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Continuous operation of enzymic reactions in liquid-surfactant membranes
Process Biochemistry 29 (1994) 575-579 Q 1994 Elsevier Science Liited Printed itt Great Britain. All tights resetved 0032-9592/94/$7.00
Continuous Operation of Enzymic Reactions in Liquid-Surfactant Membranes Tadashi Hano,* Michiaki Matsumoto & Takaaki Ohtakef Department of Applied Chemistry, Oita University, Oita 870-l 1, Japan (Received 18 September 1993; accepted 10 November 1993)
The production of L-amino acid by the stereoselective hydrolysis of a D, L.-amino acid ester by means of a-chyrnotrypsin was studied as a model of an enzymic reaction in an emulsion. Continuous operation involving the use of an emulsion of m’octylmethylammonium chloride/Span 8O/kerosene, reported previously, was found to become unusable afer nine hours because of swelling of the emulsion. To prepare a more stable emulsion, the various combinations of the membrane phase were examined. A mixture of equal weights of Span 80 and Paranox loOgave the highest production rate and the most stable emulsion. The life of the emulsion in the present system was longer than that in the system reported previously.
Schiigerl et cd.‘_5 demonstrated that enzymes in emulsion could be used in a bioreactor system accompanied with separation by combining a specific enzymic reaction and selective permeation. They studied three different systems: production of L-amino acid by the hydrolysis of n,L-amino acid ester by using encapsulated achymotrypsin,3 bio-conversion of penicillin G to 6-amino-penicillanic acid by penicillin-G acylase,4 and the reductive amination of a-ketoisocaproate to leucine by L-lecuine dehydrogenase.5 Recently, Preitschopf and Marr,6 Ha and Hong,’ and Chang and Lees studied the production of ~-amino acid as a model system of an enzyme in emulsion. Ha and Hong’ investigated the effects of the operating conditions on emulsion stability and transport rates and concluded that the enzymic reaction in an internal phase was the rate-determining step. Chang and Lee8 developed a mathematical model for the production of L-amino acid in the system proposed by Scheper et aL3
INTRODUCTION The liquid-surfactant-membrane (LSM) technique has been developed as a new method of separation based on solvent extraction. The compounds separated by LSM are mainly metallic ions, phenols, ammonia, and fermented products, such as organic acids, amino acids, and antibiotics. In addition, attempts to utilize water-in-oil (W/ 0) emulsions for the immobilization of enzyme has become of interest in recent years. Mohan and Li,‘,* who reported the first enzyme system in an emulsion, found that enzymes and microbes in a liquid membrane could retain their activities in spite of vigorous agitation during an emulsification step. *To whom correspondence should be addressed. *Present address: Kagoshima National College of Technology, Hayato, Kagoshima 899-5 1, Japan.
575
576
Tadashi Hano, Michiaki Matsumoto,
In a present work, we studied the same system as a model of an enzyme in emulsion to investigate the continuous operation in detail, since most of the previous work was carried out by a batch operation. Although Scheper et aL3 performed continuous runs of short duration (2 h), they reported simply that the reactor used was inadequate for a long run because the emulsion globules tended to stick to the reactor walls. We carried out continuous operation for a long time to evaluate the feasibility of the present system for commercial use.
EXPERIMENTAL Water-in-oil emulsion Trioctyhnethylammonium chloride (abbreviated as TOMACl) was employed as a carrier of amino acid produced in an internal phase. Paranox 100, a polyamine emulsifier developed by Li9 Span 80, or mixture of the two was employed as an emulsifier. The organic-membrane solution was prepared by dissolving a carrier and emulsifier in various diluents. a-Chymotrypsin from bovine pancreas, phenylalanine, and phenyialanine methylester were purchased from Sigma. An internal aqueous solution was prepared by dissolving a-chymotrypsin in the phosphate-buffer solution adjusted to pH 6. The W/O emulsions were prepared by mixing each 15 ml of an organic and an internal aqueous phase in a homogenizer (Nihon Seiki AM-8) at 167 s-r for 10 min. An external aqueous solution was prepared by dissolving racemic phenylalanine methylester in the phosphate-buffer solution adjusted to pH 6.
Takaaki Ohtake
Batch run Batch runs were performed at 298 K by pouring 20 ml of W/O emulsion into 70 ml of the external aqueous solution. The agitation speed was 4 s-l. The samples were withdrawn at timed intervals. Concentrations of substrate and product in the external-solution phase were analyzed by HPLC with a C- 18 column. The usual operating conditions are listed in Table 1. Continuous run The experimental apparatus for a continuous run is shown in Fig. 1, A water-jacketed reactor of 90ml effective volume was maintained at 298 K. The continuous external phase was fed at 0.5 ml min-I, and the emulsion phase of 20 ml was retained in the reactor. The agitation speed of the reactor was 4 s-l. Concentrations of substrate and product in the effluent were analyzed at timed intervals. Motor Tube
pump RopOt0r
Fig. 1.
Experimental
Table 1. Standard condition for batch or continuous
apparatus for continuous
operation
Internalphase Volume Enz(yme. concentration pH adjusted by phosphate
lOJIll O-67 g liter-’ 6
buffer)
Membrane phase Volume TOMACl concentration for Span 80 or Paranox 100 for mixture of Span 80 and Paranox Surfactant concentration for Span 80 or Paranox 100 for mixture of Span 80 and Paranox
1oml 100
1 wt% 3 wt%
100
5 wt% lOwt%
Externalphase Volume D,r-phenylahmine methyl ester concentration pH (adjusted by phosphate buffer)
70 ml 2.32 mol me3 6
operation.
577
Continuous operation of enzymic reactions
Li DE:D-Phanyhlanlm, LE:L-Phenyhlanlne U:L-PhMyld&%Ill~O O?Trhvoctylmethyl
methyl ester methyl ester ammonium
Schematic of enzyme reaction system in emulsion for the production of ~-amino acid from o,L-esters.
Scheme 1.
Operation time[h]
s
0
Fig. 2. Concentration change of amino acid and ester in continuous operation (TOMACl/Span SO/kerosene system).
RESULTS AND DISCUSSION Swelling of emulsion Scheme 1 shows the enzymic-reaction system in emulsion proposed by Scheper et aL3 In this system, the undissociated form of n,L-ester diffuses through the membrane to an internal phase. a-Chymotrypsin selectively hydrolyzes L-ester into amino acid and methanol in an internal phase. The ~-amino acid produced is transported by an anion-facilitated carrier (TOMACl) to an external phase. We examined the continuous production of ~-amino acid by the emulsion system consisting of TOMACl/Span 80/kerosene proposed by Scheper et aZ.3 After 9 h, the continuous operation became impossible because of swelling of the emulsion. The increase in enzyme concentration was found to reduce the time of stable operation. Such a swelling was considered to be caused by osmosis resulting from the difference in the solute concentration between internal and external aqueous solutions. Since the initial concentrations of the internal and external aqueous solutions were equal, the experimental results suggest that the amino acid produced was accumulated in an internal phase. The production rate is therefore assumed to be limited by the permeation of amino acid mediated by TOMACl from internal to external solutions. Ha and Hong’ claimed that the enzymic reaction in an internal phase was a rate-determining step. To accelerate the permeation rate, we increased the carrier concentration. Figure 2 shows the time course of the concentrations in an external phase at various carrier concentrations. The rate of decrease in ester concentration was independent of the carrier concentration, whereas the formation rate of L-
20
40
60
60
100
120
Reaction time[min]
Fig. 3. Effect of Paranox 100 concentration on production rate in batch operation.
phenylalanine increased. However, the stable operating time decreased with the carrier concentration because of membrane break-up. As pointed out by Simmons et aZ.,‘O the increase in carrier concentration makes the emulsion unstable owing to the increase in the hydrophilelipophile balance (HLB) value. We modified the method of preparing the membrane to obtain a more stable emulsion in a batch reactor. Membrane stability Li9 proposed the use of polyamine derivatives as a surfactant of LSM. We used the surfactant Paranox 100 instead of Span 80. Figure 3 shows the concentration changes of amino acid and its ester in an external phase. &-octane was used as a membrane solvent in these runs. The production rate decreased with the surfactant concentration,
578
Tadashi Hano, Michiaki Matsumoto,
probably owing to the increase in the membrane viscosity. In a lower concentration range (4-5 wt%), however, the membrane was completely broken after 3 h. We then examined the mixture of Span 80 and Paranox 100 as a surfactant. Figure 4 shows the effect of the surfactant composition on the production rate. A mixture of equal weights gave the highest production rate. As mentioned above, Paranox 100 caused membrane break-up. In a separate paper, we found that surfactants of polyamine derivatives act as a carrier.’ ’ From these results, we suspected that Span 80 contributes to the membrane stability and Paranox 100 promotes the permeation rate of amino acid from internal to external aqueous solutions. With the optimum surfactant composition, the carrier concentration could be increased from 1 wt% up to 3 wt%. However, the carrier above 3 wt% made the membrane unstable. Continuous operation Continuous operations were examined under the optimum condition obtained in a batch reactor. Figure 5 compares the reactor performance observed in the membrane system proposed by us with that reported by Scheper et aI.” The life of emulsion in the present system was long, although swelling occurred abruptly after 71 h and continuous operation became impossible. In order to make a prolonged operation possible, the flow of feed solution was intermittently stopped, and a batch operation was carried out to facilitate the transport of amino acid accumulated in an in-
Takaaki Ohtake
temal solution. Figure 6 shows the time course of such an operation. The difference in productivity was small between Figs 5 and 6, and finally swelling occurred. In principle, the permeation of amino acid produced in an internal solution was driven by the facilitated transport mechanism as shown in Scheme 1. The concentration of amino acid in an internal solution is therefore always higher than that in an external solution, and water continuously permeates from external to internal solutions according to the gradient of osmotic pressure. Rapid swelling may occur above the threshold level of water content. It is therefore concluded that the continuous operation of an enzyme emulsion is difficult because of the swelling, and a batch operation is preferred. Continuous operation requires the development of a new and cheap carrier of amino acid based on the active transport mechanism, in which amino acid can be transported against a concentration
E T 2. f 2
1.
PI 0
4 0
D,L-E 1L-A] Span601 ParanoxlOOlTOMACl 0 I.1 5wt%I owt% I lwt%
0.
6 e s
Operation (Batch
Comparison Fig. 5. go/kerosene system.
run)
time [ h
]
of present system and TOMACl/Span
--
13.0~
: !g
I
Reaction Fig. 4. Effect of surfactant in batch operation.
;
I
I
V,,:V,,=2Oml:7Oril
time[min]
composition
flow rated.5
[ml/min]
Operation time [h] on production
rate
Fig. 6. feed.
Continuous
operation
with
intermittent
flow
of
Continuous operation of enzymic reactions
gradient. Unfortunately, carriers for amino-acid transport have been developed not from the viewpoint of commercial use, but as biological models.
CONCLUSIONS of ~-amino acid by the hydrolysis of o,r_-amino-acid ester by using achymotrypsin was studied as a model system of an enzyme in emulsion and the following results were obtained. (i) In the emulsion system consisting of TOMACl/Span 80/kerosene proposed by Scheper et aZ.,3 continuous operation became
The continuous production
impossible in a relatively short time because of swelling of the emulsion. (ii) The formulation of the membrane phase was examined to prepare a stable emulsion, and a mixture of equal weights of Span 80 and Paranox 100 was found to be the best surfactant. (iii) The life of an emulsion prepared by the optimum system was longer than that observed in systems examined to date.
ACKNOWLEDGMENT The authors express their thanks to Mr Y. Takeuchi, Mr S. Uenoyama, Mr H. Murakami, and Mr S. Uchinomiya and their valuable experimental assistance.
579
REFERENCES 1. Mohan, R. R. & Li, N. N., Reduction and separation of nitrate and nitrite by liquid surfactant membrane~~tp;;lated enzymes. Biotechnol. Bioengng, 16 (1974) 2. Mohan, R. R. & Li, N. N., Nitrate and nitrite reduction by liquid surfactant membrane-encapsulated whole cells. Biotechnof. Bioengng, 17 (1975) 1137-56. 3. Scheper, T., Halwachs, W. & Schiigerl, K., Production of L-amino acid by continuous enzymatic hydrolysis of DLamino acid methvl ester bv the liquid membrane technique. Chem. E&gJ., 29 (1984) B31-7. 4. Barenschee, T., Scheper, T. & Schiigerl, K., An integrated process for the production and biotransformation of penicillin. J. Biotechrwl., 26 (1992) 143-54. 5. Makryaleas, K., Scheper, T., Schiigerl, K. & Kula, M. R., Enzymatic production of ~-amino acid with continuous coenzyme regeneration by liquid membrane technique. Ger. Chem. Engng, 8 (1985) 345-50. 6. Preitschopf, W. & Marr, R., Immobilized enzymes in liquid membrane permeation product separation and product brushing in biotechnology. In Solvent Extmction 1990. ed. T. Sekine. Elsevier. Amsterdam, 1992, DD. .. 1619;-23. 7. Ha, H. Y. & Hong, S. A., A study on enzymatic reaction using a liquid emulsion membrane technique. Biotechno/. Bioengng, 39 (1992) 125-31. 8. Chang, J. H. & Lee, W. K., Modeling of enzymatic reaction with emulsion liquid membrane. Chem. Engng Sci., 48 (1993) 2357-66. 9. Li, N. N., Novel liquid membrane formulations. U.S. Patent 4,259,189 (31 March 1981). 10. Simmons, D. K., May, S. W. & Agrawal, P. K., Enzymes in liquid membranes. In Downstream Processing and Biosepamtion, ed. J. D. Hamel, J. B. Hunter & S. K. Sikdar (ACS Symposium Series No. 419). American yk;hej;al Society, Washington, DC, USA, 1990, pp. 11 Hano, T., Ohtake, T., Matsumoto, M. & Ogawa, S., Application of a liquid surfactant membrane for the recovery of penicillin G. J. Membr. Sci., 84 (1993) 271-8.
Continuous Operation of Enzymic Reactions in Liquid-Surfactant Membranes Tadashi Hano,* Michiaki Matsumoto & Takaaki Ohtakef Department of Applied Chemistry, Oita University, Oita 870-l 1, Japan (Received 18 September 1993; accepted 10 November 1993)
The production of L-amino acid by the stereoselective hydrolysis of a D, L.-amino acid ester by means of a-chyrnotrypsin was studied as a model of an enzymic reaction in an emulsion. Continuous operation involving the use of an emulsion of m’octylmethylammonium chloride/Span 8O/kerosene, reported previously, was found to become unusable afer nine hours because of swelling of the emulsion. To prepare a more stable emulsion, the various combinations of the membrane phase were examined. A mixture of equal weights of Span 80 and Paranox loOgave the highest production rate and the most stable emulsion. The life of the emulsion in the present system was longer than that in the system reported previously.
Schiigerl et cd.‘_5 demonstrated that enzymes in emulsion could be used in a bioreactor system accompanied with separation by combining a specific enzymic reaction and selective permeation. They studied three different systems: production of L-amino acid by the hydrolysis of n,L-amino acid ester by using encapsulated achymotrypsin,3 bio-conversion of penicillin G to 6-amino-penicillanic acid by penicillin-G acylase,4 and the reductive amination of a-ketoisocaproate to leucine by L-lecuine dehydrogenase.5 Recently, Preitschopf and Marr,6 Ha and Hong,’ and Chang and Lees studied the production of ~-amino acid as a model system of an enzyme in emulsion. Ha and Hong’ investigated the effects of the operating conditions on emulsion stability and transport rates and concluded that the enzymic reaction in an internal phase was the rate-determining step. Chang and Lee8 developed a mathematical model for the production of L-amino acid in the system proposed by Scheper et aL3
INTRODUCTION The liquid-surfactant-membrane (LSM) technique has been developed as a new method of separation based on solvent extraction. The compounds separated by LSM are mainly metallic ions, phenols, ammonia, and fermented products, such as organic acids, amino acids, and antibiotics. In addition, attempts to utilize water-in-oil (W/ 0) emulsions for the immobilization of enzyme has become of interest in recent years. Mohan and Li,‘,* who reported the first enzyme system in an emulsion, found that enzymes and microbes in a liquid membrane could retain their activities in spite of vigorous agitation during an emulsification step. *To whom correspondence should be addressed. *Present address: Kagoshima National College of Technology, Hayato, Kagoshima 899-5 1, Japan.
575
576
Tadashi Hano, Michiaki Matsumoto,
In a present work, we studied the same system as a model of an enzyme in emulsion to investigate the continuous operation in detail, since most of the previous work was carried out by a batch operation. Although Scheper et aL3 performed continuous runs of short duration (2 h), they reported simply that the reactor used was inadequate for a long run because the emulsion globules tended to stick to the reactor walls. We carried out continuous operation for a long time to evaluate the feasibility of the present system for commercial use.
EXPERIMENTAL Water-in-oil emulsion Trioctyhnethylammonium chloride (abbreviated as TOMACl) was employed as a carrier of amino acid produced in an internal phase. Paranox 100, a polyamine emulsifier developed by Li9 Span 80, or mixture of the two was employed as an emulsifier. The organic-membrane solution was prepared by dissolving a carrier and emulsifier in various diluents. a-Chymotrypsin from bovine pancreas, phenylalanine, and phenyialanine methylester were purchased from Sigma. An internal aqueous solution was prepared by dissolving a-chymotrypsin in the phosphate-buffer solution adjusted to pH 6. The W/O emulsions were prepared by mixing each 15 ml of an organic and an internal aqueous phase in a homogenizer (Nihon Seiki AM-8) at 167 s-r for 10 min. An external aqueous solution was prepared by dissolving racemic phenylalanine methylester in the phosphate-buffer solution adjusted to pH 6.
Takaaki Ohtake
Batch run Batch runs were performed at 298 K by pouring 20 ml of W/O emulsion into 70 ml of the external aqueous solution. The agitation speed was 4 s-l. The samples were withdrawn at timed intervals. Concentrations of substrate and product in the external-solution phase were analyzed by HPLC with a C- 18 column. The usual operating conditions are listed in Table 1. Continuous run The experimental apparatus for a continuous run is shown in Fig. 1, A water-jacketed reactor of 90ml effective volume was maintained at 298 K. The continuous external phase was fed at 0.5 ml min-I, and the emulsion phase of 20 ml was retained in the reactor. The agitation speed of the reactor was 4 s-l. Concentrations of substrate and product in the effluent were analyzed at timed intervals. Motor Tube
pump RopOt0r
Fig. 1.
Experimental
Table 1. Standard condition for batch or continuous
apparatus for continuous
operation
Internalphase Volume Enz(yme. concentration pH adjusted by phosphate
lOJIll O-67 g liter-’ 6
buffer)
Membrane phase Volume TOMACl concentration for Span 80 or Paranox 100 for mixture of Span 80 and Paranox Surfactant concentration for Span 80 or Paranox 100 for mixture of Span 80 and Paranox
1oml 100
1 wt% 3 wt%
100
5 wt% lOwt%
Externalphase Volume D,r-phenylahmine methyl ester concentration pH (adjusted by phosphate buffer)
70 ml 2.32 mol me3 6
operation.
577
Continuous operation of enzymic reactions
Li DE:D-Phanyhlanlm, LE:L-Phenyhlanlne U:L-PhMyld&%Ill~O O?Trhvoctylmethyl
methyl ester methyl ester ammonium
Schematic of enzyme reaction system in emulsion for the production of ~-amino acid from o,L-esters.
Scheme 1.
Operation time[h]
s
0
Fig. 2. Concentration change of amino acid and ester in continuous operation (TOMACl/Span SO/kerosene system).
RESULTS AND DISCUSSION Swelling of emulsion Scheme 1 shows the enzymic-reaction system in emulsion proposed by Scheper et aL3 In this system, the undissociated form of n,L-ester diffuses through the membrane to an internal phase. a-Chymotrypsin selectively hydrolyzes L-ester into amino acid and methanol in an internal phase. The ~-amino acid produced is transported by an anion-facilitated carrier (TOMACl) to an external phase. We examined the continuous production of ~-amino acid by the emulsion system consisting of TOMACl/Span 80/kerosene proposed by Scheper et aZ.3 After 9 h, the continuous operation became impossible because of swelling of the emulsion. The increase in enzyme concentration was found to reduce the time of stable operation. Such a swelling was considered to be caused by osmosis resulting from the difference in the solute concentration between internal and external aqueous solutions. Since the initial concentrations of the internal and external aqueous solutions were equal, the experimental results suggest that the amino acid produced was accumulated in an internal phase. The production rate is therefore assumed to be limited by the permeation of amino acid mediated by TOMACl from internal to external solutions. Ha and Hong’ claimed that the enzymic reaction in an internal phase was a rate-determining step. To accelerate the permeation rate, we increased the carrier concentration. Figure 2 shows the time course of the concentrations in an external phase at various carrier concentrations. The rate of decrease in ester concentration was independent of the carrier concentration, whereas the formation rate of L-
20
40
60
60
100
120
Reaction time[min]
Fig. 3. Effect of Paranox 100 concentration on production rate in batch operation.
phenylalanine increased. However, the stable operating time decreased with the carrier concentration because of membrane break-up. As pointed out by Simmons et aZ.,‘O the increase in carrier concentration makes the emulsion unstable owing to the increase in the hydrophilelipophile balance (HLB) value. We modified the method of preparing the membrane to obtain a more stable emulsion in a batch reactor. Membrane stability Li9 proposed the use of polyamine derivatives as a surfactant of LSM. We used the surfactant Paranox 100 instead of Span 80. Figure 3 shows the concentration changes of amino acid and its ester in an external phase. &-octane was used as a membrane solvent in these runs. The production rate decreased with the surfactant concentration,
578
Tadashi Hano, Michiaki Matsumoto,
probably owing to the increase in the membrane viscosity. In a lower concentration range (4-5 wt%), however, the membrane was completely broken after 3 h. We then examined the mixture of Span 80 and Paranox 100 as a surfactant. Figure 4 shows the effect of the surfactant composition on the production rate. A mixture of equal weights gave the highest production rate. As mentioned above, Paranox 100 caused membrane break-up. In a separate paper, we found that surfactants of polyamine derivatives act as a carrier.’ ’ From these results, we suspected that Span 80 contributes to the membrane stability and Paranox 100 promotes the permeation rate of amino acid from internal to external aqueous solutions. With the optimum surfactant composition, the carrier concentration could be increased from 1 wt% up to 3 wt%. However, the carrier above 3 wt% made the membrane unstable. Continuous operation Continuous operations were examined under the optimum condition obtained in a batch reactor. Figure 5 compares the reactor performance observed in the membrane system proposed by us with that reported by Scheper et aI.” The life of emulsion in the present system was long, although swelling occurred abruptly after 71 h and continuous operation became impossible. In order to make a prolonged operation possible, the flow of feed solution was intermittently stopped, and a batch operation was carried out to facilitate the transport of amino acid accumulated in an in-
Takaaki Ohtake
temal solution. Figure 6 shows the time course of such an operation. The difference in productivity was small between Figs 5 and 6, and finally swelling occurred. In principle, the permeation of amino acid produced in an internal solution was driven by the facilitated transport mechanism as shown in Scheme 1. The concentration of amino acid in an internal solution is therefore always higher than that in an external solution, and water continuously permeates from external to internal solutions according to the gradient of osmotic pressure. Rapid swelling may occur above the threshold level of water content. It is therefore concluded that the continuous operation of an enzyme emulsion is difficult because of the swelling, and a batch operation is preferred. Continuous operation requires the development of a new and cheap carrier of amino acid based on the active transport mechanism, in which amino acid can be transported against a concentration
E T 2. f 2
1.
PI 0
4 0
D,L-E 1L-A] Span601 ParanoxlOOlTOMACl 0 I.1 5wt%I owt% I lwt%
0.
6 e s
Operation (Batch
Comparison Fig. 5. go/kerosene system.
run)
time [ h
]
of present system and TOMACl/Span
--
13.0~
: !g
I
Reaction Fig. 4. Effect of surfactant in batch operation.
;
I
I
V,,:V,,=2Oml:7Oril
time[min]
composition
flow rated.5
[ml/min]
Operation time [h] on production
rate
Fig. 6. feed.
Continuous
operation
with
intermittent
flow
of
Continuous operation of enzymic reactions
gradient. Unfortunately, carriers for amino-acid transport have been developed not from the viewpoint of commercial use, but as biological models.
CONCLUSIONS of ~-amino acid by the hydrolysis of o,r_-amino-acid ester by using achymotrypsin was studied as a model system of an enzyme in emulsion and the following results were obtained. (i) In the emulsion system consisting of TOMACl/Span 80/kerosene proposed by Scheper et aZ.,3 continuous operation became
The continuous production
impossible in a relatively short time because of swelling of the emulsion. (ii) The formulation of the membrane phase was examined to prepare a stable emulsion, and a mixture of equal weights of Span 80 and Paranox 100 was found to be the best surfactant. (iii) The life of an emulsion prepared by the optimum system was longer than that observed in systems examined to date.
ACKNOWLEDGMENT The authors express their thanks to Mr Y. Takeuchi, Mr S. Uenoyama, Mr H. Murakami, and Mr S. Uchinomiya and their valuable experimental assistance.
579
REFERENCES 1. Mohan, R. R. & Li, N. N., Reduction and separation of nitrate and nitrite by liquid surfactant membrane~~tp;;lated enzymes. Biotechnol. Bioengng, 16 (1974) 2. Mohan, R. R. & Li, N. N., Nitrate and nitrite reduction by liquid surfactant membrane-encapsulated whole cells. Biotechnof. Bioengng, 17 (1975) 1137-56. 3. Scheper, T., Halwachs, W. & Schiigerl, K., Production of L-amino acid by continuous enzymatic hydrolysis of DLamino acid methvl ester bv the liquid membrane technique. Chem. E&gJ., 29 (1984) B31-7. 4. Barenschee, T., Scheper, T. & Schiigerl, K., An integrated process for the production and biotransformation of penicillin. J. Biotechrwl., 26 (1992) 143-54. 5. Makryaleas, K., Scheper, T., Schiigerl, K. & Kula, M. R., Enzymatic production of ~-amino acid with continuous coenzyme regeneration by liquid membrane technique. Ger. Chem. Engng, 8 (1985) 345-50. 6. Preitschopf, W. & Marr, R., Immobilized enzymes in liquid membrane permeation product separation and product brushing in biotechnology. In Solvent Extmction 1990. ed. T. Sekine. Elsevier. Amsterdam, 1992, DD. .. 1619;-23. 7. Ha, H. Y. & Hong, S. A., A study on enzymatic reaction using a liquid emulsion membrane technique. Biotechno/. Bioengng, 39 (1992) 125-31. 8. Chang, J. H. & Lee, W. K., Modeling of enzymatic reaction with emulsion liquid membrane. Chem. Engng Sci., 48 (1993) 2357-66. 9. Li, N. N., Novel liquid membrane formulations. U.S. Patent 4,259,189 (31 March 1981). 10. Simmons, D. K., May, S. W. & Agrawal, P. K., Enzymes in liquid membranes. In Downstream Processing and Biosepamtion, ed. J. D. Hamel, J. B. Hunter & S. K. Sikdar (ACS Symposium Series No. 419). American yk;hej;al Society, Washington, DC, USA, 1990, pp. 11 Hano, T., Ohtake, T., Matsumoto, M. & Ogawa, S., Application of a liquid surfactant membrane for the recovery of penicillin G. J. Membr. Sci., 84 (1993) 271-8.