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Effects of incubation conditions on the release of a recombinant product in immobilized Escherichia coli cells
JOURNAL OF FERMENTATION ANDBIOEN~INEBRING Vol. 80, No. 6, 616-618. 1995
Effects of Incubation Conditions on the Release of a Recombinant Product in Immobilized Escherichia cob Cells OSAMU ARIGA,‘*
HISASHI TOYOFUKU,’ KAZUYUKI MATSUDAIRA,’ AND IWAO KUROIWA2
YOSHIKI SANO,’
Department of Fine Materials Engineering, Faculty of Textile Science and Technology, Shinshu University, 3-15-l Tokida, Ueda, Nagano 388 and Tiyoda Manufacturing Co. Ltd., 75-5 Imojiya, Koshoku, Nagano 387,2 Japan Received 10 May 199WAccepted 14 September 1995
Recombinant Escherichia coli cells which produce a thermostable a-amylase were immobilized in a z-carrageenan gel and the effects of incubation conditions on release of the enzyme in a glycine supplemented growth medium were investigated using a T-shape flask and a bioreactor. Glycine promoted the release of the enzyme during T-shape flask cultivation, although the viability of the immobilized cells was reduced significantly with an increase in glycine concentration. In the presence of 1% glycine, mazimum enzyme release from the free cells occurred at pH 6.5, while the increase in the initial pH promoted release of the enzyme from the immobilized cells and increased the viability of the cells in the beads. Although the enzyme release ln the bioreactor under pH control was faster at higher pH, the optimum condition was obtained with the pH controlled at 7-7.5, because enzyme stability was lower at higher pH values. [Key words: glycine, immobilized
recombinant
cells, carrageenan,
thermostable
a-amylase]
shaker. A commercially available bioreactor (TBR-2, Tiyoda Manufacturing Co. Ltd., Nagano) developed for the cultivation of cells entrapped in gel beads was used. In all experiments, the impeller was rotated at 1OOrpm and the temperature was maintained at 37°C. After preculturing IOOml of the beads were cultivated in the bioreactor containing 900 ml of DAK medium supplemented with glycine. When the pH of the culture medium was to be controlled, it was adjusted by adding 1 N sodium hydroxide solution using a hand-made pH controller. Rotation of the impeller was stopped and aliquots of the culture medium and gel beads were periodically and aseptically removed in order to maintain the bead to culture medium volume ratio at a constant level. Enzyme activity in the supernatant after centrifugation was measured as the medium fraction. The gel beads were dissolved in physiological saline and the cells collected by centrifugation. The pellets were suspended in cold distilled water and sonicated. Enzyme activity in the saline and supernatant fractions after sonication were measured as the gel bead and cell fractions, respectively. Total activity is defined as the sum of the activities in the medium, cells and beads. a-Amylase activity was assayed at 37°C according to the method of Fuwa (6). One unit of activity was defined as that causing a 10% reduction in blue color at 700 nm for 1 min. The number of viable cells in the gel beads was measured using a plate method described previously (4, 7). Immobilized cells were shaken in a T-shape flask containing DAK media supplemented with various concentrations of glycine in order to assess the effects of glycine concentration on the release of a thermostable cu-amylase. Figure la shows the effects of glycine concentration on enzyme activity released into the culture medium and the relative cell viability of the immobilized cells at 8 h versus that initially. Enzyme activity attributed to leaked free cells was neglected because growth of the leaked free cells was insignificant due to the inhibitory effects of glycine and the low pH in the culture medium (Fig. lb). Enzyme activity in the culture medium increased almost
Recovery of a target protein during the commercial production of intracellular proteins is a serious problem and often requires mechanical cell disruption. However, this technique cannot be applied to immobilized cell systems. In an attempt to overcome this difficulty, the addition of glycine to the culture medium to permeabilize the cells has been investigated and reported to be effective (1, 2). We have also studied the utilization of glycine for the production of a thermostable cu-amylase by a recombinant Escherichia coli (3). Furthermore, glycine supplementation has been successfully applied to repeated production of enzyme by recombinant cells immobilized with a n-carrageenan (4). Recently, Aristidou et al. described the importance of controlling the pH of a growth medium in the production of cr-amylase from recombinant E. coli using glycine supplementation (5). Thus far, the effects of environmental factors such as pH on the release of proteins from immobilized cells due to glycine supplementation have not been investigated. To enhance the production and release of enzyme proteins from an immobilized recombinant E. coli by supplementing the medium with glycine, the effects of incubation conditions such as glycine concentration, pH and an aeration on the release of a thermostable a-amylase were examined as a model system in the present study. The recombinant E. coli HBlOl (pHI301), which produces a thermostable cY-amylasefrom Bacillus stearothermophilus DY-5, was grown as previously described and used throughout the experiments (3). Immobilization was performed using a n-carrageenan described previously (4, 7). The gel beads were solidified in cold 2% KC1 solution for 5 h after the immobilization. As a preculture, 20ml of the immobilized cells were shaken in 180 ml of LAK broth (L-broth supplemented with 4g/l glucose, 50 mg/l ampicillin and 20 g/f KCl) for 5 h and then in 180 ml of DAK medium (Davis medium supplemented with 4 g/l glucose, 50 mg/l ampicillin and 20 g/l KCl) for 14 h using a T-shape flask with a Monod-type * Corresponding author. 616
NOTES
VOL. 80, 1995 7
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2 3 4 Glycine concentration
1
5 (%)
6
6
in proportion
to the glycine concentration up to 2%. Although the difference in enzyme activity at 4 h and 8 h was small up to a glycine concentration of 2%, it was almost double at 5%. The right coordinate indicates the relative viable cell number in a bead versus that initially (2.2 x 109 cells/ml-bead). The increase in glycine concentration resulted in a significant reduction in cell viability, and the relationship is approximated by an inverselogarithmic relation. Figure lb is a time course of changes in medium pH. At a glycine concentration less than O.S%, an increase in the number of cells in a bead was observed and the pH decreased significantly, most likely due to the production of acids in the gel beads. On the other hand, at a 5% glycine concentration the reduction in viability of the immobilized cells was drastic, and consequently the pH of the medium only decreased moderately. The effects of different medium pHs on the release of enzyme by glycine supplemention have not been reported thus far. Therefore, the effects of initial pH on the release of enzyme from free cells was examined using T20
I
15
% 4 10 I
5 0
0
FIG.
cells.
1
2
3
8
PH (-)
FIG. 1. Effects of glycine concentration on enzyme production and immobilized cells viability (T-shape flask experiment). (a) Enzyme release and cell viability. Symbols, incubation time (h): 0, 4; 0, 8; n , cell viability. (b) Time course of pH in culture medium. Symbols, glycine concentration (%): 0, 0; A, 0.2; 0, 0.5; 0, 1; A, 2; n , 5.
h z3
6.5
4
5
Time (h)
2. Effect of initial pH on the release of enzyme from free Symbols, pH (-_): 0, 7; A, 6.5; n , 6.
FIG. 3. Effect of initial pH mobilized cells (T-shape flask Symbols, incubation time (h): stability of enzyme. Symbols: zyme.
on the release of enzyme from imexperiment). (a) Enzyme release. 0, 4; A, 8. (b) Cell viability and 0, cell viability; 0, stability of en-
shape flasks. After overnight cultivation in Davis medium, the cells were collected by centrifugation and resuspended in fresh medium at various pHs containing 1% glycine. The culture was shaken in a T-shape flask and an aliquot of the culture medium was periodically and aseptically removed. Figure 2 presents the effects of initial pH on the time course of enzyme activity in culture medium. No change in medium pH was observed during the experiment. Clearly, the release was affected by the pH of the medium, with the optimum pH for release being 6.5. Thus, the effect of the pH of the medium is significant, however, such an observation has not yet been reported so far and the mechanism is not yet clear. Based on the above results, the effects of the initial pH on the release of enzyme from immobilized cells were investigated using T-shape flasks. Figure 3 shows the effects of initial medium pH on enzyme activity released for 4 and 8 h into DAK medium containing 1% glycine as well as the relative viability of the immobilized cells at 8 h. In the inset in Fig. 3a, the coordinate is expressed in logarithmic scale in order to simplify comparison of the data at lower pH values. As shown in Fig. 3a, at an initial pH of 6 the enzyme was not released, although a small amount of enzyme leaked out even without glycine at an initial pH of 7 (data not shown). At pH values of 6.5 and 7, the release of enzyme appeared to cease within 4 h because no increases in activity were observed even at 8 h. The higher the initial pH, the greater the amount of enzyme that was released from the immobilized cells. The relative viability of the immobilized cells increased with an increase in pH. As shown in Fig. lb, the pH in the medium declined rapidly from the initial value, and the pH in the beads may have been even lower than that in the culture medium. The optimum pH for release of the enzyme from free cells was 6.5. Therefore, the optimum pH for the immobilized cells may shift to an alkaline pH. For immobilized cells, the reduction in pH was large, so pH control may
618
J. FERMENT.BIOENO.,
ARIGA ET AL. TABLE 1.
Experiment Bioreactor Bioreactor Bioreactor Bioreactor T-shape flask
Comparison of performance between the bioreactor and T-shape flask experiments
Initial PH
PH control
7 6.5 7 7.5 I
_ + + + -
Total activity (U/ml) Oh
8h
Increase (%)
251 280 430 309 322
360 343 654 505 263
40 23 52 63 -18
be needed for release of the enzyme and the maintenance of cell viability. Figure 3b shows the effects of medium pH on remaining enzyme activity after 8 h incubation at 37°C in DAK medium containing 1% glycine. The stability was lower at higher pH values. The effects of cultivation conditions on the release of enzyme from immobilized cells were investigated using the bioreactor. Table 1 summarizes the results obtained for DAK medium containing 1% glycine under various incubation conditions. The last column represents the percentage of enzyme activity in the culture medium versus total activity at 8 h. Initial total activities (4th column) fluctuated under various conditions due to some fluctuation in the initial cell number in the beads. The increase in total enzyme activity (6th column) and the percentage released improved significantly using the bioreactor compared to T-shape flasks, in which 18% of the reduction in total activity was observed at 8 h (last row). The reason for the improved performance in the bioreactor experiments is not yet clear. The bioreactor experiments were carried out at pH values of 6.5, 7 and 7.5, and the results are summarized in Table 1. The increases in total activity and released percentage increased with the increase in pH. Taking the effect of pH on the stability of the enzyme into account (Fig. 3b), the optimal pH for the bioreactor experiments is estimated to be 7-7.5. In the present study, the production of an enzyme from an immobilized recombinant E. coli was investigated using a bioreactor and the optimal cultivation conditions determined. At a pH of 7.5 with pH control, maxi-
Activity in culture medium at 8 h (U/ml) 225 163 359 387 60
P;;;zst$e (%) 63 48 55 77 23
mum enzyme release and maximum enzyme production were attained through glycine supplementation. This work was supported in part by Nagano Prefecture TechnoHighland Development Organization and Asama Techno-Polis Development Organization. REFERENCES
1. Miyasbiio, S., Enei, H., Hirose, Y., and Udaka, S.: of glycine on protein by Bacillus brevis no. 47. Agric. Biol. Chem., 44, 105-112 (1980). 2. lkura, Y.: Effect of glycine and its derivatives on production and release of ,&galactosidase by Escherichia coli. Agric. Biol. Chem., 50, 2747-2753 (1986). 3. Ariga, O., Watari, T., Ando, Y., Fojisbtta, Y., and Sane, Y.: Release of thermophilic a-amylase from transformed Escherichia coli by addition of glycine. J. Ferment. Bioeng., 68, 243-246 (1989). 4. Arign, O., Ando, Y., Fuji&ha, Y., Wntari, T., and Snno, Y.: Production of thermophilic a-amylase using immobilized transformed Escherichia cofi by addition of glycine. J. Ferment. Bioeng., 71, 397-402 (1991). 5. Aristidoo, A. A., Yu, P., and San, K. Y.: Effect of glycine supplement on protein production and release in recombinant Escherichia coli. Biotechnol. Lett., 15, 331-336 (1993). 6. Fowa, H.: A new method for microdetermination of amylase activity by use of arnylose as the substrate. J. Biochem., 41, 583-603 (1954). I. Mga, O., Watari, T., Taken&i, IL., Andoh, M., Takagi, H., Sane, Y., Itjima, S., and Kobayasbi, T.: Effect of immobilization on stability of recombinant plasmid in gene-engineered microorganisms. J. Chem. Eng. Japan, 21, 117-122 (1988).
Effects of Incubation Conditions on the Release of a Recombinant Product in Immobilized Escherichia cob Cells OSAMU ARIGA,‘*
HISASHI TOYOFUKU,’ KAZUYUKI MATSUDAIRA,’ AND IWAO KUROIWA2
YOSHIKI SANO,’
Department of Fine Materials Engineering, Faculty of Textile Science and Technology, Shinshu University, 3-15-l Tokida, Ueda, Nagano 388 and Tiyoda Manufacturing Co. Ltd., 75-5 Imojiya, Koshoku, Nagano 387,2 Japan Received 10 May 199WAccepted 14 September 1995
Recombinant Escherichia coli cells which produce a thermostable a-amylase were immobilized in a z-carrageenan gel and the effects of incubation conditions on release of the enzyme in a glycine supplemented growth medium were investigated using a T-shape flask and a bioreactor. Glycine promoted the release of the enzyme during T-shape flask cultivation, although the viability of the immobilized cells was reduced significantly with an increase in glycine concentration. In the presence of 1% glycine, mazimum enzyme release from the free cells occurred at pH 6.5, while the increase in the initial pH promoted release of the enzyme from the immobilized cells and increased the viability of the cells in the beads. Although the enzyme release ln the bioreactor under pH control was faster at higher pH, the optimum condition was obtained with the pH controlled at 7-7.5, because enzyme stability was lower at higher pH values. [Key words: glycine, immobilized
recombinant
cells, carrageenan,
thermostable
a-amylase]
shaker. A commercially available bioreactor (TBR-2, Tiyoda Manufacturing Co. Ltd., Nagano) developed for the cultivation of cells entrapped in gel beads was used. In all experiments, the impeller was rotated at 1OOrpm and the temperature was maintained at 37°C. After preculturing IOOml of the beads were cultivated in the bioreactor containing 900 ml of DAK medium supplemented with glycine. When the pH of the culture medium was to be controlled, it was adjusted by adding 1 N sodium hydroxide solution using a hand-made pH controller. Rotation of the impeller was stopped and aliquots of the culture medium and gel beads were periodically and aseptically removed in order to maintain the bead to culture medium volume ratio at a constant level. Enzyme activity in the supernatant after centrifugation was measured as the medium fraction. The gel beads were dissolved in physiological saline and the cells collected by centrifugation. The pellets were suspended in cold distilled water and sonicated. Enzyme activity in the saline and supernatant fractions after sonication were measured as the gel bead and cell fractions, respectively. Total activity is defined as the sum of the activities in the medium, cells and beads. a-Amylase activity was assayed at 37°C according to the method of Fuwa (6). One unit of activity was defined as that causing a 10% reduction in blue color at 700 nm for 1 min. The number of viable cells in the gel beads was measured using a plate method described previously (4, 7). Immobilized cells were shaken in a T-shape flask containing DAK media supplemented with various concentrations of glycine in order to assess the effects of glycine concentration on the release of a thermostable cu-amylase. Figure la shows the effects of glycine concentration on enzyme activity released into the culture medium and the relative cell viability of the immobilized cells at 8 h versus that initially. Enzyme activity attributed to leaked free cells was neglected because growth of the leaked free cells was insignificant due to the inhibitory effects of glycine and the low pH in the culture medium (Fig. lb). Enzyme activity in the culture medium increased almost
Recovery of a target protein during the commercial production of intracellular proteins is a serious problem and often requires mechanical cell disruption. However, this technique cannot be applied to immobilized cell systems. In an attempt to overcome this difficulty, the addition of glycine to the culture medium to permeabilize the cells has been investigated and reported to be effective (1, 2). We have also studied the utilization of glycine for the production of a thermostable cu-amylase by a recombinant Escherichia coli (3). Furthermore, glycine supplementation has been successfully applied to repeated production of enzyme by recombinant cells immobilized with a n-carrageenan (4). Recently, Aristidou et al. described the importance of controlling the pH of a growth medium in the production of cr-amylase from recombinant E. coli using glycine supplementation (5). Thus far, the effects of environmental factors such as pH on the release of proteins from immobilized cells due to glycine supplementation have not been investigated. To enhance the production and release of enzyme proteins from an immobilized recombinant E. coli by supplementing the medium with glycine, the effects of incubation conditions such as glycine concentration, pH and an aeration on the release of a thermostable a-amylase were examined as a model system in the present study. The recombinant E. coli HBlOl (pHI301), which produces a thermostable cY-amylasefrom Bacillus stearothermophilus DY-5, was grown as previously described and used throughout the experiments (3). Immobilization was performed using a n-carrageenan described previously (4, 7). The gel beads were solidified in cold 2% KC1 solution for 5 h after the immobilization. As a preculture, 20ml of the immobilized cells were shaken in 180 ml of LAK broth (L-broth supplemented with 4g/l glucose, 50 mg/l ampicillin and 20 g/f KCl) for 5 h and then in 180 ml of DAK medium (Davis medium supplemented with 4 g/l glucose, 50 mg/l ampicillin and 20 g/l KCl) for 14 h using a T-shape flask with a Monod-type * Corresponding author. 616
NOTES
VOL. 80, 1995 7
I
I
100
I g
6.5 t $ cl.
I
100 -
$
40-
h8
.4
I
200 I
I
20-0
I
I
I 0
0 0 I
I
.& 60 ‘2 8
-
40 .E
-
: 20 ‘; D
7
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OK
300
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617
2 r 100 a
0
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0
2 3 4 Glycine concentration
1
5 (%)
6
6
in proportion
to the glycine concentration up to 2%. Although the difference in enzyme activity at 4 h and 8 h was small up to a glycine concentration of 2%, it was almost double at 5%. The right coordinate indicates the relative viable cell number in a bead versus that initially (2.2 x 109 cells/ml-bead). The increase in glycine concentration resulted in a significant reduction in cell viability, and the relationship is approximated by an inverselogarithmic relation. Figure lb is a time course of changes in medium pH. At a glycine concentration less than O.S%, an increase in the number of cells in a bead was observed and the pH decreased significantly, most likely due to the production of acids in the gel beads. On the other hand, at a 5% glycine concentration the reduction in viability of the immobilized cells was drastic, and consequently the pH of the medium only decreased moderately. The effects of different medium pHs on the release of enzyme by glycine supplemention have not been reported thus far. Therefore, the effects of initial pH on the release of enzyme from free cells was examined using T20
I
15
% 4 10 I
5 0
0
FIG.
cells.
1
2
3
8
PH (-)
FIG. 1. Effects of glycine concentration on enzyme production and immobilized cells viability (T-shape flask experiment). (a) Enzyme release and cell viability. Symbols, incubation time (h): 0, 4; 0, 8; n , cell viability. (b) Time course of pH in culture medium. Symbols, glycine concentration (%): 0, 0; A, 0.2; 0, 0.5; 0, 1; A, 2; n , 5.
h z3
6.5
4
5
Time (h)
2. Effect of initial pH on the release of enzyme from free Symbols, pH (-_): 0, 7; A, 6.5; n , 6.
FIG. 3. Effect of initial pH mobilized cells (T-shape flask Symbols, incubation time (h): stability of enzyme. Symbols: zyme.
on the release of enzyme from imexperiment). (a) Enzyme release. 0, 4; A, 8. (b) Cell viability and 0, cell viability; 0, stability of en-
shape flasks. After overnight cultivation in Davis medium, the cells were collected by centrifugation and resuspended in fresh medium at various pHs containing 1% glycine. The culture was shaken in a T-shape flask and an aliquot of the culture medium was periodically and aseptically removed. Figure 2 presents the effects of initial pH on the time course of enzyme activity in culture medium. No change in medium pH was observed during the experiment. Clearly, the release was affected by the pH of the medium, with the optimum pH for release being 6.5. Thus, the effect of the pH of the medium is significant, however, such an observation has not yet been reported so far and the mechanism is not yet clear. Based on the above results, the effects of the initial pH on the release of enzyme from immobilized cells were investigated using T-shape flasks. Figure 3 shows the effects of initial medium pH on enzyme activity released for 4 and 8 h into DAK medium containing 1% glycine as well as the relative viability of the immobilized cells at 8 h. In the inset in Fig. 3a, the coordinate is expressed in logarithmic scale in order to simplify comparison of the data at lower pH values. As shown in Fig. 3a, at an initial pH of 6 the enzyme was not released, although a small amount of enzyme leaked out even without glycine at an initial pH of 7 (data not shown). At pH values of 6.5 and 7, the release of enzyme appeared to cease within 4 h because no increases in activity were observed even at 8 h. The higher the initial pH, the greater the amount of enzyme that was released from the immobilized cells. The relative viability of the immobilized cells increased with an increase in pH. As shown in Fig. lb, the pH in the medium declined rapidly from the initial value, and the pH in the beads may have been even lower than that in the culture medium. The optimum pH for release of the enzyme from free cells was 6.5. Therefore, the optimum pH for the immobilized cells may shift to an alkaline pH. For immobilized cells, the reduction in pH was large, so pH control may
618
J. FERMENT.BIOENO.,
ARIGA ET AL. TABLE 1.
Experiment Bioreactor Bioreactor Bioreactor Bioreactor T-shape flask
Comparison of performance between the bioreactor and T-shape flask experiments
Initial PH
PH control
7 6.5 7 7.5 I
_ + + + -
Total activity (U/ml) Oh
8h
Increase (%)
251 280 430 309 322
360 343 654 505 263
40 23 52 63 -18
be needed for release of the enzyme and the maintenance of cell viability. Figure 3b shows the effects of medium pH on remaining enzyme activity after 8 h incubation at 37°C in DAK medium containing 1% glycine. The stability was lower at higher pH values. The effects of cultivation conditions on the release of enzyme from immobilized cells were investigated using the bioreactor. Table 1 summarizes the results obtained for DAK medium containing 1% glycine under various incubation conditions. The last column represents the percentage of enzyme activity in the culture medium versus total activity at 8 h. Initial total activities (4th column) fluctuated under various conditions due to some fluctuation in the initial cell number in the beads. The increase in total enzyme activity (6th column) and the percentage released improved significantly using the bioreactor compared to T-shape flasks, in which 18% of the reduction in total activity was observed at 8 h (last row). The reason for the improved performance in the bioreactor experiments is not yet clear. The bioreactor experiments were carried out at pH values of 6.5, 7 and 7.5, and the results are summarized in Table 1. The increases in total activity and released percentage increased with the increase in pH. Taking the effect of pH on the stability of the enzyme into account (Fig. 3b), the optimal pH for the bioreactor experiments is estimated to be 7-7.5. In the present study, the production of an enzyme from an immobilized recombinant E. coli was investigated using a bioreactor and the optimal cultivation conditions determined. At a pH of 7.5 with pH control, maxi-
Activity in culture medium at 8 h (U/ml) 225 163 359 387 60
P;;;zst$e (%) 63 48 55 77 23
mum enzyme release and maximum enzyme production were attained through glycine supplementation. This work was supported in part by Nagano Prefecture TechnoHighland Development Organization and Asama Techno-Polis Development Organization. REFERENCES
1. Miyasbiio, S., Enei, H., Hirose, Y., and Udaka, S.: of glycine on protein by Bacillus brevis no. 47. Agric. Biol. Chem., 44, 105-112 (1980). 2. lkura, Y.: Effect of glycine and its derivatives on production and release of ,&galactosidase by Escherichia coli. Agric. Biol. Chem., 50, 2747-2753 (1986). 3. Ariga, O., Watari, T., Ando, Y., Fojisbtta, Y., and Sane, Y.: Release of thermophilic a-amylase from transformed Escherichia coli by addition of glycine. J. Ferment. Bioeng., 68, 243-246 (1989). 4. Arign, O., Ando, Y., Fuji&ha, Y., Wntari, T., and Snno, Y.: Production of thermophilic a-amylase using immobilized transformed Escherichia cofi by addition of glycine. J. Ferment. Bioeng., 71, 397-402 (1991). 5. Aristidoo, A. A., Yu, P., and San, K. Y.: Effect of glycine supplement on protein production and release in recombinant Escherichia coli. Biotechnol. Lett., 15, 331-336 (1993). 6. Fowa, H.: A new method for microdetermination of amylase activity by use of arnylose as the substrate. J. Biochem., 41, 583-603 (1954). I. Mga, O., Watari, T., Taken&i, IL., Andoh, M., Takagi, H., Sane, Y., Itjima, S., and Kobayasbi, T.: Effect of immobilization on stability of recombinant plasmid in gene-engineered microorganisms. J. Chem. Eng. Japan, 21, 117-122 (1988).