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Intermolecular transglycosylating reaction of cyclodextrin glucanotransferase immobilized on capillary membrane
JOURNAL OF FERMENTATIONAND BIOENGINEERING Vol. 77, No. 3, 264-267. 1994
Intermolecular Transglycosylating Reaction of Cyclodextrin Glucanotransferase Immobilized on Capillary Membrane TAKESHI OKADA, MASAAKI ITO, AND KEN HIBINO* Medical Research Laboratory, Nitto Denko Corporation, 1-1-2, Shimohozumi, Ibaraki, O~aka 567, Japan Received 15 June 1993/Accepted 10 November 1993 The intermolecular transglycosylating reaction of cyclodextrin glucanotransferase ([EC 2.4.1.191; CGTase) immobilized on a capillary membrane was investigated using low molecular weight substrates such as cyclodextrin (CD), maltooligosaceharide (MOS), and a CD-MOS mixture. The immobilized CGTase catalyzed the conversion reaction of a-CD to ~-CD and MOS or ~-CD to a-CD and MOS within a short residence time. The conversion ratio increased as the amount of immobilized CGTase increased. The addition of glucose, maltose, and sucrose as acceptors in the substrate solution containing CD resulted in the acceleration of CD degradation compared with only CD substrate. Furthermore, the MOS substrate (degree of polymerization=2-6) was disproportionated with a conversion ratio exceeding 70~o by the immobilized CGTase. These data demonstrate that immobilized CGTase can catalyze intermolecular transglycosylation between low molecular substrates in a few minutes by regulating the amount of immobilized enzyme and the residence time. This might contribute to our comprehension of CGTase-immobilized bioreactors for CD production as well as to the development of new glycosides through its excellent transglycosylation ability.
in detail. This study deals with the intermolecular transglycosylation of CGTase immobilized on a capillary membrane using low molecular substrates such as CD, MOS, and a CD-MOS mixture.
Cyclodextrins (CDs) are able to form inclusion complexes with hydrophobic substances (1). This property, named "molecular encapsulation", is useful for improving the properties of certain unstable, volatile, readily oxidable, or poorly soluble substances. In Japan, CDs are widely used in various industrial fields such as food processing, medicine, agricultural chemicals, and cosmetics. It is well known that CD can be synthesized from such carbohydrates as starch, glycogen, and maltooligosaccharides (MSOs) by cyclodextrin glucanotransferase ([EC 2.4.1.19]; CGTase) (2-9). At present, CDs are generally produced from potato starch using industrial scale batch operations. CGTase derived from Bacillus macerans, Bacillus megaterium, and Bacillus stearothermophilus is a very interesting enzyme which catalyzes intramolecular (cyclizing) and intermolecular (coupling, disproportionation) transglycosylation, as well as having a hydrolytic action on starch and CD (4, 10). In intermolecular transglycosylation, CGTase transfers glycosyl residues from starch or CD to a suitable acceptor like glucose or sucrose. For example, in the application of its coupling reaction, a coupling sugar which can be used as a sweetener for the prevention of dental caries is efficiently produced from starch and sucrose by CGTase. When CD is produced in a batch operation, however, the intermolecular transglycosylation should be reduced as much as possible in order to increase the CD content in the reaction mixture. In a previous paper (11), we investigated the kinetics of CGTase immobilized on a capillary membrane and demonstrated that excess immobilized CGTase could lead to a reduction in the CD content in the permeate as well as a decrease in the CD production rate due to sidereactions and low flux. Although side-reactions could be an intermolecular transglycosylation like a coupling reaction, the mechanism of the reaction has not been studied
MATERIALS AND METHODS
Crude CGTase from B. macerans was purchased from Amano Pharmaceutical Co. Ltd. (Nagoya). This solution had an enzyme activity of approximately 600 T H U / m l (30 THU per mg protein). CDs were obtained from Tokyo Kasei Kogyo Co. Ltd. (Tokyo). Glucose, maltose, sucrose and glutaraldehyde solution were purchased from Wako Pure Chemical Industries Co. Ltd. (Osaka). Maltooligosaccharides (degree of polymerization=3-6) were obtained from Nihon Shokuhin Kako Co. Ltd. (Tokyo) and Oriental Yeast Co. Ltd. (Tokyo); their purities were 96-98% according to high performance liquid chromatography (HPLC) analysis. Chemicals
Preparation of CGTase immobilized on the capillary membrane The immobilization of CGTase on the
capillary membrane, operation of the membrane type bioreactor with the immobilized CGTase, and calculation of the amount of immobilized CGTase were carried out according to the conditions described in a previous paper (11), except that in this study the reaction temperature was set at 50°C. The substrate solution was circulated for 30-60 rain since the product concentration in permeate reached a constant value during this circulation time (data not shown). Measurement of CD, glucose, and MOS Both permeate and circulate containing CD, glucose, and MOS were sampled at regular time intervals. The concentrations of CD and MOS were determined by HPLC analysis as described by Kato et al. (6) with a slight modification. A detailed description of their measurement was presented in a previous paper (11). In some samples, glucoamylase (Seikagaku Kogyo Co. Ltd., Tokyo) was
* Corresponding author. 264
VOL 77, 1994
COUPLING REACTION OF IMMOBILIZED CGTase
added in order to convert MOS to glucose before H P L C analysis. Protein assay Protein concentration was assayed according to the method of Lowry et al. (12).
120 [(A)
265
(B)
~i00
4
80 3 RESULTS
.= 60
Effect o f r e s i d e n c e t i m e o n c o n v e r s i o n
from
CD
to
MOS The a m o u n t of immobilized CGTase ',0.49 m g / cm 2) was adjusted to the level where a side-reaction seems to occur in the immobilized capillary membrane, as described previously (11). The CD and MOS concentrations in permeate versus residence time that was used as the apparent contact time between substrate and immobilized enzyme are plotted in Fig. 1A and 1C when 0.5% a-CD and ~-CD containing 25 m M acetate buffer (pH 6.0) and 1 . 0 m M CaCIE, respectively, were used as substrates. As a result, 60-70% of the a-CD was converted by the immobilized CGTase to fl-CD and MOS over the residence time range of 1.3-13.5 min. The ratio of aCD to ~-CD in the permeate was approximately 1.0 for each residence time (Fig. 1B). Although the ~-CD was also converted to a-CD and MOS, the conversion ratio of 13-CD to t~-CD was lower than the result obtained in Fig. 1A and 1B, when the residence time was short (Fig. 1C and 1D). In both reactions, the MOS had a degree of polymerization of 1-7 and the longer residence time resulted in an increase in MOS content rather than in an acceleration of the conversion from a-CD to ~-CD or ~3-CD to a-CD. These data indicate that immobilized CGTase has a strong hydrolytic effect on both a-CD and ~-CD. Thus, conversion of the substrate by immobilized CGTase could be performed with the process which after CD was hydrolyzed, the MOS obtained as first product is furthermore converted to MOS (second product) having higher and lower degree of polymerization by the disproportionating reaction and then the second products having higher degree of polymerization are used as a substrate for CD production. Effect o f a c c e p t o r c o n c e n t r a t i o n o n the c o u p l i n g reaction of immobilized CGTase A mixture of a-CD
(5 m g / m l ) and glucose ( 0 - 6 m g / m l ) was used as a substrate for immobilized CGTase (0.05 and 0 . 6 0 m g / c m 2) in order to investigate the effect of acceptors on the coupling reaction of the immobilized CGTase. Residence
2 40 r~
1
20
G)
,
0 o 120
,
I
4 8 12 16 Residence time (min)
0
t
o
5,
(C)
~100
i
t
4 8 12 16 Residence time (min) (D)
4
80 3!
e~
.= 60 2 40 20
G)
o
b
0
I
0
4 8 12 16 Residence time (min)
i
0
t
t
4 8 12 16 Residence time (min)
FIG. 1. Effectsof residence time on the conversion of a-CD and /3-CD by CGTase immobilized on a capillary membrane and the ratio of a-CD to 3-CD in permeate. The amount of immobilized CGTase was adjusted to 0.49 mg/cm2, and 0.50/00a-CD (A, B) and ~-CD (C, D) were used as substrates, respectively. Symbols: ~, a-CD; ~, t~CD; ~, MOS. time was controlled within a minimal variation time of 3.1-3.5 min. Figure 2A shows that the reduction in total CD (a-CD+I~-CD) in the permeate of both bioreactors was significantly dependent o n the concentration of glucose, which was added as an acceptor. Both permeates also contained /3-CD and MOS. The conversion ratio of immobilized CGTase with 0.60 m g / c m 2 was higher than that with 0.05 m g / c m 2, indicating that the a m o u n t of ira(B)
(A)
4
1
2
3
4
S
6
Glucoseconcentration(mg/ml)
0
I
i
I
I
I
I
1
2
3
4
5
6
Glucoseconcentration(mg/ml)
FIG. 2. Effects of glucose on the coupling reaction of CGTase immobilized on a capillary membrane. The amount of immobilized CGTase was adjusted to 0.05 and 0.60 mg/cm2, respectively. 0.5~ a-CD containing glucose was used as a substrate. The residence time was controlled within 3.1-3.5 min. Symbols: o , 0.05 mg/cm2; A, 0.60 mg/cmL
266
O K A D A ET AL.
J. FERMENT. BIOENG., 100
~4
~
so
E "~ 2
60
~ ~o
1
~ g
2o
0 6 Molar ratio of acceptor to ct- CD FIG. 3. Effects of various acceptors on the coupling reaction of CGTase immobilized on a capillary membrane. The amount of immobilized CGTase was adjusted to 0.49 m g / c m z, 0.5% a-CD containing various acceptors was used as a substrate. The residence time was controlled within 8.6-10.4min. Symbols: o , glucose; A, sucrose; • , maltose; v, m a l t o t r i o s e ; . , maltotetraose.
mobilized CGTase is important for enhancing intermolecular transglycosylation. The ratios of a-CD to /3-CD obtained at each glucose concentration are shown in Fig. 2B. The ratios obtained by bioreactors having 0.05 and 0.60mg/cm 2 of immobilized CGTase were 5.5-7.8 and approximately 1.0 respectively, regardless of glucose concentration. Effects of various aeceptors on the coupling reaction of immobilized CGTase To investigate the effects of oligosaccharides as acceptors in the conversion of a-CD to MOS by immobilized CGTase, glucose, maltose, maltotriose, maltotetraose, or sucrose were added to reaction mixtures containing 0.5% a-CD. Figure 3 depicts the relationship between the molar ratio of acceptor to a-CD and total CD concentration in permeate. Total CD concentration decreased in the order of glucose> sucrose > maltose > maltotriose > maltotetraose when each reaction mixture contained an equal amount of nonreducing residue. However, for maltotriose and maltotetraose, CD production was higher than that obtained in the reaction mixture containing only a-CD. This may be due to CD production from maltotriose and maltotetraose, as well as from MOS, having a high degree of polymerization produced by the disproportionation reaction of immobilized CGTase, as described below. Cyclizing and disproportionating reactions by immobilized CGTase using MOS as a substrate MOS is well known to be disproportionated by CGTase to yield MOS with higher and lower degrees of polymerization (1). Figure 4 shows the relationship between the degree of polymerization in MOS when used as a substrate and the ratio that was converted from the substrate to CD and MOS having a different degree of polymerization from that of the substrate. Residence times were controlled within a minimal variation time of 8.9-9.6 min. The conversion ratio depended on the degree of polymerization in substrate; ratios exceeding 90% were obtained for maltotetraose and maltohexaose. Immobilized CGTase also produced CD from MOS, and the CD content in permeate increased to 18, 32, and 45°//00 for maltotriose, maltotetraose, and maltohexaose substrates, respectively.
0 0
l
2
3
4
5
6
Number of glucose in MOS FIG. 4. Effect of the number of glucose molecules in MOS on the cyclizing and disproportionating reaction of CGTase immobilized on a capillary membrane. The amount of immobilized CGTase was adjusted to 0.49 m g / c m 2. 0.6% MOS was used as a substrate. The residence time was controlled within 8.9-9.6min. Symbols: o , conversion ratio; A, CD content in permeate.
Glucose was not disproportionated or cyclized at all. DISCUSSION In the present paper, it has been demonstrated that immobilized CGTase at a high concentration could catalyze intermolecular transglycosylations, such as coupling and disproportionating reactions, within a very short reaction time. Although such reactions have been well characterized using free CGTase derived from several Bacillus strains (4, 10), the degree of coupling and disproportionation seemed to be very low because of the relatively low concentration of free CGTase used in the enzyme reaction. Kitahata et al. (4) have shown that only 25% of the maltotriose used as a substrate was disproportionated with free CGTase from B. macerans, even though the reaction mixture was incubated for 3 h. On the other hand, immobilized CGTase converted 70-90% of the MOS used as a substrate to CD and MOS having different degrees of polymerization from that of the substrate within a short residence time. Immobilized CGTase also hydrolyzed both a-CD and ~-CD. Although CGTase is classified as a transferase, some researchers have shown that CGTase has hydrolytic activity since free CGTase from Bacillus strains liquefies starch paste rapidly and forms reducing residue (4, 10). However, it has been reported that the hydrolytic efficiency of immobilized CGTase on CD was higher than that by free CGTase (4, 10, 13). This is clearly due to differences in the enzyme concentrations used in the experiments. In general, when CD is produced with free CGTase in a batch operation, a low concentration of CGTase is added to reduce the coupling reaction (14). While CD conversion by immobilized CGTase was accelerated in accordance with increases in acceptors like glucose and sucrose added to the reaction mixture, as is also the case for free CGTase, the addition of an acceptor with a higher degree of polymerization was ineffective for CD degradation. Since immobilized CGTase can produce higher levels of MOS by the disproportionating reaction, maltotetraose or maltohexaose used as an ac-
VOL 77, 1994
COUPLING REACTION OF IMMOBILIZED CGTase
ceptor could be changed to a desirable substrate for cyclizing reaction after being d i s p r o p o r t i o n a t e d . However, these findings regarding the effects o f acceptors on coupling reaction were different f r o m those obtained with free CGTase. K o b a y a s h i (13) has indicated that the a d d i t i o n o f M O S (degree o f p o l y m e r i z a t i o n = 1--4) promotes the coupling reaction c o m p a r e d to absence o f the acceptor, and that maltose is the most effective acceptor. Thus, C G T a s e m a y exhibit interesting enzymatic p r o p e r ties as the enzyme concentration increases. The d a t a o b t a i n e d in this study s u p p o r t the hypothesis that regulation o f b o t h the a m o u n t o f immobilized C G T a s e and the residence time is i m p o r t a n t for reducing intermolecular transglycosylation and hydrolysis, as well as for improving CD productivity, as has been suggested in a previous p a p e r (11). A l t h o u g h side-reactions which cause CD degradation should be reduced for CD production from starch, an alternative use for bioreactors with high concentrations o f immobilized C G T a s e is the production o f coupling sugars or glycosides by utilizing their excellent intermolecular transglycosylation ability. Moreover, the decrease in flux in the m e m b r a n e type bioreactor using low molecular substrate was not r e m a r k a ble, even if the b i o r e a c t o r was used for repeated reactions. This merit suggests that practical application is possible. Transglycosylation by enzymes is currently a c o m m o n m e t h o d for improving the p o o r solubility o f bioactive c o m p o u n d s derived from plants on an industrial scale. We are now investigating the possibility o f using this b i o r e a c t o r for the actual p r o d u c t i o n o f bioactive c o m p o u n d s . REFERENCES
1. French, D.: The schardinger dextrins. Adv. Carbohydrate Chem., 12, 189-260 (1957). 2. DePinto, J.A. and Campbell, L.L.: Purification and proper-
3. 4.
5. 6. 7.
267
ties of the cyciodextrinase of Bacillus macerans. Biochemistry, 7, 121-125 (1968). Nakamura, N. and Horikoshi, K.: Purification and properties of cyclodextrin glycosyltransferase of an alkalophilic Bacillus sp. Agric. Biol. Chem., 40, 935-941 (1976). Kitabata, S. and Okada, S.: Comparison of action of cyclodextrin glucanotransferase from Bacillus megaterium, B. circulans, B. stearothermophilus and B. macerans. J. Jpn. Soc. Starch Sci., 29, 13-18 (1982). Kobayashi, S., Maruyama, K., and Kainuma, K.: Some properties and application of branched-cyclodextrins. J. Jpn. Soc. Starch Sci., 30, 231-239 (1983). Kato, T. and Horikoshi, K.: A new ~'-cyclodextrin forming enzyme produced by Bacillus subtilis no. 313. J. Jpn. Soc. Starch Sci., 33, 137-143 (1986). Kitahata, S., Kubota, N., and Okada, S.: Industrial production of cyclodextrin. Kagaku to Kougyou, 60, 335-338 (1990). (in Japanese)
8. Fujita, Y., Tsubouchi, H., Inagi, Y., Tomita, K., Ozaki, A., and Nakanishi, K.: Purification and properties of cyclodextrin
glycosyltransferase from Bacillus sp. AL-6. J. Ferment. Bioeng., 70, 150-154 (1990). 9. Tomita, K., Kaneda, M., Kawamura, K., and Nakanishi, K.:
Purification and properties of a cyclodextrin glucanotransferase from Bacillus autolyticus 11149 and selective formation of /3cyclodextrin. J. Ferment. Bioeng., 75, 89-92 (1993). 10. Kobayashi, S., Kainuma, K., and Suzuki, S.: Purification and some properties of Bacillus macerans cycloamylose (cyclodextrin) glucanotransferase. Carbohydr. Res., 61, 229-238 (1978). 11. Okada, T., Ito, M., and Hibino, K.: Immobilization of cyclodextrin glucanotransferase on capillary membrane. J. Ferment. Bioeng., 77, 259-263 (1994). 12. Lowry, O.H., Rocebrough, N.J., Farr, A.L., and Randall,
R.J.: Protein measurements with the Folin phenol reagent. J. Biol. Chem., 193, 265-275 (1951). 13. Kobayashi, S.: Action mechanism of Bacillus macerans enzyme and preparation of cyciodextrins. J. Jpn. Soc. Starch Sci., 22, 126-132 (1975). 14. Hashimoto, H., Hara, K., Kuwahara, N., and Hosomi, A.:
The continuous production using the ultrafiltration membrane reactor. J. Jpn. Soc. Starch Sci., 33, 25-28 (1986).
Intermolecular Transglycosylating Reaction of Cyclodextrin Glucanotransferase Immobilized on Capillary Membrane TAKESHI OKADA, MASAAKI ITO, AND KEN HIBINO* Medical Research Laboratory, Nitto Denko Corporation, 1-1-2, Shimohozumi, Ibaraki, O~aka 567, Japan Received 15 June 1993/Accepted 10 November 1993 The intermolecular transglycosylating reaction of cyclodextrin glucanotransferase ([EC 2.4.1.191; CGTase) immobilized on a capillary membrane was investigated using low molecular weight substrates such as cyclodextrin (CD), maltooligosaceharide (MOS), and a CD-MOS mixture. The immobilized CGTase catalyzed the conversion reaction of a-CD to ~-CD and MOS or ~-CD to a-CD and MOS within a short residence time. The conversion ratio increased as the amount of immobilized CGTase increased. The addition of glucose, maltose, and sucrose as acceptors in the substrate solution containing CD resulted in the acceleration of CD degradation compared with only CD substrate. Furthermore, the MOS substrate (degree of polymerization=2-6) was disproportionated with a conversion ratio exceeding 70~o by the immobilized CGTase. These data demonstrate that immobilized CGTase can catalyze intermolecular transglycosylation between low molecular substrates in a few minutes by regulating the amount of immobilized enzyme and the residence time. This might contribute to our comprehension of CGTase-immobilized bioreactors for CD production as well as to the development of new glycosides through its excellent transglycosylation ability.
in detail. This study deals with the intermolecular transglycosylation of CGTase immobilized on a capillary membrane using low molecular substrates such as CD, MOS, and a CD-MOS mixture.
Cyclodextrins (CDs) are able to form inclusion complexes with hydrophobic substances (1). This property, named "molecular encapsulation", is useful for improving the properties of certain unstable, volatile, readily oxidable, or poorly soluble substances. In Japan, CDs are widely used in various industrial fields such as food processing, medicine, agricultural chemicals, and cosmetics. It is well known that CD can be synthesized from such carbohydrates as starch, glycogen, and maltooligosaccharides (MSOs) by cyclodextrin glucanotransferase ([EC 2.4.1.19]; CGTase) (2-9). At present, CDs are generally produced from potato starch using industrial scale batch operations. CGTase derived from Bacillus macerans, Bacillus megaterium, and Bacillus stearothermophilus is a very interesting enzyme which catalyzes intramolecular (cyclizing) and intermolecular (coupling, disproportionation) transglycosylation, as well as having a hydrolytic action on starch and CD (4, 10). In intermolecular transglycosylation, CGTase transfers glycosyl residues from starch or CD to a suitable acceptor like glucose or sucrose. For example, in the application of its coupling reaction, a coupling sugar which can be used as a sweetener for the prevention of dental caries is efficiently produced from starch and sucrose by CGTase. When CD is produced in a batch operation, however, the intermolecular transglycosylation should be reduced as much as possible in order to increase the CD content in the reaction mixture. In a previous paper (11), we investigated the kinetics of CGTase immobilized on a capillary membrane and demonstrated that excess immobilized CGTase could lead to a reduction in the CD content in the permeate as well as a decrease in the CD production rate due to sidereactions and low flux. Although side-reactions could be an intermolecular transglycosylation like a coupling reaction, the mechanism of the reaction has not been studied
MATERIALS AND METHODS
Crude CGTase from B. macerans was purchased from Amano Pharmaceutical Co. Ltd. (Nagoya). This solution had an enzyme activity of approximately 600 T H U / m l (30 THU per mg protein). CDs were obtained from Tokyo Kasei Kogyo Co. Ltd. (Tokyo). Glucose, maltose, sucrose and glutaraldehyde solution were purchased from Wako Pure Chemical Industries Co. Ltd. (Osaka). Maltooligosaccharides (degree of polymerization=3-6) were obtained from Nihon Shokuhin Kako Co. Ltd. (Tokyo) and Oriental Yeast Co. Ltd. (Tokyo); their purities were 96-98% according to high performance liquid chromatography (HPLC) analysis. Chemicals
Preparation of CGTase immobilized on the capillary membrane The immobilization of CGTase on the
capillary membrane, operation of the membrane type bioreactor with the immobilized CGTase, and calculation of the amount of immobilized CGTase were carried out according to the conditions described in a previous paper (11), except that in this study the reaction temperature was set at 50°C. The substrate solution was circulated for 30-60 rain since the product concentration in permeate reached a constant value during this circulation time (data not shown). Measurement of CD, glucose, and MOS Both permeate and circulate containing CD, glucose, and MOS were sampled at regular time intervals. The concentrations of CD and MOS were determined by HPLC analysis as described by Kato et al. (6) with a slight modification. A detailed description of their measurement was presented in a previous paper (11). In some samples, glucoamylase (Seikagaku Kogyo Co. Ltd., Tokyo) was
* Corresponding author. 264
VOL 77, 1994
COUPLING REACTION OF IMMOBILIZED CGTase
added in order to convert MOS to glucose before H P L C analysis. Protein assay Protein concentration was assayed according to the method of Lowry et al. (12).
120 [(A)
265
(B)
~i00
4
80 3 RESULTS
.= 60
Effect o f r e s i d e n c e t i m e o n c o n v e r s i o n
from
CD
to
MOS The a m o u n t of immobilized CGTase ',0.49 m g / cm 2) was adjusted to the level where a side-reaction seems to occur in the immobilized capillary membrane, as described previously (11). The CD and MOS concentrations in permeate versus residence time that was used as the apparent contact time between substrate and immobilized enzyme are plotted in Fig. 1A and 1C when 0.5% a-CD and ~-CD containing 25 m M acetate buffer (pH 6.0) and 1 . 0 m M CaCIE, respectively, were used as substrates. As a result, 60-70% of the a-CD was converted by the immobilized CGTase to fl-CD and MOS over the residence time range of 1.3-13.5 min. The ratio of aCD to ~-CD in the permeate was approximately 1.0 for each residence time (Fig. 1B). Although the ~-CD was also converted to a-CD and MOS, the conversion ratio of 13-CD to t~-CD was lower than the result obtained in Fig. 1A and 1B, when the residence time was short (Fig. 1C and 1D). In both reactions, the MOS had a degree of polymerization of 1-7 and the longer residence time resulted in an increase in MOS content rather than in an acceleration of the conversion from a-CD to ~-CD or ~3-CD to a-CD. These data indicate that immobilized CGTase has a strong hydrolytic effect on both a-CD and ~-CD. Thus, conversion of the substrate by immobilized CGTase could be performed with the process which after CD was hydrolyzed, the MOS obtained as first product is furthermore converted to MOS (second product) having higher and lower degree of polymerization by the disproportionating reaction and then the second products having higher degree of polymerization are used as a substrate for CD production. Effect o f a c c e p t o r c o n c e n t r a t i o n o n the c o u p l i n g reaction of immobilized CGTase A mixture of a-CD
(5 m g / m l ) and glucose ( 0 - 6 m g / m l ) was used as a substrate for immobilized CGTase (0.05 and 0 . 6 0 m g / c m 2) in order to investigate the effect of acceptors on the coupling reaction of the immobilized CGTase. Residence
2 40 r~
1
20
G)
,
0 o 120
,
I
4 8 12 16 Residence time (min)
0
t
o
5,
(C)
~100
i
t
4 8 12 16 Residence time (min) (D)
4
80 3!
e~
.= 60 2 40 20
G)
o
b
0
I
0
4 8 12 16 Residence time (min)
i
0
t
t
4 8 12 16 Residence time (min)
FIG. 1. Effectsof residence time on the conversion of a-CD and /3-CD by CGTase immobilized on a capillary membrane and the ratio of a-CD to 3-CD in permeate. The amount of immobilized CGTase was adjusted to 0.49 mg/cm2, and 0.50/00a-CD (A, B) and ~-CD (C, D) were used as substrates, respectively. Symbols: ~, a-CD; ~, t~CD; ~, MOS. time was controlled within a minimal variation time of 3.1-3.5 min. Figure 2A shows that the reduction in total CD (a-CD+I~-CD) in the permeate of both bioreactors was significantly dependent o n the concentration of glucose, which was added as an acceptor. Both permeates also contained /3-CD and MOS. The conversion ratio of immobilized CGTase with 0.60 m g / c m 2 was higher than that with 0.05 m g / c m 2, indicating that the a m o u n t of ira(B)
(A)
4
1
2
3
4
S
6
Glucoseconcentration(mg/ml)
0
I
i
I
I
I
I
1
2
3
4
5
6
Glucoseconcentration(mg/ml)
FIG. 2. Effects of glucose on the coupling reaction of CGTase immobilized on a capillary membrane. The amount of immobilized CGTase was adjusted to 0.05 and 0.60 mg/cm2, respectively. 0.5~ a-CD containing glucose was used as a substrate. The residence time was controlled within 3.1-3.5 min. Symbols: o , 0.05 mg/cm2; A, 0.60 mg/cmL
266
O K A D A ET AL.
J. FERMENT. BIOENG., 100
~4
~
so
E "~ 2
60
~ ~o
1
~ g
2o
0 6 Molar ratio of acceptor to ct- CD FIG. 3. Effects of various acceptors on the coupling reaction of CGTase immobilized on a capillary membrane. The amount of immobilized CGTase was adjusted to 0.49 m g / c m z, 0.5% a-CD containing various acceptors was used as a substrate. The residence time was controlled within 8.6-10.4min. Symbols: o , glucose; A, sucrose; • , maltose; v, m a l t o t r i o s e ; . , maltotetraose.
mobilized CGTase is important for enhancing intermolecular transglycosylation. The ratios of a-CD to /3-CD obtained at each glucose concentration are shown in Fig. 2B. The ratios obtained by bioreactors having 0.05 and 0.60mg/cm 2 of immobilized CGTase were 5.5-7.8 and approximately 1.0 respectively, regardless of glucose concentration. Effects of various aeceptors on the coupling reaction of immobilized CGTase To investigate the effects of oligosaccharides as acceptors in the conversion of a-CD to MOS by immobilized CGTase, glucose, maltose, maltotriose, maltotetraose, or sucrose were added to reaction mixtures containing 0.5% a-CD. Figure 3 depicts the relationship between the molar ratio of acceptor to a-CD and total CD concentration in permeate. Total CD concentration decreased in the order of glucose> sucrose > maltose > maltotriose > maltotetraose when each reaction mixture contained an equal amount of nonreducing residue. However, for maltotriose and maltotetraose, CD production was higher than that obtained in the reaction mixture containing only a-CD. This may be due to CD production from maltotriose and maltotetraose, as well as from MOS, having a high degree of polymerization produced by the disproportionation reaction of immobilized CGTase, as described below. Cyclizing and disproportionating reactions by immobilized CGTase using MOS as a substrate MOS is well known to be disproportionated by CGTase to yield MOS with higher and lower degrees of polymerization (1). Figure 4 shows the relationship between the degree of polymerization in MOS when used as a substrate and the ratio that was converted from the substrate to CD and MOS having a different degree of polymerization from that of the substrate. Residence times were controlled within a minimal variation time of 8.9-9.6 min. The conversion ratio depended on the degree of polymerization in substrate; ratios exceeding 90% were obtained for maltotetraose and maltohexaose. Immobilized CGTase also produced CD from MOS, and the CD content in permeate increased to 18, 32, and 45°//00 for maltotriose, maltotetraose, and maltohexaose substrates, respectively.
0 0
l
2
3
4
5
6
Number of glucose in MOS FIG. 4. Effect of the number of glucose molecules in MOS on the cyclizing and disproportionating reaction of CGTase immobilized on a capillary membrane. The amount of immobilized CGTase was adjusted to 0.49 m g / c m 2. 0.6% MOS was used as a substrate. The residence time was controlled within 8.9-9.6min. Symbols: o , conversion ratio; A, CD content in permeate.
Glucose was not disproportionated or cyclized at all. DISCUSSION In the present paper, it has been demonstrated that immobilized CGTase at a high concentration could catalyze intermolecular transglycosylations, such as coupling and disproportionating reactions, within a very short reaction time. Although such reactions have been well characterized using free CGTase derived from several Bacillus strains (4, 10), the degree of coupling and disproportionation seemed to be very low because of the relatively low concentration of free CGTase used in the enzyme reaction. Kitahata et al. (4) have shown that only 25% of the maltotriose used as a substrate was disproportionated with free CGTase from B. macerans, even though the reaction mixture was incubated for 3 h. On the other hand, immobilized CGTase converted 70-90% of the MOS used as a substrate to CD and MOS having different degrees of polymerization from that of the substrate within a short residence time. Immobilized CGTase also hydrolyzed both a-CD and ~-CD. Although CGTase is classified as a transferase, some researchers have shown that CGTase has hydrolytic activity since free CGTase from Bacillus strains liquefies starch paste rapidly and forms reducing residue (4, 10). However, it has been reported that the hydrolytic efficiency of immobilized CGTase on CD was higher than that by free CGTase (4, 10, 13). This is clearly due to differences in the enzyme concentrations used in the experiments. In general, when CD is produced with free CGTase in a batch operation, a low concentration of CGTase is added to reduce the coupling reaction (14). While CD conversion by immobilized CGTase was accelerated in accordance with increases in acceptors like glucose and sucrose added to the reaction mixture, as is also the case for free CGTase, the addition of an acceptor with a higher degree of polymerization was ineffective for CD degradation. Since immobilized CGTase can produce higher levels of MOS by the disproportionating reaction, maltotetraose or maltohexaose used as an ac-
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ceptor could be changed to a desirable substrate for cyclizing reaction after being d i s p r o p o r t i o n a t e d . However, these findings regarding the effects o f acceptors on coupling reaction were different f r o m those obtained with free CGTase. K o b a y a s h i (13) has indicated that the a d d i t i o n o f M O S (degree o f p o l y m e r i z a t i o n = 1--4) promotes the coupling reaction c o m p a r e d to absence o f the acceptor, and that maltose is the most effective acceptor. Thus, C G T a s e m a y exhibit interesting enzymatic p r o p e r ties as the enzyme concentration increases. The d a t a o b t a i n e d in this study s u p p o r t the hypothesis that regulation o f b o t h the a m o u n t o f immobilized C G T a s e and the residence time is i m p o r t a n t for reducing intermolecular transglycosylation and hydrolysis, as well as for improving CD productivity, as has been suggested in a previous p a p e r (11). A l t h o u g h side-reactions which cause CD degradation should be reduced for CD production from starch, an alternative use for bioreactors with high concentrations o f immobilized C G T a s e is the production o f coupling sugars or glycosides by utilizing their excellent intermolecular transglycosylation ability. Moreover, the decrease in flux in the m e m b r a n e type bioreactor using low molecular substrate was not r e m a r k a ble, even if the b i o r e a c t o r was used for repeated reactions. This merit suggests that practical application is possible. Transglycosylation by enzymes is currently a c o m m o n m e t h o d for improving the p o o r solubility o f bioactive c o m p o u n d s derived from plants on an industrial scale. We are now investigating the possibility o f using this b i o r e a c t o r for the actual p r o d u c t i o n o f bioactive c o m p o u n d s . REFERENCES
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