 A decline in enzyme activity occurred, however, after about 7 runs, which resulted in slightly longer times for complete conversion. Enhancement of A~-dehydrogenaseactivity of immobilized A. simplex is possible when the cells are incubated in nutrient media. 8 This activation is due to an increase in the number of cells that are simultaneously being induced for dehydrogenase activity by the substrate hydrocortisone. Application of a similar approach here, that is, adding the nutrients to the aqueous two-phase system, helped maintain the catalytic activity of the cells. 4 It is important, however, to maintain sterile conditions in longterm operations. Remarks Table IV compares aqueous two-phase systems with some of the methods previously used for hydrocortisone transformation. It is evident that the reaction rate achieved in the two-phase system is among the highest reported. Immobilized cells have earlier been employed to protect the biocatalyst from denaturation. 1~Conversion of hydrocortisone to prednisolone is also subject to product inhibition. 12Aqueous two-phase systems offer suitable reaction conditions even when the viability of the microbial cells is to be maintained. The top phase serves as a steroid reservoir, whereas the cells are confined to the bottom phase. As the reaction proceeds the product is extracted simultaneously to the top phase and away from the biocatalyst. By manipulation of the phase volume ratios, it is possible to obtain most of the product in the top phase. Acknowledgments The authors are grateful to The National Swedish Board for Technical and Industrial Development (NUTEK) for financial support.
ii K. Sonomoto, A. Tanaka, T. Omata, T. Yamane, and S. Fukui, Fur. J. Appl. MicrobioL BiotechnoL 6, 325 (1979). ~2j. Kloosterman IV and M. D. Lilly, Enzyme Microb. Technol. 6, 113 (1984).