- Email: [email protected]
[25] Immobilization of enzymes and microbial cells in gelatin
[25]
I M M O B I L I Z A T I O N OF BIOCATALYSTS IN GELATIN
293
whey (50% solids), pH 6.5-7, 40°. The catalyst and the whey are stirred with a paddle shaft (1300 rpm). Within eight hours the hydrolysis of 80% of lactose is achieved. After each batch, the reactor system and the catalyst are cleaned for 2 hr with diluted TEGO (0.1%) and thereafter they are repeatedly washed with a neutral buffer while a new batch of substrate is prepared in the feeding tank. High substrate concentration as well as high temperature and short reaction times prevent microbial contaminations.
[25] I m m o b i l i z a t i o n o f E n z y m e s a n d M i c r o b i a l Cells in G e l a t i n By VINCENZO
SCARDI
Choice of Gelatin as an Immobilization Matrix Gelatin is an inexpensive, abundant, and safe material, largely used as a food additive. It offers further advantages over other polymeric materials, both natural and synthetic, currently employed as matrices for immobilizing enzymes and whole cells. The protein nature of gelatin, its high hydrophilicity, and, consequently, its strong swelling power provide environmental conditions very favorable for immobilized enzymes or whole cells, and reduce the mass-transfer resistance to the diffusion of substrate and reaction product. Gelatin chain lengths (average MW 60,000-80,000; axial ratio 20:1 or higher) and the well-balanced number of polar and nonpolar groups play a fundamental role in gel formation. Without going deeply into this rather complicated process, it can be said that the gelatin gels which form from the gelatin sols at temperatures below 40 ° are unique in that they are thermally reversible; furthermore, they are also mechanically unstable. For these reasons gelatin has generally been disregarded as a possible matrix for entrapping enzymes and whole cells. However, thermostability and mechanical strength can be achieved by treating gelatin gels with formaldehyde, a hardening agent that in the author's experience has proved to be the most efficient among several other agents used in industry for making gelatin insoluble and impermeable. Thus, insolubilized gelatin was first used by the author and his collaborators in 1980 for immobilizing invertase-active whole cells of Saccharomyces cerevisiae.~ Although this immobilization method gave satisfactory results, it had two disadvantages: it consisted of several steps, i L . G i a n f r e d a , P. P a r a s c a n d o l a , a n d V. S c a r d i , Eur. J. Appl. Microbiol. Biotechnol. 11, 6 (1980).
METHODS 1N ENZYMOLOGY, VOL. 135
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
294
IMMOBILIZATION TECHNIQUES FOR CELLS/ORGANELLES
[25]
some of which were not easily controlled, and it yielded irregularly shaped gel particles. The method was then simplified and made more reproducible. 2 The main improvement obtained from combining the gelation and hardening steps was the formation of a strong spongelike gel having a well-defined geometry. Subsequently, the improved method was extended to microbial cells other than those of S. cerevisiae and to enzymes as well. Standard Procedure
Immobilization of Cells Microbial cells are suspended in deionized water and dispersed in a 10% (w/v) gelatin (Deutsche Gelatine-Fabriken Stoess & Co., Eberbach, Germany)-aqueous solution at 35-40 ° to give a final cell-to-gelatin ratio of 1 : I0 on a dry weight basis. To 9.5 ml of this dispersion, 0.5 ml of a hardening solution consisting of 20% w/v formaldehyde in 50% v/v ethanol is added. The mixture is quickly poured into a cylindrical mold (0.8 cm diameter) and allowed to gel in a deep freezer (about -25°). After at least 4 hr, the mold is brought to room temperature (18-20 °) and the stiff gelatin cylinder obtained is thoroughly rinsed with tap water, kept overnight in a large volume of deionized water at refrigerator temperature (45°), and then cut into thin disks (0.2-0.4 cm). The gel disks can be stored refrigerated for months without any appreciable loss of enzyme activity (e.g., yeast cell invertase), either wet in deionized water containing a proper preservative or dry in a desiccator over silica gel.
Immobilization of Enzymes Essentially the same procedure can be employed for enzymes except that it is preferable to mix gelatin and hardening solution first and within 1 rain to add the appropriate volume of enzyme solution. This has the advantage of reducing the exposure of enzyme to formaldehyde rendering it a more active preparation. Cylindrical Molds. Acylindrical mold is constructed of two plexiglass slabs (1 x 5 x 10 cm) which are held together by two binder clips and have a 9-cm-deep hollow (0.8 cm diameter) between them. More simply, a cylindrical mold can also be constructed from a disposable hypodermic syringe with the needle end of the barrel cut off squarely. Of course, the size of the resulting gelatin cylinder is determined by the sectional area of z p. Dhulster, P. Parascandola, and V. Scardi, Enzyme Microb. Technol. 5, 65 (1983).
[25]
IMMOBILIZATION OF BIOCATALYSTS IN GELATIN
295
the syringe and by the position of the plunger, which can be used for removing the hardened gelatin from the syringe barrel. In addition to these, other geometric shapes can be obtained by using the properly shaped molds. However, if spherical beads or membranes are wanted, the following procedures are suggested. Spherical Beads. The gelation mixture prepared as described above, instead of being poured into a mold, is added dropwise by means of a pipet or syringe to a large volume (at least 10-fold larger) of a tetrachloroethylene/cyclohexane mixture (1:9, by volume) in a tall beaker or Erlenmeyer flask at - 2 5 °. Beads thus formed are kept overnight under these conditions, then are collected from the solvent mixture (e.g., on a sintered glass filter), abundantly washed with tap water, and stored in a refrigerator as described for disks. Flat Membranes. Using a flat glass plate having a 10 × l0 cm square etched with a glass pencil, l0 ml of the gelatin mixture is spread as uniformly as possible between the limits of the square. The glass plate is quickly put into a deep freezer and after at least 4 hr is gradually brought to room temperature. The thin membrane (-0.1 cm) thus formed, once removed from the glass plate, is thoroughly rinsed with tap water and stored in a refrigerator under the same conditions described for gel disks. Assay and Use of Gelatin-Immobilized Biocatalysts The catalytic activity of gelatin-immobilized enzyme or microbial cells is usually measured at 30° by suspending a number of disks, beads, or other shaped particles in the buffered substrate solution within a miniaturized stirred-batch reactor. This consists of a thermostatted glass vessel ( - 7 0 ml) equipped with a specially designed poly(vinyl chloride) (PVC) cover bearing the magnetic stirrer assembly. 3 At fixed time intervals, stirring is interrupted and samples of the supernatant phase are withdrawn and assayed for the reaction product. The total volume of the samples should not exceed 1% of the reaction volume. A reaction curve is then plotted and the slope is measured at the origin; thus the catalytic activity and the apparent specific activity may be calculated. Toward the end of the activity assay, the immobilized phase is removed from the miniaturized reactor while the sampling of the supernatant phase is continued for at least 1 hr or less depending on the reaction rate. If no further increase in the product concentration is observed, enzyme or cell leakage from the gelatin matrix can be excluded. Then the activity yield can be calculated 3 p. Parascandola and V. Scardi, Biotechnol. Lett. 4, 753 (1982).
296
IMMOBILIZATION TECHNIQUES FOR CELLS/ORGANELLES
[25]
as the ratio of the activity of the immobilized phase to that of the corresponding free enzyme or cells before being immobilized. The gelatin-immobilized enzymes or cells can also be assayed and used in continuous reactors. For example, owing to its special PVC cover, the miniaturized stirred-batch reactor can be easily converted into a continuous stirred-tank reactor (CSTR). 3 Depending on the size and shape of the gel particles, a packed-bed reactor such as that previously described 4 can be used. In both cases the reaction rate, v, is evaluated by measuring the concentration of the reaction product in the effluent when a steady-state condition is established. Then, v = Q(So - S), where Q is the volumetric flow rate (ml/min), and So and S are the substrate concentration in the feed and effluent (/.tmol/ml), respectively. Properties of the Gelatin-Immobilized Biocatalysts There is a more or less restricted limit to the amount of enzyme (mg) that can be immobilized per gram of gelatin without changes in the apparent specific activity. For example, in the case of acid phosphatase, the apparent specific activity did not vary up to 8 mg enzyme/g of gelatin (dry weight), after which it rapidly decreased. 5 Therefore, to achieve the best results, the optimal enzyme-to-gelatin ratio (as rag/g) should be chosen in relation to the particular type of enzyme. This is why no quantitative instruction has been given under "Immobilization of Enzymes." On the contrary, a cell-to-gelatin ratio of 1:10 is suggested since this value, which has been determined experimentally for yeast cells, 1 was shown to be essentially dependent on the cell size, 5 hence it may be adopted for all microbial species. However, for bacterial cells a lower ratio can be used. In any case, an excess of cells can cause the formation of a mechanically poor gelatin gel. Kinetic Behavior. The kinetic parameters of the gelatin-immobilized enzymes are different from the corresponding ones of the free enzymes. For instance, the apparent Km values are generally higher. This is mainly due to the internal diffusional limitations, as confirmed by the fact that the specific activity of the gelatin-immobilized enzymes markedly increases upon homogenization of the gel particles. Although it never reaches the corresponding value of the free enzyme, the specific activity of the gelatin-immobilized enzyme after homogenization represents the maximal value experimentally obtained and, with a good approximation, it may be considered as the activity exhibited by the gelatin-immobilized enzyme in the absence of diffusional limitations. The ratio of the actual activity of a gelatin-immobilized enzyme to that of the same preparation after homoge4 p. Parascandola and V. Scardi, Biotechnol. Lett. 3, 369 (1981). 5 E. de Alteriis, P. Parascandola, and V. Scardi, unpublished results (1984).
[25]
IMMOBILIZATION OF BIOCATALYSTS IN GELATIN
297
TABLE I HYDROLASES IMMOBILIZED IN STANDARD GELATIN DISKS a
Specific activity (/zmol/min mg)
Activity Immo-
yiel&
(%)
Enzyme
Free
bilized
a-Glucosidase Urease Invertase
11.6 1.4 122.0 2.9
1.1 0.4 47.5 1.4
Acid p h o s p h a -
9.6, 27.5, 39.0, 48.0,
20.0 57.0 73.0 89.0
Effectiveness" factor, "0
(HCHO)5o d (M)
0.48 0.48 0.53 0.54
0.003 0.07 0.4 8.0
tase a D i a m e t e r 0.8 cm; t h i c k n e s s 0.4 cm.
Activity yield = (immobilized enzyme activity/free enzyme activity) × 100. The values in italics are those measured after having homogenized the gelatin disks. ' rl = Activity of the immobilized enzyme/same, after homogenization of disks. d The formaldehyde concentration which causes 50% disactivation of the free enzyme after 5 min incubation at 30 °.
nization approaches the definition of the effectiveness factor, ~, given for porous catalysts. 6,7 This is illustrated in Table I in the case of four hydrolases. When the effectiveness factor is used to correct the values of the reaction rate obtained experimentally with a gelatin-immobilized enzyme, the Km value calculated by a double-reciprocal plot or other graphical methods is very close to that of the free enzyme. The portion of enzyme activity which cannot be recovered even after homogenization of the gelatin-immobilized preparation represents the activity lost by interaction with formaldehyde. This may be responsible of either denaturation of the enzyme or co-cross-linking the latter to gelatin chains in the stabilized gel network, thus causing steric hindrance. In fact, according to the opinion of most experts in the field, the hardening effect of formaldehyde on proteins is probably due to formation of cross-linking methylene bridges (---CH2--) between amino groups on the one side and primary amide or guanidyl groups on the other. 8 The effect of formaldehyde on enzymes can be preliminarily evaluated by incubating at 30 ° for 5 min a fixed amount of enzyme with increasing concentration of formaldehyde. The residual enzyme activity plotted 6 K. G. Denbigh and J. C. R. Turner, " C h e m i c a l Reactor Theory." Cambridge Univ. Press, London and New York, 1971. v W. H. Pitcher, Jr., in "Immobilized Enzymes for Industrial R e a c t o r s " (R. A. Messing ed.), p. 151. Academic Press, N e w York, 1975. 8 H. Fraenkel-Conrat and H. S. Olcott, J. Am. Chem. Soc. 70, 2673 (1948).
298
IMMOBILIZATION TECHNIQUES FOR CELLS/ORGANELLES
[25]
against the log of the formaldehyde concentration allows the determination of (HCHO)50, i.e., the formaldehyde concentration giving 50% inactivation of enzyme. This is an approximate value which should be carefully considered as it has been obtained under conditions that are much more drastic than those of an enzyme immobilized in gelatin, where the time of contact is shorter, temperature is much lower, and, above all, a large excess of gelatin is present which neutralizes formaldehyde reactivity. In the case of gelatin-immobilized microbial cells, the effect of formaldehyde seems to be limited only to their viability. For example, yeast cells lose their fermenting capacity but retain most of the enzyme activity. 2 Activity yield are generally high, ranging from 70 to 90%, and do not increase appreciably by homogenizing the gel particles. Additional Considerations The characteristic properties of the hardened gelatin gel used in the present immobilization procedure are mechanical strength, some elasticity, and an enormous internal surface area, owing to the spongelike structure, which compensate for the decreased permeability of the gelatin matrix. The spongelike structure (Fig. 1) is a consequence of the combined gelation and hardening processes at - 2 5 °. In fact, if gelation/hardening is performed at 4-5 ° or at room temperature, the resulting gel shows a very homogeneous internal structure when examined in the electron microscope. Furthermore, its mechanical strength is poor and the activity yield is low, owing to the effect of formaldehyde on the enzyme or cells. The mechanical strength can be measured by a pocket pressure dynamometer. For example, a standard gelatin disk (0.8 cm diameter, 0.4 cm thickness) can stand a pressure of about 2.5 kg on its base before crushing. Disks represent the most efficient geometrical shape for gelatin-immobilized biocatalysts operating in stirred-batch reactor and CSTR. 2 However, the gelatin membrane, which is mechanically strong and flexible, has the advantage of displaying a larger surface area per unit volume than other geometries. It can be most conveniently used only in continuous operations as a spirally wound catalytic module such as the one described by Vieth and Venkatasubramanian. 9 Ethanolic formaldehyde can be replaced by aqueous glutaraldehyde as a hardening agent, which should be added to gelatin to give a final concentration of 0.35 w/v. However, comparative experiments have shown that with formaldehyde higher activity yields are obtained. 2,5 9 W. R. Vieth and K. Venkatasubramanian, this series, Vol. 44, p. 243.
[25]
IMMOBILIZATION OF BIOCATALYSTS IN GELATIN
299
FIG. 1. Details of a standard gelatin disk viewed through a scanning electron microscope (SEM).
Ethanol as a solvent for formaldehyde seems to play a role in the hardening process, probably because of a synergism. When ethanol has been replaced by glycerol, ethylene glycol, or water, activity yields were always lower, especially in the case of water. This is particularly true for gelatin-immobilized enzymes and less for gelatin-immobilized microbial cells. The present gelatin-immobilization procedure is simple and generally gives satisfactory results in terms of activity yield, and of both storage and operational stabilities. However, to obtain good and reproducible results it is of the outmost importance to always use the same quality of gelatin. In fact, the term gelatin is used commercially to indicate products derived from mammalian collagen which can be dispersed in water and show reversible sol-gel change with temperature. Since the conditions employed during the production of gelatins determine their characteristics, it is advisable to test several gelatins from different commercial sources and to select the one giving the best results.
I M M O B I L I Z A T I O N OF BIOCATALYSTS IN GELATIN
293
whey (50% solids), pH 6.5-7, 40°. The catalyst and the whey are stirred with a paddle shaft (1300 rpm). Within eight hours the hydrolysis of 80% of lactose is achieved. After each batch, the reactor system and the catalyst are cleaned for 2 hr with diluted TEGO (0.1%) and thereafter they are repeatedly washed with a neutral buffer while a new batch of substrate is prepared in the feeding tank. High substrate concentration as well as high temperature and short reaction times prevent microbial contaminations.
[25] I m m o b i l i z a t i o n o f E n z y m e s a n d M i c r o b i a l Cells in G e l a t i n By VINCENZO
SCARDI
Choice of Gelatin as an Immobilization Matrix Gelatin is an inexpensive, abundant, and safe material, largely used as a food additive. It offers further advantages over other polymeric materials, both natural and synthetic, currently employed as matrices for immobilizing enzymes and whole cells. The protein nature of gelatin, its high hydrophilicity, and, consequently, its strong swelling power provide environmental conditions very favorable for immobilized enzymes or whole cells, and reduce the mass-transfer resistance to the diffusion of substrate and reaction product. Gelatin chain lengths (average MW 60,000-80,000; axial ratio 20:1 or higher) and the well-balanced number of polar and nonpolar groups play a fundamental role in gel formation. Without going deeply into this rather complicated process, it can be said that the gelatin gels which form from the gelatin sols at temperatures below 40 ° are unique in that they are thermally reversible; furthermore, they are also mechanically unstable. For these reasons gelatin has generally been disregarded as a possible matrix for entrapping enzymes and whole cells. However, thermostability and mechanical strength can be achieved by treating gelatin gels with formaldehyde, a hardening agent that in the author's experience has proved to be the most efficient among several other agents used in industry for making gelatin insoluble and impermeable. Thus, insolubilized gelatin was first used by the author and his collaborators in 1980 for immobilizing invertase-active whole cells of Saccharomyces cerevisiae.~ Although this immobilization method gave satisfactory results, it had two disadvantages: it consisted of several steps, i L . G i a n f r e d a , P. P a r a s c a n d o l a , a n d V. S c a r d i , Eur. J. Appl. Microbiol. Biotechnol. 11, 6 (1980).
METHODS 1N ENZYMOLOGY, VOL. 135
Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
294
IMMOBILIZATION TECHNIQUES FOR CELLS/ORGANELLES
[25]
some of which were not easily controlled, and it yielded irregularly shaped gel particles. The method was then simplified and made more reproducible. 2 The main improvement obtained from combining the gelation and hardening steps was the formation of a strong spongelike gel having a well-defined geometry. Subsequently, the improved method was extended to microbial cells other than those of S. cerevisiae and to enzymes as well. Standard Procedure
Immobilization of Cells Microbial cells are suspended in deionized water and dispersed in a 10% (w/v) gelatin (Deutsche Gelatine-Fabriken Stoess & Co., Eberbach, Germany)-aqueous solution at 35-40 ° to give a final cell-to-gelatin ratio of 1 : I0 on a dry weight basis. To 9.5 ml of this dispersion, 0.5 ml of a hardening solution consisting of 20% w/v formaldehyde in 50% v/v ethanol is added. The mixture is quickly poured into a cylindrical mold (0.8 cm diameter) and allowed to gel in a deep freezer (about -25°). After at least 4 hr, the mold is brought to room temperature (18-20 °) and the stiff gelatin cylinder obtained is thoroughly rinsed with tap water, kept overnight in a large volume of deionized water at refrigerator temperature (45°), and then cut into thin disks (0.2-0.4 cm). The gel disks can be stored refrigerated for months without any appreciable loss of enzyme activity (e.g., yeast cell invertase), either wet in deionized water containing a proper preservative or dry in a desiccator over silica gel.
Immobilization of Enzymes Essentially the same procedure can be employed for enzymes except that it is preferable to mix gelatin and hardening solution first and within 1 rain to add the appropriate volume of enzyme solution. This has the advantage of reducing the exposure of enzyme to formaldehyde rendering it a more active preparation. Cylindrical Molds. Acylindrical mold is constructed of two plexiglass slabs (1 x 5 x 10 cm) which are held together by two binder clips and have a 9-cm-deep hollow (0.8 cm diameter) between them. More simply, a cylindrical mold can also be constructed from a disposable hypodermic syringe with the needle end of the barrel cut off squarely. Of course, the size of the resulting gelatin cylinder is determined by the sectional area of z p. Dhulster, P. Parascandola, and V. Scardi, Enzyme Microb. Technol. 5, 65 (1983).
[25]
IMMOBILIZATION OF BIOCATALYSTS IN GELATIN
295
the syringe and by the position of the plunger, which can be used for removing the hardened gelatin from the syringe barrel. In addition to these, other geometric shapes can be obtained by using the properly shaped molds. However, if spherical beads or membranes are wanted, the following procedures are suggested. Spherical Beads. The gelation mixture prepared as described above, instead of being poured into a mold, is added dropwise by means of a pipet or syringe to a large volume (at least 10-fold larger) of a tetrachloroethylene/cyclohexane mixture (1:9, by volume) in a tall beaker or Erlenmeyer flask at - 2 5 °. Beads thus formed are kept overnight under these conditions, then are collected from the solvent mixture (e.g., on a sintered glass filter), abundantly washed with tap water, and stored in a refrigerator as described for disks. Flat Membranes. Using a flat glass plate having a 10 × l0 cm square etched with a glass pencil, l0 ml of the gelatin mixture is spread as uniformly as possible between the limits of the square. The glass plate is quickly put into a deep freezer and after at least 4 hr is gradually brought to room temperature. The thin membrane (-0.1 cm) thus formed, once removed from the glass plate, is thoroughly rinsed with tap water and stored in a refrigerator under the same conditions described for gel disks. Assay and Use of Gelatin-Immobilized Biocatalysts The catalytic activity of gelatin-immobilized enzyme or microbial cells is usually measured at 30° by suspending a number of disks, beads, or other shaped particles in the buffered substrate solution within a miniaturized stirred-batch reactor. This consists of a thermostatted glass vessel ( - 7 0 ml) equipped with a specially designed poly(vinyl chloride) (PVC) cover bearing the magnetic stirrer assembly. 3 At fixed time intervals, stirring is interrupted and samples of the supernatant phase are withdrawn and assayed for the reaction product. The total volume of the samples should not exceed 1% of the reaction volume. A reaction curve is then plotted and the slope is measured at the origin; thus the catalytic activity and the apparent specific activity may be calculated. Toward the end of the activity assay, the immobilized phase is removed from the miniaturized reactor while the sampling of the supernatant phase is continued for at least 1 hr or less depending on the reaction rate. If no further increase in the product concentration is observed, enzyme or cell leakage from the gelatin matrix can be excluded. Then the activity yield can be calculated 3 p. Parascandola and V. Scardi, Biotechnol. Lett. 4, 753 (1982).
296
IMMOBILIZATION TECHNIQUES FOR CELLS/ORGANELLES
[25]
as the ratio of the activity of the immobilized phase to that of the corresponding free enzyme or cells before being immobilized. The gelatin-immobilized enzymes or cells can also be assayed and used in continuous reactors. For example, owing to its special PVC cover, the miniaturized stirred-batch reactor can be easily converted into a continuous stirred-tank reactor (CSTR). 3 Depending on the size and shape of the gel particles, a packed-bed reactor such as that previously described 4 can be used. In both cases the reaction rate, v, is evaluated by measuring the concentration of the reaction product in the effluent when a steady-state condition is established. Then, v = Q(So - S), where Q is the volumetric flow rate (ml/min), and So and S are the substrate concentration in the feed and effluent (/.tmol/ml), respectively. Properties of the Gelatin-Immobilized Biocatalysts There is a more or less restricted limit to the amount of enzyme (mg) that can be immobilized per gram of gelatin without changes in the apparent specific activity. For example, in the case of acid phosphatase, the apparent specific activity did not vary up to 8 mg enzyme/g of gelatin (dry weight), after which it rapidly decreased. 5 Therefore, to achieve the best results, the optimal enzyme-to-gelatin ratio (as rag/g) should be chosen in relation to the particular type of enzyme. This is why no quantitative instruction has been given under "Immobilization of Enzymes." On the contrary, a cell-to-gelatin ratio of 1:10 is suggested since this value, which has been determined experimentally for yeast cells, 1 was shown to be essentially dependent on the cell size, 5 hence it may be adopted for all microbial species. However, for bacterial cells a lower ratio can be used. In any case, an excess of cells can cause the formation of a mechanically poor gelatin gel. Kinetic Behavior. The kinetic parameters of the gelatin-immobilized enzymes are different from the corresponding ones of the free enzymes. For instance, the apparent Km values are generally higher. This is mainly due to the internal diffusional limitations, as confirmed by the fact that the specific activity of the gelatin-immobilized enzymes markedly increases upon homogenization of the gel particles. Although it never reaches the corresponding value of the free enzyme, the specific activity of the gelatin-immobilized enzyme after homogenization represents the maximal value experimentally obtained and, with a good approximation, it may be considered as the activity exhibited by the gelatin-immobilized enzyme in the absence of diffusional limitations. The ratio of the actual activity of a gelatin-immobilized enzyme to that of the same preparation after homoge4 p. Parascandola and V. Scardi, Biotechnol. Lett. 3, 369 (1981). 5 E. de Alteriis, P. Parascandola, and V. Scardi, unpublished results (1984).
[25]
IMMOBILIZATION OF BIOCATALYSTS IN GELATIN
297
TABLE I HYDROLASES IMMOBILIZED IN STANDARD GELATIN DISKS a
Specific activity (/zmol/min mg)
Activity Immo-
yiel&
(%)
Enzyme
Free
bilized
a-Glucosidase Urease Invertase
11.6 1.4 122.0 2.9
1.1 0.4 47.5 1.4
Acid p h o s p h a -
9.6, 27.5, 39.0, 48.0,
20.0 57.0 73.0 89.0
Effectiveness" factor, "0
(HCHO)5o d (M)
0.48 0.48 0.53 0.54
0.003 0.07 0.4 8.0
tase a D i a m e t e r 0.8 cm; t h i c k n e s s 0.4 cm.
Activity yield = (immobilized enzyme activity/free enzyme activity) × 100. The values in italics are those measured after having homogenized the gelatin disks. ' rl = Activity of the immobilized enzyme/same, after homogenization of disks. d The formaldehyde concentration which causes 50% disactivation of the free enzyme after 5 min incubation at 30 °.
nization approaches the definition of the effectiveness factor, ~, given for porous catalysts. 6,7 This is illustrated in Table I in the case of four hydrolases. When the effectiveness factor is used to correct the values of the reaction rate obtained experimentally with a gelatin-immobilized enzyme, the Km value calculated by a double-reciprocal plot or other graphical methods is very close to that of the free enzyme. The portion of enzyme activity which cannot be recovered even after homogenization of the gelatin-immobilized preparation represents the activity lost by interaction with formaldehyde. This may be responsible of either denaturation of the enzyme or co-cross-linking the latter to gelatin chains in the stabilized gel network, thus causing steric hindrance. In fact, according to the opinion of most experts in the field, the hardening effect of formaldehyde on proteins is probably due to formation of cross-linking methylene bridges (---CH2--) between amino groups on the one side and primary amide or guanidyl groups on the other. 8 The effect of formaldehyde on enzymes can be preliminarily evaluated by incubating at 30 ° for 5 min a fixed amount of enzyme with increasing concentration of formaldehyde. The residual enzyme activity plotted 6 K. G. Denbigh and J. C. R. Turner, " C h e m i c a l Reactor Theory." Cambridge Univ. Press, London and New York, 1971. v W. H. Pitcher, Jr., in "Immobilized Enzymes for Industrial R e a c t o r s " (R. A. Messing ed.), p. 151. Academic Press, N e w York, 1975. 8 H. Fraenkel-Conrat and H. S. Olcott, J. Am. Chem. Soc. 70, 2673 (1948).
298
IMMOBILIZATION TECHNIQUES FOR CELLS/ORGANELLES
[25]
against the log of the formaldehyde concentration allows the determination of (HCHO)50, i.e., the formaldehyde concentration giving 50% inactivation of enzyme. This is an approximate value which should be carefully considered as it has been obtained under conditions that are much more drastic than those of an enzyme immobilized in gelatin, where the time of contact is shorter, temperature is much lower, and, above all, a large excess of gelatin is present which neutralizes formaldehyde reactivity. In the case of gelatin-immobilized microbial cells, the effect of formaldehyde seems to be limited only to their viability. For example, yeast cells lose their fermenting capacity but retain most of the enzyme activity. 2 Activity yield are generally high, ranging from 70 to 90%, and do not increase appreciably by homogenizing the gel particles. Additional Considerations The characteristic properties of the hardened gelatin gel used in the present immobilization procedure are mechanical strength, some elasticity, and an enormous internal surface area, owing to the spongelike structure, which compensate for the decreased permeability of the gelatin matrix. The spongelike structure (Fig. 1) is a consequence of the combined gelation and hardening processes at - 2 5 °. In fact, if gelation/hardening is performed at 4-5 ° or at room temperature, the resulting gel shows a very homogeneous internal structure when examined in the electron microscope. Furthermore, its mechanical strength is poor and the activity yield is low, owing to the effect of formaldehyde on the enzyme or cells. The mechanical strength can be measured by a pocket pressure dynamometer. For example, a standard gelatin disk (0.8 cm diameter, 0.4 cm thickness) can stand a pressure of about 2.5 kg on its base before crushing. Disks represent the most efficient geometrical shape for gelatin-immobilized biocatalysts operating in stirred-batch reactor and CSTR. 2 However, the gelatin membrane, which is mechanically strong and flexible, has the advantage of displaying a larger surface area per unit volume than other geometries. It can be most conveniently used only in continuous operations as a spirally wound catalytic module such as the one described by Vieth and Venkatasubramanian. 9 Ethanolic formaldehyde can be replaced by aqueous glutaraldehyde as a hardening agent, which should be added to gelatin to give a final concentration of 0.35 w/v. However, comparative experiments have shown that with formaldehyde higher activity yields are obtained. 2,5 9 W. R. Vieth and K. Venkatasubramanian, this series, Vol. 44, p. 243.
[25]
IMMOBILIZATION OF BIOCATALYSTS IN GELATIN
299
FIG. 1. Details of a standard gelatin disk viewed through a scanning electron microscope (SEM).
Ethanol as a solvent for formaldehyde seems to play a role in the hardening process, probably because of a synergism. When ethanol has been replaced by glycerol, ethylene glycol, or water, activity yields were always lower, especially in the case of water. This is particularly true for gelatin-immobilized enzymes and less for gelatin-immobilized microbial cells. The present gelatin-immobilization procedure is simple and generally gives satisfactory results in terms of activity yield, and of both storage and operational stabilities. However, to obtain good and reproducible results it is of the outmost importance to always use the same quality of gelatin. In fact, the term gelatin is used commercially to indicate products derived from mammalian collagen which can be dispersed in water and show reversible sol-gel change with temperature. Since the conditions employed during the production of gelatins determine their characteristics, it is advisable to test several gelatins from different commercial sources and to select the one giving the best results.