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Immobilization of yeast cells in acrylamide gel matrix
Immobilization of yeast cells in awylamidegel matrix G.Aykut,V.N.Has&i and G.Aheddinoglu Middle East Technical University, Department of Biological Sciences, Ankara, Turkey (Received 8 December 1986; accepted 25 March 1987)
Entrapment of yeast cells in a three-dimensional polymer matrix was achieved, and various properties of the polymer matrix as well as the invertase activity of the yeast cells were studied. When the matrix was highly cross-linked or synthesized from concentrated polymer solutions, its swelling ratio decreased. lnvertase activity was found to increase with water content of the matrix. Cell content of the gel was found to affect adversely enzyme activity. The enzyme was found to retain its activity after seven runs with the same sample. Keywords: Saccharomyces
ceravisiae, invertase, acrylamide gel matrix enzyme acth4ty
Immobilization of yeast Saccharomyces cerevisiae through application of various techniques (e.g. covalent bonding, encapsulation, entrapment in a gel matrix) has been reported ‘-* . The main emphasis in these studies has been on the process of immobilization and the extent to which enzyme activity is retained. The aim of immobilization is to obtain reusable, stable enzyme stocks that can be handled with ease and without the need for any isolation or purification procedure. Apart from the inactivation of the cells during immobilization, the success of immobilization lies in the efficient intake of substrate and removal of product via a simple diffusion process. Diffusion, to a very large extent, depends on the tightness of the matrix as well as on its interaction with the product and/or the substrate. By studying these features in gel matrices and establishing the trends, optimization of the experimental conditions becomes possible. In this study, the polymer matrix, the various parameters affecting its properties and the effect of the matrix on the activity of the cell were investigated.
(Table I) in 5 ml of isotonic phosphate buffer (pH 7.6) and incubating the resultant solution in an uncovered glass tube of fixed dimension, at 37°C for 90 min. After recovery from the glass tube, each gel was stored in water, ethanol and chloroform for a minimum of 10 days and then weighed. The dry weights of the gels were obtained after drying in a vacuum oven at 50°C. Swelling ratio (0) and the volume of absorbed solvent (VAS) for each gel in solvents of varying polarity were calculated according to the following equations: Q = w,/wd
(1)
VAS = (w, - Wd)/&Wd
(2)
where w, is weight of swollen gel, wd is weight of dry gel and ps is the density of the solvent at room temperature. The weight of polymer chain length between crosslinks (M,) was calculated as follows: MC (theoretical) = M/2X,
EXPERIMENTAL Preparation of gels Gels were prepared by dissolving acrylamide, /V,N’-methylene bisacrylamide and ammonium persulphate in different ratios
Here, X1 is the mole fraction of the cross-linker, and M, is the molecular weight of the polymeric unit of the cross-linked polymer and is calculated as follows: M, = X, . MW,
Correspondence to: Professor V.N. Hasirci
+X2
. MW2
Table 1 Input concentrations of acrylamide (A4). N.N’-methylene bisacrylamide (BAA) and ammonium persulphate lengths between cross-links M, (theoretical) in the synthesis of polyacrylemide gels
Gelno. pA5 ml) 1 2 3 4 5
0.75 0.75 0.75 1.50 0.38
0.04 0.08 0.02 0.08 0.02
Biomaterials
1988. Vol9 March
(4)
(APS), and weight of polymer chain
APS
% Polymer (w/v)
BAA/AA (% molar)
M,
(915 ml) 0.013 0.013 0.013 0.025 0.006
15.0 16.6 15.4 31.6 7.9
2.46 4.95 1.22 2.46 2.46
1482 760 2963 1482 1482
0 1988 168
(3)
Butterworth 8 Co (Publishers) Ltd. 0142-9612/88/020168-05$03.00
Immobilization
where 1 and 2 denote the cross-linker and the monomer, respectively.
Preparation of cells and growth media The yeast used in gel entrapment was isolated from Pak Maya (Istanbul), and reidentified as a strain of S. cerevisiae. The cells were grown aerobically in a growth medium containing N broth (Oxoid), 1% sodium lactate and 0.2% glucose2 and incubated at 30°C for 48 h. After growth, cells were centrifuged at 60009 and resuspended in cold isotonic phosphate buffer. Appropriate amounts of this cell suspension were used as the source of invertase for free and immobilized cells. The amount of cells used is presented as the wet weight of the packed cells. In the immobilization of cells, the same procedure for gel preparation was employed but with the addition of cell suspension to the polymerization mixture and 10 min vortexing before incubation.
Determination
The Nelson method9 was applied in the determination of invertase activity. This procedure makes use of the reaction in which glucose and fructose reduce the CIJ+~ complex to Cu,O, and convert arsenomolybdic acid (yellow) to arsenomolybdous acid (blue). This is then spectrophotometrically determined at 540 nm. Assay reaction time was 30 min and the reaction volume was 7 ml. One unit of enzyme activity was defined as pmol glucose formed per minute.
k-
0
3-
s
2l0
I 500
. *I
%i 1000
A
1500
I
I
2000
2500
3000
M, Figure 1 Volume of absorbed solvent by gels with varying M, values: I*) water; (A) erhanol; (W) chloroform.
987-
= 2 E 0
6-
9
4-
Effect of temperature The effect of temperature on the rate of inversion of sucrose was investigated-over the range 4-80°C at pH 3.6 with a sucrose concentration of 200 mM.
Effect of pH To determine the effect of pH, reactions were carried out at 25°C with 200 mM sucroseconcentration and atvarious pH values.
Activitv after reoeated use . Gel pellets with 1 and 20% cell loading were prepared in triplicate. Each pellet was washed with acetate buffer (pH 5) and resuspended in a fresh reaction mixture, assayed separately, repeating the process five times with no time delay between the assays.
RESULTS AND DISCUSSION
of invertase activity
2;::
of yeast cells: G. Aykut et al.
5-
Properties of the polymer matrix Swelling was almost complete within the first 24 h for all solvents and was constant after the third day for all the gels. The effect of the cross-linking agent on the amount of absorbed solvent is presented in Figure 1. As the distance between the cross-links (M,) increases, the amount of solvent absorption also increases. This was predicted by Flory”and observed byothers”-‘3, as the restraint imposed on swelling by the cross-links is inversely related to M,. The effect is less prominent when a solvent of lesser polarity than that of water is used, indicating that the gel is hydrophilic. In Figure 2, the effect of the concentration of the polymerization mixture on swelling can be observed (gels 1, 4 and 5). Here the bisacrylamide to acrylamide ratio was kept constant and the polymerization mixture concentration was varied. It was found that the volume of absorbed water decreases with increasing concentration, in accordance with the findings in the literature14,15; the volume of absorbed ethanol and chloroform increases only insignificantly. The substantial change in thevolume of absorbed solvent may be due to the transfer of radicals to solvent becoming more important than that to the cross-linker, thus leading to a gel with a lower degree of cross-linking. It might also be partially because growing chains are sufficiently separated from each other in a good solvent, leading to low cross-linking degree, but when monomer concentration increases, the medium is no longer a good solvent for the growing chains which are now more ordered and are subject to more cross-linking. Using gel 1 as a typical gel, the effect of temperature on the swelling ratio (Q) was determined (Figure 3). Increases in the temperature of the medium from 4 to 80°C gradually increased the swelling ratio but this increase was quite small. This might indicate that the entropy of swelling for acrylamide in water is quite low and only slightly alters the free energy of swelling. Since the enthalpy of swelling
3-
Polymerization
mixture
concentration
(M)
Figure 2 Volume of absorbed solvent by gels 1. 4 and 5: (@) water: (A) ethanol; @J chloroform.
10
20
30
40
50
Temperature Figure 3
60
70
80
90
(“C)
Effect of remperarure on the swelling ratio, 0 of gel 1.
Biomarerials
1988, Vol 9 March
169
Immobih’zation of yeast cells: G. Aykut et al.
should be constant in this temperature range, a substantial change in swelling with temperature is not observed. Swelling ratios determined at different pH values indicated that changes in pH did not affect the swelling ability of the gel. This is to be expected from the non-ionic nature of acrylamide.
Enzymatic
properties
of free and immobilized
cells
The response of free cells to parameters such as substrate concentration, pH and temperature usually differ from those of the immobilized cells. The difference resides in the physical and chemical properties of the carriers that alter the microenvironment of the bound cells. Therefore each parameter has to be studied in both bound and free cells. The following results are from tests that were performed on 1 and 20% cell loaded samples as well as on free cells. To determine the effect of the substrate concentration on the activity of the enzyme, reactions were carried out in the range 32-940 InM sucrose concentration. As shown in Figure 4, invertase activities of both free cells and cells immobilized in gel 5 were inhibited by sucrose concentrations higher than 350 mM. No considerable effect was observed in the range 32-313 mM when free cells were used. Immobilized cell invertase activity, regardless of loading, increased ninefold within the same concentration range and led to a maximum at about 315 mM. The following explanation can be proposed for this observation. Acrylamide is highly polar and a large fraction of the water in the gel is bound water that is unable to dissolve solutes. The sucrose concentration in the gel is then expected to be much lower than that in solution and the enzymes will not be saturated. Upon increase of sucrose concentration, its concentration in the gel would be higher and an increase in enzyme activity observed. For free cells, little change is observed or expected as they will be operating at V,,,. Optimal invertase activity for free cells was exhibited at pH 3.6 (Figure 5). For the immobilized cells with both loadings, a more neutral pH (pH 5) was obtained as the optimum value. Alterations in the pH profiles of enzymes immobilized on charged carriers are believed to occur due to a difference in the electrostatic field around the immobilized
100
90 80 70 60 50 40 30 20 10
t 1
I
2
3
4
5
6
7
8
PH Figure 5 Effect of pH on invertase activity: fW) free cells; (e) loaded gel 5; (X) 20% cell loaded gel 5.
1% cell
100
90
80
70
60
50
40 100 30
20
10
-/ I
I
I
I
I
I
I
10
20
30
40
50
60
70
Temperature
(“C)
Figure 6 Effect of temperature on invertase activiv: (m) free cells; (e) 1% cell loaded gel 5; (X) 20% cell loaded gel 5.
III
III
200
400
I
600
Sucrose concentration
III 800
1000
(mtd)
Figure 4 Effect of substrate concentration on invertase activity; (m) free cells; (@) 1% cell loaded gel 5; (X) 20% cell loaded gel 5.
170
Biomaterials
1988, Vol 9 March
enzyme caused by the inherent charge of the carrier16. In an earlier study on the invertase activity of acrylamide-entrapped S. cerevisiae2, no change in the optimum pH was observed upon immobilization. Shifts in the optimum pH upon immobilization are not uncommon and have been reported for phosphoglycerate mutase and aspartase of fscherichia co/i entrapped in polyacrylamide’6* I’. The shift observed in this study can be explained by the protonation of the matrix
Immobilization
Table 2
I
I
I
3
4
I
I 5
I 7
6
I
I
I
8
910
al.
Reuse of immobilized cells
Run no.
I 12
of yeast ceils: G. Aykut et
1 2 3 4 5
Units per gram packed ceils 1% load
20% load _
278 530 530 530 586
107 129 136 129 143
Q Figure 7 g cells)
100
Effectof swelling ratio, Q on the cellinveltese
activity (in units per
-~
90 80 70 60 50 40 30
\,
20 10
-a I 5
I
I
I
10 15 20
Celi concentration
(X w/v)
I
I
I
I
.
5
10
15
20
Cell concentration
with immobilization of more concentrated cell suspensions or solutions has been reported’*, lg. These phenomena can be explained as follows. When the cells are entrapped, the diffusions of the substrate and product are restricted, there might be inactivation due to the entrapment procedure, and an increase in loading alters the microenvironment more (i.e. accumulation of more products in the closevicinityof the cells, change in pH) and also leads to a deficiency of substrate within the gel. Retention of activity after repeated use of the immobilized cell system is very important economically. As observed in Table 2, with a 1% cell load, doubling of invertase activity was detected on the second use. A less pronounced increase was also found with 20% loading. In the third, fourth and fifth runs the enzyme activity was not significantly altered. Similar results have been reported and the changes after the first wash have generally been attributed to an increase in the cell membrane permeability for the substrate and/or product”20.
(% w/v)
Figure 8 Effect of cellloading on immobilizedinvertase activity: (a) specific activity (in unitsperg of ceils) and (b) totalactivity (in unitsper5 mlgel).
aminogroups at low pH, thus creating a very different (highly charged) environment for the immobilized cells. For the free cells the highest activity was found to be at the highest temperature tested (SOOC) (Figure 6). With 20% loading, the cells behaved in the same way as the free cells. An unexpected observation, however, was that, with 1% loading, the cells displayed maximum activity at 35°C and the activity decreased when the temperature was increased further. in selecting a gel for use in the cell activity experiments the gel with the highest activity is preferred, to obtain the most significant change. The gel with the maximum possible swelling without any cell loss due to leakage is expected to yield maximum activity because of the close resemblance to the environment of the free cell. lnvertase activity, obtained per gram of cell material, was plotted against the swelling ratio (Figure 7). It can be seen that gel 5 yields the highest activity, as expected from its swelling ratio. The results of leakage experiments (even for extended periods of soaking) indicated that there was no significant leakage from any of the gels tested, thus gel 5 was chosen for use in the following experiments. The activity of gel 5 prepared with different concentrations of cells at 200 mM sucrose concentration is presented in Figure 8. The results indicate that when the loading is increased from 1 to 20%, the specific activity per gram cell is decreased by about 70%. Another expression of this activity decrease upon increase in loading can be seen in the total activities. In comparison with the free cells, the specific activities are about 60 and 20% when the loadings are 1 and 20%, respectively. A drop in the specific activity
CONCLUSIONS In general, within the published literature suitableconditions for immobilization and enzymatic properties of the cells are determined, but no information on the physical properties of the support is reported and characterization of the physical properties of the supports used in immobilization is not a common practice. In the present study, acrylamide gels have been characterized before immobilization and the invertase activities of the cells immobilized in these gels under various conditions have been determined. As a result of the characterization and optimization study of the gel itself and with the immobilized cells, it is possible to modify the entrapment conditions to suit the requirements of a specific application. It will also be possible to identify any observed changes in the activity as being of gel or cell origin.
REFERENCES Chibata, I., Tosa, T. and Sato, T., Immobilized aspartase containing microbial ceils: preparation and enzymatic properties,Appl. Microbial. 1974, 27(5), 878-885 D’Souza. S.F. and Nadkami, G.B., Continuous inversion of sucrose by gel entrapment of yeast cells, Enzyme Microb. Technol. 1980, 2. 2 17-222 DSouza, S.F. and Nadkarni, G.B., Continuous conversion of sucrose to fructose and gluconic acid by Immobilized yeast cell multienzyme complex, Biotechnol. Bioeng. 1980, 22, 2 179-2189 Isaeve, V.S. and Kolpakchi, A.P.. Fixation of Brewer’s yeast to polymer materials, Prikl. Biokhim. Mikrobiol. 1976, 12(6), 866-870 Kennedy, J.F., Barker, S.A. and Humphreys, J.D., Microbial cells living immobilized on metal hydroxides, Nature 1976, 261, 242-244 Navarro. J.M., Fermentation en continua a I’aide de microorganismes fixes, 1975, Thesis Doct. Ing., Univ. Toulouse
Biomaterials
1988, Vol9 March
171
Immobilization
7
8
9 10 11 12 13
172
of yeast cells: G. Aykut et al.
Samejima, H., Kimura, K., Ado, Y., Suzuki, Y. and Tadokoro, T., Regeneration of ATP by immobilized microbial cells and its utilization for synthesis of nucleotides, Enzyme Eng. 1978, 4, 237-244 Sate, T., Nishida, Y., Taso, T. and Chibata, I., Immobilization of E. co/i cells containing aspartase activity, Biochem. Biophys. Acta 1979, 570, 179-l 86 Wharton, D.C. and McCarty, R.E., Experiments in Biochemistry, Macmillan, New York, 1972, pp 313 Flory, P.J., Principles of Polymer Chemistry, Cornell University Press, New York, 1953, p 494 Galina, H. and Kolarz, B.N., Studies of porous polymer gels, ./. Appl. PolymerSci. 1979, 24.891-900 Errede, L.A., Polymer swelling, Macromolecules 1986,19,654-658 Miller, D.R. and Peppas, N.A., Bulk characterization and scanning electron microscopy of hydrogels of P(VA-co-NVP), Biomateriels 1986,7.329-339
Biomaterials
1988, Voi 9 March
14
15 16 17 18 19 20
Refojo. M.F. and Yasuda, H., Hydrogels from 2-hydroxyethyl methacrylate and propylene glycol monoacrylate, J. App/. Polymer Sci. 1965,9,2425-2435 Jacobelli, H., Bartholin, M. and Guyot, A., Styrene divinyl benzene copolymers, J. Appl. Polymer Sci. 1979, 23, 927-939 Smiley, K.L. and Strandberg, G.W., Immobilized Enzymes, (Ed. R.C. Weast) CRC Press, OH, 1973, pp 13-38 Zaborsky, 0.. Entrapment within crosslinked polymers, in Immobilized Enzymes, (Ed. R.C. Weast) CRC Press, OH, 1973 Durand, G. and Navarro, J.M., Immobilized microbial cells, Process Biochem. 1978, 13(g), 14-23 Onyezili, F.N. and Onitiri, A.C., Immobilization of invertase on modified nylon tubes, Anal. Biochem, 198 1, 113, 203-206 Chibata, I., Tosa, T. and Sato. T.. Production of L-aspartic acid by microbial cells entrapped in polyacrylamide gels, in Methods in Enzymology, Vol. 44. Academic Press, New York, 1976
Entrapment of yeast cells in a three-dimensional polymer matrix was achieved, and various properties of the polymer matrix as well as the invertase activity of the yeast cells were studied. When the matrix was highly cross-linked or synthesized from concentrated polymer solutions, its swelling ratio decreased. lnvertase activity was found to increase with water content of the matrix. Cell content of the gel was found to affect adversely enzyme activity. The enzyme was found to retain its activity after seven runs with the same sample. Keywords: Saccharomyces
ceravisiae, invertase, acrylamide gel matrix enzyme acth4ty
Immobilization of yeast Saccharomyces cerevisiae through application of various techniques (e.g. covalent bonding, encapsulation, entrapment in a gel matrix) has been reported ‘-* . The main emphasis in these studies has been on the process of immobilization and the extent to which enzyme activity is retained. The aim of immobilization is to obtain reusable, stable enzyme stocks that can be handled with ease and without the need for any isolation or purification procedure. Apart from the inactivation of the cells during immobilization, the success of immobilization lies in the efficient intake of substrate and removal of product via a simple diffusion process. Diffusion, to a very large extent, depends on the tightness of the matrix as well as on its interaction with the product and/or the substrate. By studying these features in gel matrices and establishing the trends, optimization of the experimental conditions becomes possible. In this study, the polymer matrix, the various parameters affecting its properties and the effect of the matrix on the activity of the cell were investigated.
(Table I) in 5 ml of isotonic phosphate buffer (pH 7.6) and incubating the resultant solution in an uncovered glass tube of fixed dimension, at 37°C for 90 min. After recovery from the glass tube, each gel was stored in water, ethanol and chloroform for a minimum of 10 days and then weighed. The dry weights of the gels were obtained after drying in a vacuum oven at 50°C. Swelling ratio (0) and the volume of absorbed solvent (VAS) for each gel in solvents of varying polarity were calculated according to the following equations: Q = w,/wd
(1)
VAS = (w, - Wd)/&Wd
(2)
where w, is weight of swollen gel, wd is weight of dry gel and ps is the density of the solvent at room temperature. The weight of polymer chain length between crosslinks (M,) was calculated as follows: MC (theoretical) = M/2X,
EXPERIMENTAL Preparation of gels Gels were prepared by dissolving acrylamide, /V,N’-methylene bisacrylamide and ammonium persulphate in different ratios
Here, X1 is the mole fraction of the cross-linker, and M, is the molecular weight of the polymeric unit of the cross-linked polymer and is calculated as follows: M, = X, . MW,
Correspondence to: Professor V.N. Hasirci
+X2
. MW2
Table 1 Input concentrations of acrylamide (A4). N.N’-methylene bisacrylamide (BAA) and ammonium persulphate lengths between cross-links M, (theoretical) in the synthesis of polyacrylemide gels
Gelno. pA5 ml) 1 2 3 4 5
0.75 0.75 0.75 1.50 0.38
0.04 0.08 0.02 0.08 0.02
Biomaterials
1988. Vol9 March
(4)
(APS), and weight of polymer chain
APS
% Polymer (w/v)
BAA/AA (% molar)
M,
(915 ml) 0.013 0.013 0.013 0.025 0.006
15.0 16.6 15.4 31.6 7.9
2.46 4.95 1.22 2.46 2.46
1482 760 2963 1482 1482
0 1988 168
(3)
Butterworth 8 Co (Publishers) Ltd. 0142-9612/88/020168-05$03.00
Immobilization
where 1 and 2 denote the cross-linker and the monomer, respectively.
Preparation of cells and growth media The yeast used in gel entrapment was isolated from Pak Maya (Istanbul), and reidentified as a strain of S. cerevisiae. The cells were grown aerobically in a growth medium containing N broth (Oxoid), 1% sodium lactate and 0.2% glucose2 and incubated at 30°C for 48 h. After growth, cells were centrifuged at 60009 and resuspended in cold isotonic phosphate buffer. Appropriate amounts of this cell suspension were used as the source of invertase for free and immobilized cells. The amount of cells used is presented as the wet weight of the packed cells. In the immobilization of cells, the same procedure for gel preparation was employed but with the addition of cell suspension to the polymerization mixture and 10 min vortexing before incubation.
Determination
The Nelson method9 was applied in the determination of invertase activity. This procedure makes use of the reaction in which glucose and fructose reduce the CIJ+~ complex to Cu,O, and convert arsenomolybdic acid (yellow) to arsenomolybdous acid (blue). This is then spectrophotometrically determined at 540 nm. Assay reaction time was 30 min and the reaction volume was 7 ml. One unit of enzyme activity was defined as pmol glucose formed per minute.
k-
0
3-
s
2l0
I 500
. *I
%i 1000
A
1500
I
I
2000
2500
3000
M, Figure 1 Volume of absorbed solvent by gels with varying M, values: I*) water; (A) erhanol; (W) chloroform.
987-
= 2 E 0
6-
9
4-
Effect of temperature The effect of temperature on the rate of inversion of sucrose was investigated-over the range 4-80°C at pH 3.6 with a sucrose concentration of 200 mM.
Effect of pH To determine the effect of pH, reactions were carried out at 25°C with 200 mM sucroseconcentration and atvarious pH values.
Activitv after reoeated use . Gel pellets with 1 and 20% cell loading were prepared in triplicate. Each pellet was washed with acetate buffer (pH 5) and resuspended in a fresh reaction mixture, assayed separately, repeating the process five times with no time delay between the assays.
RESULTS AND DISCUSSION
of invertase activity
2;::
of yeast cells: G. Aykut et al.
5-
Properties of the polymer matrix Swelling was almost complete within the first 24 h for all solvents and was constant after the third day for all the gels. The effect of the cross-linking agent on the amount of absorbed solvent is presented in Figure 1. As the distance between the cross-links (M,) increases, the amount of solvent absorption also increases. This was predicted by Flory”and observed byothers”-‘3, as the restraint imposed on swelling by the cross-links is inversely related to M,. The effect is less prominent when a solvent of lesser polarity than that of water is used, indicating that the gel is hydrophilic. In Figure 2, the effect of the concentration of the polymerization mixture on swelling can be observed (gels 1, 4 and 5). Here the bisacrylamide to acrylamide ratio was kept constant and the polymerization mixture concentration was varied. It was found that the volume of absorbed water decreases with increasing concentration, in accordance with the findings in the literature14,15; the volume of absorbed ethanol and chloroform increases only insignificantly. The substantial change in thevolume of absorbed solvent may be due to the transfer of radicals to solvent becoming more important than that to the cross-linker, thus leading to a gel with a lower degree of cross-linking. It might also be partially because growing chains are sufficiently separated from each other in a good solvent, leading to low cross-linking degree, but when monomer concentration increases, the medium is no longer a good solvent for the growing chains which are now more ordered and are subject to more cross-linking. Using gel 1 as a typical gel, the effect of temperature on the swelling ratio (Q) was determined (Figure 3). Increases in the temperature of the medium from 4 to 80°C gradually increased the swelling ratio but this increase was quite small. This might indicate that the entropy of swelling for acrylamide in water is quite low and only slightly alters the free energy of swelling. Since the enthalpy of swelling
3-
Polymerization
mixture
concentration
(M)
Figure 2 Volume of absorbed solvent by gels 1. 4 and 5: (@) water: (A) ethanol; @J chloroform.
10
20
30
40
50
Temperature Figure 3
60
70
80
90
(“C)
Effect of remperarure on the swelling ratio, 0 of gel 1.
Biomarerials
1988, Vol 9 March
169
Immobih’zation of yeast cells: G. Aykut et al.
should be constant in this temperature range, a substantial change in swelling with temperature is not observed. Swelling ratios determined at different pH values indicated that changes in pH did not affect the swelling ability of the gel. This is to be expected from the non-ionic nature of acrylamide.
Enzymatic
properties
of free and immobilized
cells
The response of free cells to parameters such as substrate concentration, pH and temperature usually differ from those of the immobilized cells. The difference resides in the physical and chemical properties of the carriers that alter the microenvironment of the bound cells. Therefore each parameter has to be studied in both bound and free cells. The following results are from tests that were performed on 1 and 20% cell loaded samples as well as on free cells. To determine the effect of the substrate concentration on the activity of the enzyme, reactions were carried out in the range 32-940 InM sucrose concentration. As shown in Figure 4, invertase activities of both free cells and cells immobilized in gel 5 were inhibited by sucrose concentrations higher than 350 mM. No considerable effect was observed in the range 32-313 mM when free cells were used. Immobilized cell invertase activity, regardless of loading, increased ninefold within the same concentration range and led to a maximum at about 315 mM. The following explanation can be proposed for this observation. Acrylamide is highly polar and a large fraction of the water in the gel is bound water that is unable to dissolve solutes. The sucrose concentration in the gel is then expected to be much lower than that in solution and the enzymes will not be saturated. Upon increase of sucrose concentration, its concentration in the gel would be higher and an increase in enzyme activity observed. For free cells, little change is observed or expected as they will be operating at V,,,. Optimal invertase activity for free cells was exhibited at pH 3.6 (Figure 5). For the immobilized cells with both loadings, a more neutral pH (pH 5) was obtained as the optimum value. Alterations in the pH profiles of enzymes immobilized on charged carriers are believed to occur due to a difference in the electrostatic field around the immobilized
100
90 80 70 60 50 40 30 20 10
t 1
I
2
3
4
5
6
7
8
PH Figure 5 Effect of pH on invertase activity: fW) free cells; (e) loaded gel 5; (X) 20% cell loaded gel 5.
1% cell
100
90
80
70
60
50
40 100 30
20
10
-/ I
I
I
I
I
I
I
10
20
30
40
50
60
70
Temperature
(“C)
Figure 6 Effect of temperature on invertase activiv: (m) free cells; (e) 1% cell loaded gel 5; (X) 20% cell loaded gel 5.
III
III
200
400
I
600
Sucrose concentration
III 800
1000
(mtd)
Figure 4 Effect of substrate concentration on invertase activity; (m) free cells; (@) 1% cell loaded gel 5; (X) 20% cell loaded gel 5.
170
Biomaterials
1988, Vol 9 March
enzyme caused by the inherent charge of the carrier16. In an earlier study on the invertase activity of acrylamide-entrapped S. cerevisiae2, no change in the optimum pH was observed upon immobilization. Shifts in the optimum pH upon immobilization are not uncommon and have been reported for phosphoglycerate mutase and aspartase of fscherichia co/i entrapped in polyacrylamide’6* I’. The shift observed in this study can be explained by the protonation of the matrix
Immobilization
Table 2
I
I
I
3
4
I
I 5
I 7
6
I
I
I
8
910
al.
Reuse of immobilized cells
Run no.
I 12
of yeast ceils: G. Aykut et
1 2 3 4 5
Units per gram packed ceils 1% load
20% load _
278 530 530 530 586
107 129 136 129 143
Q Figure 7 g cells)
100
Effectof swelling ratio, Q on the cellinveltese
activity (in units per
-~
90 80 70 60 50 40 30
\,
20 10
-a I 5
I
I
I
10 15 20
Celi concentration
(X w/v)
I
I
I
I
.
5
10
15
20
Cell concentration
with immobilization of more concentrated cell suspensions or solutions has been reported’*, lg. These phenomena can be explained as follows. When the cells are entrapped, the diffusions of the substrate and product are restricted, there might be inactivation due to the entrapment procedure, and an increase in loading alters the microenvironment more (i.e. accumulation of more products in the closevicinityof the cells, change in pH) and also leads to a deficiency of substrate within the gel. Retention of activity after repeated use of the immobilized cell system is very important economically. As observed in Table 2, with a 1% cell load, doubling of invertase activity was detected on the second use. A less pronounced increase was also found with 20% loading. In the third, fourth and fifth runs the enzyme activity was not significantly altered. Similar results have been reported and the changes after the first wash have generally been attributed to an increase in the cell membrane permeability for the substrate and/or product”20.
(% w/v)
Figure 8 Effect of cellloading on immobilizedinvertase activity: (a) specific activity (in unitsperg of ceils) and (b) totalactivity (in unitsper5 mlgel).
aminogroups at low pH, thus creating a very different (highly charged) environment for the immobilized cells. For the free cells the highest activity was found to be at the highest temperature tested (SOOC) (Figure 6). With 20% loading, the cells behaved in the same way as the free cells. An unexpected observation, however, was that, with 1% loading, the cells displayed maximum activity at 35°C and the activity decreased when the temperature was increased further. in selecting a gel for use in the cell activity experiments the gel with the highest activity is preferred, to obtain the most significant change. The gel with the maximum possible swelling without any cell loss due to leakage is expected to yield maximum activity because of the close resemblance to the environment of the free cell. lnvertase activity, obtained per gram of cell material, was plotted against the swelling ratio (Figure 7). It can be seen that gel 5 yields the highest activity, as expected from its swelling ratio. The results of leakage experiments (even for extended periods of soaking) indicated that there was no significant leakage from any of the gels tested, thus gel 5 was chosen for use in the following experiments. The activity of gel 5 prepared with different concentrations of cells at 200 mM sucrose concentration is presented in Figure 8. The results indicate that when the loading is increased from 1 to 20%, the specific activity per gram cell is decreased by about 70%. Another expression of this activity decrease upon increase in loading can be seen in the total activities. In comparison with the free cells, the specific activities are about 60 and 20% when the loadings are 1 and 20%, respectively. A drop in the specific activity
CONCLUSIONS In general, within the published literature suitableconditions for immobilization and enzymatic properties of the cells are determined, but no information on the physical properties of the support is reported and characterization of the physical properties of the supports used in immobilization is not a common practice. In the present study, acrylamide gels have been characterized before immobilization and the invertase activities of the cells immobilized in these gels under various conditions have been determined. As a result of the characterization and optimization study of the gel itself and with the immobilized cells, it is possible to modify the entrapment conditions to suit the requirements of a specific application. It will also be possible to identify any observed changes in the activity as being of gel or cell origin.
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