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Preparation of immobilized enzyme surfaces by radiation technique
Int..I. AppL Radiat. Isot. Vol. 35. No. 10, pp, 969-970, 1984 Printed in Great Britain. All rights reserved
0020-708X/84S3.00+0.00 Copyright ~ 1984 Pergamon Press Ltd
Preparation of Immobilized Enzyme Surfaces by Radiation Technique MINORU
KUMAKURA
and ISAO KAETSU
Takasaki Radiation Chemistry Research Establishment, Japan Atomic Energy Research Institute, Takasaki, Gunma, Japan
(Receired 6 February 1984) Immobilized cellulase surfaces were prepared by a simple radiation technique, coating 2-hydroxyethyl methacrylate and tetraethyleneglycol diacrylate on the inner surface of a polyethylene tube. The durability of the surfaces was examined by repeated batch enzyme reactions as a function of monomer and enzyme concentration. The amount of the enzymes located on the surface was about 0.5 mg/cm e, in which the enzymes did not leak from the polymer matrix. The optimum monomer concentration in the immobilization was 60-70%.
Introduction Immobilized enzymes are gaining importance in many industrial and biomedical applications. In the various immobilization methods of enzymes on carriers, covalent binding is considered the best method for obtaining products with surface enzyme activity.(" The importance of using the graft polymerization method as well as the covalent binding method has been noticed recently, and radiation graft polymerization methods have been studied by many workers. (2"~) However, for many reasons it is dii~cult in the graft polymerization method to obtain the immobilized enzymes with high enzyme contents, giving high concentrations of bound enzyme per gram. Therefore, in this work, preparation of immobilized enzyme surfaces by radiation polymerization technique of monomer containing enzymes coated on the inner surface of a polyethylene tube was studied. With this method, immobilized enzymes trapped with high contents on the surface of the polymer matrix will be obtained by controlling the enzyme concentration in the preparation.
Preparation of immobilized enzyme surfaces Monomer and enzyme were dissolved in 0.t M acetate buffer solution, pH 4.5. This monomer solution was coated on the inner surface of a polyethylene test tube (1.8cm in diameter and 17cm in length), and it was immediately cooled to - 7 8 ° C by immersion into a dry ice-methanol mixture. After freezing the coated monomer, the tube was irradiated with 1 × l06 tad and a dose rate of 1 x 106 R/h by y-rays from a e C o source. The irradiation temperature was maintained at - 7 8 ° C . After irradiation, the monomer coating on the inner surface of the tube was polymerized giving the immobilized enzyme surfaces.
M a t e r i a l s and M e t h o d s
Enzyme reaction The enzyme reactions with the immobilized enzyme surfaces were carried out using the tube conmining the substrate, by repeating a batch enzyme reaction, in which the temperature and time of the reaction were 40°C and 1.0 h respectively. Sodium carboxymethyicellulose (0.5%) dissolved in 0.I M acetate buffer solution was used as substrate throughout this work. The concentration of glucose formed at each batch enzyme reaction was measured with a giucose-specific reagent. The enzyme activity (%) of the immobilized enzyme surfaces remaining in the repeated batch enzyme reactions was obtained from the glucose-formation ratio of the immobilized and original enzymes at each batch enzyme reaction.
Materials 2-Hydroxyethy! methacry|ate (HEMA) and tetraethylenegiycol diacrylate (A-4G) obtained from Shin Nakamura Chemical Co. Ltd was used as a Results and Discussion monomer. The durability of the immobilized enzyme surfaces Ce[lulase (EC3.2.1.4, from Trichoderma vir_ide) was obtained from Yakult Biochemicals Co. Ltd, its was examined by repetition of the batch enzyme activity being I x 104 units/g. reaction. The relationship between enzyme activity 969
970
ML',;ORUKUMAKUV, A and ISAoKJmrsu
100
m
w
.~
-_
:_S 50
bJ
o
5
1o
15
Number of balcl~ enzyme reactions (times)
I 02
I 0.4
I 0.6
Enzyme concentrotion (%)
Fig. 1. Relationship between enzyme activity and number of batch enzyme reactions in the immobilized enzyme surfaces prepared from various monomer concentrations. Monomer ratio (A-4G/HEMA): 0.1. Monomer concentration (%): 020, e50, AT0, &90.
and number of batch enzyme reactions is shown in Fig. I. The enzyme activity of the immobilized enzyme surfaces prepared from low monomer concentrations (20%) decreased, but that of high monomer concentrations above 50% was almost constant with increasing number of batch enzyme reactions. The decrease of the enzyme activity in low monomer concentrations could be caused by leakage of the enzyme from the polymer matrix. The enzyme activity in 70°,/0 monomer concentration was higher than that in 90% monomer concentration. This result suggests that as the monomer concentration increases a part of the enzymes is trapped in the polymer matrix and then the enzyme activity is decreased. Furthermore, as the monomer concentration increases, the diffusion resistance of the substrate in the polymer matrix would be increased by the increase of the thickness of the polymer matrix and by the decrease of the pore size in the polymer matrix. The thickness of the immobilized enzyme surfaces prepared from 70% monomer concentration was about 300/zm. Thus, an optimum monomer concentration in the immobilization appeared to be 60--70%, in which the enzymes are firmly trapped on the surface of the coated polymer matrix. The effect of enzyme concentration on the immobilization was examined. The relationship between enzyme concentration and enzyme activity at the later stage of the repeated batch enzyme reactions (15 times) is shown in Fig. 2. The enzyme activity was constant till about 0.5% enzyme concentration and after that was slightly decreased, indicating that the limit of the permitted enzyme concentration in the
Fig. 2. Relationship between enzyme activity and enzyme concentration. Monomer ratio (A-4G/HEMA): 0.1. Monomer concentration (%): 70. immobilization with 70% monomer concentration was about 0.5%. From this result, the amount of the enzymes located on the surface of the polymer matrix obtained was found to be about 0.5 mg/cm 2, since the enzyme activity in the monomer concentration of 0.5% was about 70% as seen in Fig. 2. The concentration limit of the enzyme in the present immobilization method would be high in comparison to that in other methods. It is suggested that the polymer matrix of the immobilized enzyme surfaces has a porous structure and its polymer chains are crosslinked by the addition of A-4G. Furthermore, the immobilized enzyme surfaces produced a liquid state resulting from a cross-linkage reaction and were firmly adhered to the polyethylene tube. The enzyme surfaces could not be stripped from the inner surface of the tube throughout repeated batch enzyme reactions. The preparation process of the immobilized enzyme surfaces is simple and the immobilized enzyme surfaces prepared using various enzymes can be used as reactors for various reactions.
References
I. Messing R. A. (Ed). Immobilized Enzymesfor Industrial Reactors (Academic Press, New York, 1975), 2. D'Angiuro L., Vccchio G., Mazzola G., Cantati R. and Cremonesi P. Angew. Makromol. Chem. 104, 129 (1982). 3. Libby M. J., Gannett J. L. and Kenyon R. S. J. Polym. ScL Syrup. No. 49, 109 (1975). 4. Venkataraman S., Horbett T. A. and Hoffman A. S. J. Moi. Catal. 2, 273 (1977). 5. Hay F. A., Beddow S. G. and Guthrie J. T. Makromolek. Chem. 182, 717 (1981). 6. Beddows C. (3. and Guthrie J. T. Biotechnol. Bioeng. 24, 1371 (1982).
0020-708X/84S3.00+0.00 Copyright ~ 1984 Pergamon Press Ltd
Preparation of Immobilized Enzyme Surfaces by Radiation Technique MINORU
KUMAKURA
and ISAO KAETSU
Takasaki Radiation Chemistry Research Establishment, Japan Atomic Energy Research Institute, Takasaki, Gunma, Japan
(Receired 6 February 1984) Immobilized cellulase surfaces were prepared by a simple radiation technique, coating 2-hydroxyethyl methacrylate and tetraethyleneglycol diacrylate on the inner surface of a polyethylene tube. The durability of the surfaces was examined by repeated batch enzyme reactions as a function of monomer and enzyme concentration. The amount of the enzymes located on the surface was about 0.5 mg/cm e, in which the enzymes did not leak from the polymer matrix. The optimum monomer concentration in the immobilization was 60-70%.
Introduction Immobilized enzymes are gaining importance in many industrial and biomedical applications. In the various immobilization methods of enzymes on carriers, covalent binding is considered the best method for obtaining products with surface enzyme activity.(" The importance of using the graft polymerization method as well as the covalent binding method has been noticed recently, and radiation graft polymerization methods have been studied by many workers. (2"~) However, for many reasons it is dii~cult in the graft polymerization method to obtain the immobilized enzymes with high enzyme contents, giving high concentrations of bound enzyme per gram. Therefore, in this work, preparation of immobilized enzyme surfaces by radiation polymerization technique of monomer containing enzymes coated on the inner surface of a polyethylene tube was studied. With this method, immobilized enzymes trapped with high contents on the surface of the polymer matrix will be obtained by controlling the enzyme concentration in the preparation.
Preparation of immobilized enzyme surfaces Monomer and enzyme were dissolved in 0.t M acetate buffer solution, pH 4.5. This monomer solution was coated on the inner surface of a polyethylene test tube (1.8cm in diameter and 17cm in length), and it was immediately cooled to - 7 8 ° C by immersion into a dry ice-methanol mixture. After freezing the coated monomer, the tube was irradiated with 1 × l06 tad and a dose rate of 1 x 106 R/h by y-rays from a e C o source. The irradiation temperature was maintained at - 7 8 ° C . After irradiation, the monomer coating on the inner surface of the tube was polymerized giving the immobilized enzyme surfaces.
M a t e r i a l s and M e t h o d s
Enzyme reaction The enzyme reactions with the immobilized enzyme surfaces were carried out using the tube conmining the substrate, by repeating a batch enzyme reaction, in which the temperature and time of the reaction were 40°C and 1.0 h respectively. Sodium carboxymethyicellulose (0.5%) dissolved in 0.I M acetate buffer solution was used as substrate throughout this work. The concentration of glucose formed at each batch enzyme reaction was measured with a giucose-specific reagent. The enzyme activity (%) of the immobilized enzyme surfaces remaining in the repeated batch enzyme reactions was obtained from the glucose-formation ratio of the immobilized and original enzymes at each batch enzyme reaction.
Materials 2-Hydroxyethy! methacry|ate (HEMA) and tetraethylenegiycol diacrylate (A-4G) obtained from Shin Nakamura Chemical Co. Ltd was used as a Results and Discussion monomer. The durability of the immobilized enzyme surfaces Ce[lulase (EC3.2.1.4, from Trichoderma vir_ide) was obtained from Yakult Biochemicals Co. Ltd, its was examined by repetition of the batch enzyme activity being I x 104 units/g. reaction. The relationship between enzyme activity 969
970
ML',;ORUKUMAKUV, A and ISAoKJmrsu
100
m
w
.~
-_
:_S 50
bJ
o
5
1o
15
Number of balcl~ enzyme reactions (times)
I 02
I 0.4
I 0.6
Enzyme concentrotion (%)
Fig. 1. Relationship between enzyme activity and number of batch enzyme reactions in the immobilized enzyme surfaces prepared from various monomer concentrations. Monomer ratio (A-4G/HEMA): 0.1. Monomer concentration (%): 020, e50, AT0, &90.
and number of batch enzyme reactions is shown in Fig. I. The enzyme activity of the immobilized enzyme surfaces prepared from low monomer concentrations (20%) decreased, but that of high monomer concentrations above 50% was almost constant with increasing number of batch enzyme reactions. The decrease of the enzyme activity in low monomer concentrations could be caused by leakage of the enzyme from the polymer matrix. The enzyme activity in 70°,/0 monomer concentration was higher than that in 90% monomer concentration. This result suggests that as the monomer concentration increases a part of the enzymes is trapped in the polymer matrix and then the enzyme activity is decreased. Furthermore, as the monomer concentration increases, the diffusion resistance of the substrate in the polymer matrix would be increased by the increase of the thickness of the polymer matrix and by the decrease of the pore size in the polymer matrix. The thickness of the immobilized enzyme surfaces prepared from 70% monomer concentration was about 300/zm. Thus, an optimum monomer concentration in the immobilization appeared to be 60--70%, in which the enzymes are firmly trapped on the surface of the coated polymer matrix. The effect of enzyme concentration on the immobilization was examined. The relationship between enzyme concentration and enzyme activity at the later stage of the repeated batch enzyme reactions (15 times) is shown in Fig. 2. The enzyme activity was constant till about 0.5% enzyme concentration and after that was slightly decreased, indicating that the limit of the permitted enzyme concentration in the
Fig. 2. Relationship between enzyme activity and enzyme concentration. Monomer ratio (A-4G/HEMA): 0.1. Monomer concentration (%): 70. immobilization with 70% monomer concentration was about 0.5%. From this result, the amount of the enzymes located on the surface of the polymer matrix obtained was found to be about 0.5 mg/cm 2, since the enzyme activity in the monomer concentration of 0.5% was about 70% as seen in Fig. 2. The concentration limit of the enzyme in the present immobilization method would be high in comparison to that in other methods. It is suggested that the polymer matrix of the immobilized enzyme surfaces has a porous structure and its polymer chains are crosslinked by the addition of A-4G. Furthermore, the immobilized enzyme surfaces produced a liquid state resulting from a cross-linkage reaction and were firmly adhered to the polyethylene tube. The enzyme surfaces could not be stripped from the inner surface of the tube throughout repeated batch enzyme reactions. The preparation process of the immobilized enzyme surfaces is simple and the immobilized enzyme surfaces prepared using various enzymes can be used as reactors for various reactions.
References
I. Messing R. A. (Ed). Immobilized Enzymesfor Industrial Reactors (Academic Press, New York, 1975), 2. D'Angiuro L., Vccchio G., Mazzola G., Cantati R. and Cremonesi P. Angew. Makromol. Chem. 104, 129 (1982). 3. Libby M. J., Gannett J. L. and Kenyon R. S. J. Polym. ScL Syrup. No. 49, 109 (1975). 4. Venkataraman S., Horbett T. A. and Hoffman A. S. J. Moi. Catal. 2, 273 (1977). 5. Hay F. A., Beddow S. G. and Guthrie J. T. Makromolek. Chem. 182, 717 (1981). 6. Beddows C. (3. and Guthrie J. T. Biotechnol. Bioeng. 24, 1371 (1982).