BIOTECHNOLOGY AND BIOENGINEERING, VOL. XVIII, PAGES 1507-1516 (1976)
Physical Characteristics of Porous Cellulose Beads as Supporting Material for Immobilized Enzymes LI FU CHEN and GEORGE T. TSAO, School of Chemical Engineering, Purdue trniversity, West Lafayette, Indiana 479G7
Summary Cellulose beads prepared in this report have high porosity (75-80%) and evenly distributed pores. The pore size is about 1000 A. The cellulose beads are physically strong and contain large amounts of reactive groups, making them suitable for use as carriers for immobilized enzymes.
INTRODUCTION A good carrier for immobilizing enzymes must be a stable material which possesses versatile chemical properties. It must be inexpensive especially when it is used in commercial processes. The success of an immobilized enzyme for use in practical application depends upon the properties of the carriers employed. Accordingly, a good carrier should be of such physical shape that it is easy to be employed in a reactor. I n this regard, the shape of a bead is particularly desirable, since it is useful in a packed bed, fluidized bed, expanded bed, stirred tank, or other commonly found designs of chemical reactors. A carrier should also have the proper physical and mechanical strength such that it will not be crushed or deformed when packcd in a tall column. A suitable carrier should also possess versatile chemical properties such that the immobilization of enzymes onto the carrier through ionic or chemical covalent bonding, as well as surface absorption, can be readily achieved. The carrier should have a high capacity for forming a large number of bonds such that each unit of the carrier can immobilize large amounts of enzymes, if desired. A carrier having a high degree of porosity and a large internal surface area is particularly desirable. Carriers should also be chemically stable and inert towards microbiological contamination so that they will not deteriorate due to attack by microorganisms and will thereby provide a n immobilized enzyme system having a prolonged life of activity. 1507 @ 1976 by John Wiley& Sons, Inc.
CHEN AND TSAO
1508
This study presents some characteristics of inexpensive physically strong, spherical, and highly porous cellulose particles having versatile chemical properties. They have been found useful as carriers for immobilized enzymes.
MATERIALS AND METHODS Materials DEAE cellulose and blue dextran were purchased from Sigma Chemical Company. Formaldehyde, 2-~hlorotriethylamine, and tolylene 2,4-diisocyanate were purchased from Aldrich Chemical Company, Inc. Glucoamylase was obtained from Novo Enzyme Company. Glucose isomerase was a gift from Miles Laboratories, Inc. A scanning electron microscope Model JSM-U3 was used t o take the electron micrographs of the porous cellulose beads.
Preparation of Cellulose Beads Cellulose acetate was dissolved in a water miscible organic solvent
(a 6: 4 mixture of acetone and DMSO t o form a 12.5y0 w/v solution). The solution was then dispersed into a water phase. When the dispersed droplets came in contact with water, the liquid droplets coagulated and porous particles were formed. The particles were collected and washed. The cellulose acetate was then regenerated to cellulose by hydrolysis.
Preparation of DEAE Cellulose Beads The porous cellulose beads prepared as described above were crosslinked with 360/, formaldehyde and 370/, hydrochloric acid as described by Bullock and Guthrie.1 The crosslinked beads were then treated with 2-chlorotriethylamine to form DEAE cellulose.'
Isocyanate Treatment Two grams of cellulose beads were first dispersed in 50 ml of acetone. Two milliliters of triethylamine were added as a catalyst. Finally, 2 ml of tolylene 2,4-diisocyanate were then added. After 45 min, the solution was decanted. The beads were washed by acetone t o remove the excess diisocyanate.
Immobilization of Enzyme
Diisocyanate method The beads treated with tolylene 2,4-diisocyanate were suspended in 100 ml of a glucoamylase solution (25 mg protein ml) a t PH 4.75.
POROUS CELLULOSE BEADS
The suspension was stored in the refrigerator overnight. washing with water, they were then ready for use.
1509
After
D U E method Three-tenths grams of the DEAE cellulose beads were washed with 1N NaOH and distilled water and next were washed with 0.1M phosphate buffer solution. Ten milliliters of a glucose isomerase solution (5 mg protein/ml) were added to the DEAE cellulose beads. The mixture stood a t 4°C overnight. After washing with water, they were ready for use.
Determination of Porosity The porosity of the bead was determined by a technique commonly employed in liquid chromatography. The beads were packed in a column with a known diameter. The percentage of porosity was determined by the following equation:
yoporosity= total porous volume in column - void volume outside beads (1) bed volume - void volume outside beads where the bed volume is the n D 2 X L, D is the diameter of the column, and L is the height of the packed bed. Total porous volume in the column was determined by passing a phenolphthalein or glucose solution through the column. When the dye or glucose started to come out of the column the volume was taken to be the total porous volume. Void volume outside the beads were determined by passing blue dextran through the column. When blue dextran came out of the column the volume was taken to be the void volume outside of the beads.
Size of the Pore The size of the pores on the beads was measured from scanning electron micrographs of these beads. Since the beads shrank when they were dried in air, critical point drying with liquid carbon dioxide was used. The beads were mounted on an aluminum plate. After the sample was coated with carbon, it was then scanned.
Pressure Drop in the Packed Column The two ends of the column were connected to a mercury u-tube manometer. Water was pumped through the column by a Cole-
1510
CHEN AND TSAO
Palmer peristaltic pump at various nominal linear flow rates defined by the equation nominal linear flow rate =
vol/min cross-sectional area of the column
(2)
RESULTS AND DISCUSSION Size Distribution of Beads Formation A typical size distribution pattern is shown in Figure 1. Undesirable particle size can be recycled before the cellulose derivative was regenerated to cellulose. The desired particle size for enzyme immobilization depends on the kind of enzymes to be immobilized. For instance, the K , value of the immobilized enzyme complex, its effectiveness factor, and other parameters can all be affected by particle size.2 Structure of Cellulose Beads The electron micrographs show that the beads are mostly spherical. The regenerated cellulose forming the beads seems to exist in the shape of threads. The interior and the surface of the beads have the same structure. The pore sizes are quite uniform and the pores are distributed evenly throughout the whole bead (Fig. 2).
Pore Size of the Beads The pore size of the beads was measured from scanning electron micrographs (Fig. 2). The scanning micrographing requires dry samples. Drying the beads in air will result in a size shrinkage. Critical point drying of the beads with liquid carbon dioxide was therefore used. Since the cellulose is in the shape of threads, the beads are similar to a small cotton ball. The pore size is defined as the distance between two strings of cellulose threads in the beads. From the scanning electron micrographs, the pore size was measured to be about 1000 8. The thickne6 of the cellulose thread is about 500 A.
Porosity of the Beads The beads shrink when they are dried, so t h a t the technique commonly employed in liquid chromatography for determining porosity was applied. DEAE beads possess positive charges on the particle surface, they tend to absorb the dye in dextran at neutral pH.
POROUS CELLULOSE BEADS
1511
0.11
I 0.15
0.15
I 0.21
0.21
I 0.3
0.30
I 0.42
0.42
I 0.59
0.59
0.84
over
1 1 1 . 6 8 0.84 1.60
Diameter o f Beads (mn)
Fig. 1. Typical size distribution of cellulose beads.
Therefore, the pH of eluent water was adjusted at pH 11.0 to prevent this adsorption. In eq. (l), the term “void volume outside beads” was determined with the blue dextran. The term “total porous volume in column” was determined with glucose for the DEAE beads, and the total porous volume in the column was determined with phenolphthalein for the diisocyanate treated beads. The bead porosity thus calculated with eq. (1) was 78y0 and 75y0 for DEAE cellulose beads and diisocyanate treated beads, respectively. By
1512
CHEN AND TSAO
x
0 0
8 e4
e9 a
POROUS CELLULOSE BEADS
1513
controlling the conditions employed in forming the cellulose beads, the porosity can be varied.
Pressure Drop Across the Column The pressure drop is expressed in cm of Hg per cm of column length. A comparison between microcrystalline DEAE cellulose and the porous DEAE cellulose beads in Figure 3 shows that the porous beads exhibit stronger physical strength than microcrystalline DEAE cellulose. When the flow rate is less than 2 cm/sec, the DEAE beads have a linear relationship between the pressure drop and nominal linear flow rate. When the flow rate is higher than 2 cm/sec, the curve slope increases, indicating that the beads are compressed, deformed, and eventually collapsed. Apparently there seems to be an irreversible process taking place when the linear flow rate was increased and then decreased, the pressure did not drop to the original point corresponding to the same linear flow rate. A hysterisis phe-
1
3
2
4
Nominal Linear Flow Rate, cm/sec
Fig. 3.
Pressure drop in the column a t various nominal linear flow rate. (x), Crystalline DEAE cellulose; DEAE cellulose beads. ( e ) ,
1514
CHEN AND TSAO
nomenon exists in both microcrystalline DEAE and the porous DEAE cellulose particles. Figure 4 shows the same type of plot for the porous cellulose beads treated with diisocyanate and enzyme. The curve did not show a hysterisis behavior. The slope of the curve increased slightly when the linear flow rate increased. The beads were able to stand up to the pressure when the linear flow rate was increased. Untreated cellulose beads were easily compressed. However, when the nominal linear flow rate is less than 0.5 cm/sec, the curve slope is more or less constant. This means that the beads are not compressed and
1 2 3 4 Nominal Linear Flow Rate (cm per second)
Fig. 4. Pressure drop in the column a t various nominal linear flow rate. (x) Cellulose beads (48-65 mesh particle size); (.) cellulose beads treated with
cellulose beads treated with diisodiisocyanate arid enzyme (4&65 mesh) ; (H) cyanate and enzyme (20-3.5 mesh particle size).
POIlOUS CELLULOSE BEADS
1515
deformed. In the industrial process the linear flow rate is usually less than 0.5 cm/sec. These untreated beads would be suitable for the industrial processes. In Figure 4 the hysterisis effect of untreated cellulose beads was not investigated.
Immobilization of the Enzyme on the Beads Glucoamylase was covalently bonded to the porous cellulose beads with tolylene 2,4-diisocyanate. The enzyme loading was 2000 units of activity per gram of cellulose beads. One unit of glucoamylase activity was defined as the production of 1 pmol glucose per minute a t 55"C, using a 30% partially hydrolyzed (DE 10) starch solution as the substrate. When DEAE beads were used t o immobilize glucose isomerase, the enzyme activity on 1 g of porous DEAE cellulose bead was 400 units. A unit was defined as the production of 1 pmol glucose per minute a t 60"C, using a 2M fructose solution as its substrate.
CONCLUSIONS The cellulose beads appear to qualify as an excellent carrier for enzyme immobilization. The basic porous beads can be made easily and cost about $1.50 per pound. Cellulose having three hydroxyl groups on each anhydroglucose unit is highly versatile in chemical reactions and high in immobilization capacity. In this study, only two chemical procedures of enzyme immobilization were reported: the diisocyanate and the DEAE methods. In our laboratory, seven different procedures have all been successfully tested and will be reported separately. After treatment with, for instance, tolylene 2,4-diisocyanate1 the porous beads become very strong and rigid having good flow properties as described above. The porosity of the beads can be controlled easily. The pore size distribution is quite uniform and pores are more or less uniformly distributed throughout a bead as shown by the electron micrographs. The enzyme immobilization loading on the porous cellulose beads is quite high as reported. Further details of the chemical properties of immobilized glucose isomerase, glucoamylase, invertase, and lactase on the cellulose beads will be reported Separately. I n a test with glucoamylase, 220 mg of protein were attached to 1 g of the cellulose beads. Immobilized glucoamylase on the porous beads by diisocyanate attachment was found t o have an activity half-life of 70 hr a t 60°C, 130 hr a t 55OC, and an extrapolated 2800 hr a t 45°C.
1516
CHEN AND TSAO
The porous DEAE cellulose beads can be attached to glucose isomerase as well as a number of other enzymes. DEAE cellulose has been used as an enzyme carrier in commercial operations. Because of the flow properties of the ordinary DEAE cellulose, only shallow packed beds can be used for large-scale operation to avoid excessive pressure build-up. The porous DEAE cellulose beads reported have very good flow properties as well as high enzyme loading capacity.
References 1. A. L. Bullock and J . D. Guthrie, in Methods in Carbohydrate Chemistry, R. L. Whistler, Ed., Academic Press, New York, 1965, pp. 408-411. 2. Y. Y. Lee and George T. Tsao, J . Food Sci.,39,667 (1974).
Accepted for Publication June 25, 1976
Physical Characteristics of Porous Cellulose Beads as Supporting Material for Immobilized Enzymes LI FU CHEN and GEORGE T. TSAO, School of Chemical Engineering, Purdue trniversity, West Lafayette, Indiana 479G7
Summary Cellulose beads prepared in this report have high porosity (75-80%) and evenly distributed pores. The pore size is about 1000 A. The cellulose beads are physically strong and contain large amounts of reactive groups, making them suitable for use as carriers for immobilized enzymes.
INTRODUCTION A good carrier for immobilizing enzymes must be a stable material which possesses versatile chemical properties. It must be inexpensive especially when it is used in commercial processes. The success of an immobilized enzyme for use in practical application depends upon the properties of the carriers employed. Accordingly, a good carrier should be of such physical shape that it is easy to be employed in a reactor. I n this regard, the shape of a bead is particularly desirable, since it is useful in a packed bed, fluidized bed, expanded bed, stirred tank, or other commonly found designs of chemical reactors. A carrier should also have the proper physical and mechanical strength such that it will not be crushed or deformed when packcd in a tall column. A suitable carrier should also possess versatile chemical properties such that the immobilization of enzymes onto the carrier through ionic or chemical covalent bonding, as well as surface absorption, can be readily achieved. The carrier should have a high capacity for forming a large number of bonds such that each unit of the carrier can immobilize large amounts of enzymes, if desired. A carrier having a high degree of porosity and a large internal surface area is particularly desirable. Carriers should also be chemically stable and inert towards microbiological contamination so that they will not deteriorate due to attack by microorganisms and will thereby provide a n immobilized enzyme system having a prolonged life of activity. 1507 @ 1976 by John Wiley& Sons, Inc.
CHEN AND TSAO
1508
This study presents some characteristics of inexpensive physically strong, spherical, and highly porous cellulose particles having versatile chemical properties. They have been found useful as carriers for immobilized enzymes.
MATERIALS AND METHODS Materials DEAE cellulose and blue dextran were purchased from Sigma Chemical Company. Formaldehyde, 2-~hlorotriethylamine, and tolylene 2,4-diisocyanate were purchased from Aldrich Chemical Company, Inc. Glucoamylase was obtained from Novo Enzyme Company. Glucose isomerase was a gift from Miles Laboratories, Inc. A scanning electron microscope Model JSM-U3 was used t o take the electron micrographs of the porous cellulose beads.
Preparation of Cellulose Beads Cellulose acetate was dissolved in a water miscible organic solvent
(a 6: 4 mixture of acetone and DMSO t o form a 12.5y0 w/v solution). The solution was then dispersed into a water phase. When the dispersed droplets came in contact with water, the liquid droplets coagulated and porous particles were formed. The particles were collected and washed. The cellulose acetate was then regenerated to cellulose by hydrolysis.
Preparation of DEAE Cellulose Beads The porous cellulose beads prepared as described above were crosslinked with 360/, formaldehyde and 370/, hydrochloric acid as described by Bullock and Guthrie.1 The crosslinked beads were then treated with 2-chlorotriethylamine to form DEAE cellulose.'
Isocyanate Treatment Two grams of cellulose beads were first dispersed in 50 ml of acetone. Two milliliters of triethylamine were added as a catalyst. Finally, 2 ml of tolylene 2,4-diisocyanate were then added. After 45 min, the solution was decanted. The beads were washed by acetone t o remove the excess diisocyanate.
Immobilization of Enzyme
Diisocyanate method The beads treated with tolylene 2,4-diisocyanate were suspended in 100 ml of a glucoamylase solution (25 mg protein ml) a t PH 4.75.
POROUS CELLULOSE BEADS
The suspension was stored in the refrigerator overnight. washing with water, they were then ready for use.
1509
After
D U E method Three-tenths grams of the DEAE cellulose beads were washed with 1N NaOH and distilled water and next were washed with 0.1M phosphate buffer solution. Ten milliliters of a glucose isomerase solution (5 mg protein/ml) were added to the DEAE cellulose beads. The mixture stood a t 4°C overnight. After washing with water, they were ready for use.
Determination of Porosity The porosity of the bead was determined by a technique commonly employed in liquid chromatography. The beads were packed in a column with a known diameter. The percentage of porosity was determined by the following equation:
yoporosity= total porous volume in column - void volume outside beads (1) bed volume - void volume outside beads where the bed volume is the n D 2 X L, D is the diameter of the column, and L is the height of the packed bed. Total porous volume in the column was determined by passing a phenolphthalein or glucose solution through the column. When the dye or glucose started to come out of the column the volume was taken to be the total porous volume. Void volume outside the beads were determined by passing blue dextran through the column. When blue dextran came out of the column the volume was taken to be the void volume outside of the beads.
Size of the Pore The size of the pores on the beads was measured from scanning electron micrographs of these beads. Since the beads shrank when they were dried in air, critical point drying with liquid carbon dioxide was used. The beads were mounted on an aluminum plate. After the sample was coated with carbon, it was then scanned.
Pressure Drop in the Packed Column The two ends of the column were connected to a mercury u-tube manometer. Water was pumped through the column by a Cole-
1510
CHEN AND TSAO
Palmer peristaltic pump at various nominal linear flow rates defined by the equation nominal linear flow rate =
vol/min cross-sectional area of the column
(2)
RESULTS AND DISCUSSION Size Distribution of Beads Formation A typical size distribution pattern is shown in Figure 1. Undesirable particle size can be recycled before the cellulose derivative was regenerated to cellulose. The desired particle size for enzyme immobilization depends on the kind of enzymes to be immobilized. For instance, the K , value of the immobilized enzyme complex, its effectiveness factor, and other parameters can all be affected by particle size.2 Structure of Cellulose Beads The electron micrographs show that the beads are mostly spherical. The regenerated cellulose forming the beads seems to exist in the shape of threads. The interior and the surface of the beads have the same structure. The pore sizes are quite uniform and the pores are distributed evenly throughout the whole bead (Fig. 2).
Pore Size of the Beads The pore size of the beads was measured from scanning electron micrographs (Fig. 2). The scanning micrographing requires dry samples. Drying the beads in air will result in a size shrinkage. Critical point drying of the beads with liquid carbon dioxide was therefore used. Since the cellulose is in the shape of threads, the beads are similar to a small cotton ball. The pore size is defined as the distance between two strings of cellulose threads in the beads. From the scanning electron micrographs, the pore size was measured to be about 1000 8. The thickne6 of the cellulose thread is about 500 A.
Porosity of the Beads The beads shrink when they are dried, so t h a t the technique commonly employed in liquid chromatography for determining porosity was applied. DEAE beads possess positive charges on the particle surface, they tend to absorb the dye in dextran at neutral pH.
POROUS CELLULOSE BEADS
1511
0.11
I 0.15
0.15
I 0.21
0.21
I 0.3
0.30
I 0.42
0.42
I 0.59
0.59
0.84
over
1 1 1 . 6 8 0.84 1.60
Diameter o f Beads (mn)
Fig. 1. Typical size distribution of cellulose beads.
Therefore, the pH of eluent water was adjusted at pH 11.0 to prevent this adsorption. In eq. (l), the term “void volume outside beads” was determined with the blue dextran. The term “total porous volume in column” was determined with glucose for the DEAE beads, and the total porous volume in the column was determined with phenolphthalein for the diisocyanate treated beads. The bead porosity thus calculated with eq. (1) was 78y0 and 75y0 for DEAE cellulose beads and diisocyanate treated beads, respectively. By
1512
CHEN AND TSAO
x
0 0
8 e4
e9 a
POROUS CELLULOSE BEADS
1513
controlling the conditions employed in forming the cellulose beads, the porosity can be varied.
Pressure Drop Across the Column The pressure drop is expressed in cm of Hg per cm of column length. A comparison between microcrystalline DEAE cellulose and the porous DEAE cellulose beads in Figure 3 shows that the porous beads exhibit stronger physical strength than microcrystalline DEAE cellulose. When the flow rate is less than 2 cm/sec, the DEAE beads have a linear relationship between the pressure drop and nominal linear flow rate. When the flow rate is higher than 2 cm/sec, the curve slope increases, indicating that the beads are compressed, deformed, and eventually collapsed. Apparently there seems to be an irreversible process taking place when the linear flow rate was increased and then decreased, the pressure did not drop to the original point corresponding to the same linear flow rate. A hysterisis phe-
1
3
2
4
Nominal Linear Flow Rate, cm/sec
Fig. 3.
Pressure drop in the column a t various nominal linear flow rate. (x), Crystalline DEAE cellulose; DEAE cellulose beads. ( e ) ,
1514
CHEN AND TSAO
nomenon exists in both microcrystalline DEAE and the porous DEAE cellulose particles. Figure 4 shows the same type of plot for the porous cellulose beads treated with diisocyanate and enzyme. The curve did not show a hysterisis behavior. The slope of the curve increased slightly when the linear flow rate increased. The beads were able to stand up to the pressure when the linear flow rate was increased. Untreated cellulose beads were easily compressed. However, when the nominal linear flow rate is less than 0.5 cm/sec, the curve slope is more or less constant. This means that the beads are not compressed and
1 2 3 4 Nominal Linear Flow Rate (cm per second)
Fig. 4. Pressure drop in the column a t various nominal linear flow rate. (x) Cellulose beads (48-65 mesh particle size); (.) cellulose beads treated with
cellulose beads treated with diisodiisocyanate arid enzyme (4&65 mesh) ; (H) cyanate and enzyme (20-3.5 mesh particle size).
POIlOUS CELLULOSE BEADS
1515
deformed. In the industrial process the linear flow rate is usually less than 0.5 cm/sec. These untreated beads would be suitable for the industrial processes. In Figure 4 the hysterisis effect of untreated cellulose beads was not investigated.
Immobilization of the Enzyme on the Beads Glucoamylase was covalently bonded to the porous cellulose beads with tolylene 2,4-diisocyanate. The enzyme loading was 2000 units of activity per gram of cellulose beads. One unit of glucoamylase activity was defined as the production of 1 pmol glucose per minute a t 55"C, using a 30% partially hydrolyzed (DE 10) starch solution as the substrate. When DEAE beads were used t o immobilize glucose isomerase, the enzyme activity on 1 g of porous DEAE cellulose bead was 400 units. A unit was defined as the production of 1 pmol glucose per minute a t 60"C, using a 2M fructose solution as its substrate.
CONCLUSIONS The cellulose beads appear to qualify as an excellent carrier for enzyme immobilization. The basic porous beads can be made easily and cost about $1.50 per pound. Cellulose having three hydroxyl groups on each anhydroglucose unit is highly versatile in chemical reactions and high in immobilization capacity. In this study, only two chemical procedures of enzyme immobilization were reported: the diisocyanate and the DEAE methods. In our laboratory, seven different procedures have all been successfully tested and will be reported separately. After treatment with, for instance, tolylene 2,4-diisocyanate1 the porous beads become very strong and rigid having good flow properties as described above. The porosity of the beads can be controlled easily. The pore size distribution is quite uniform and pores are more or less uniformly distributed throughout a bead as shown by the electron micrographs. The enzyme immobilization loading on the porous cellulose beads is quite high as reported. Further details of the chemical properties of immobilized glucose isomerase, glucoamylase, invertase, and lactase on the cellulose beads will be reported Separately. I n a test with glucoamylase, 220 mg of protein were attached to 1 g of the cellulose beads. Immobilized glucoamylase on the porous beads by diisocyanate attachment was found t o have an activity half-life of 70 hr a t 60°C, 130 hr a t 55OC, and an extrapolated 2800 hr a t 45°C.
1516
CHEN AND TSAO
The porous DEAE cellulose beads can be attached to glucose isomerase as well as a number of other enzymes. DEAE cellulose has been used as an enzyme carrier in commercial operations. Because of the flow properties of the ordinary DEAE cellulose, only shallow packed beds can be used for large-scale operation to avoid excessive pressure build-up. The porous DEAE cellulose beads reported have very good flow properties as well as high enzyme loading capacity.
References 1. A. L. Bullock and J . D. Guthrie, in Methods in Carbohydrate Chemistry, R. L. Whistler, Ed., Academic Press, New York, 1965, pp. 408-411. 2. Y. Y. Lee and George T. Tsao, J . Food Sci.,39,667 (1974).
Accepted for Publication June 25, 1976
