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Biocatalysis by sol-gel entrapped enzymes
/OURNA
Journal of Non-Crystalline Solids 147&148 (1992) 739-743 North-Holland
L OF
NON-CRYSTALLINE SOLIDS
Biocatalysis by sol-gel entrapped enzymes Sergei B r a u n a, S a r a h S h t e l z e r a, Sara R a p p o p o r t a, D a v i d A v n i r b and M i c h a e l O t t o l e n g h i b a A. Silberman Institute of Life Sciences, The Hebrew University, Jerusalem 91904, Israel b Institute of Chemistry, The Hebrew University, Jerusalem 91904, Israel
Attachment of enzymes to insoluble matrix is an essential step in the development of biocatalysts. Transparent xerogels containing various enzymes were obtained by mixing a solution of an enzyme with tetra-methoxy orthosilicate (TMOS) at room temperature followed by gelation and drying. Effective immobilization was usually obtained at initial pH values > 7, where there is a change in the gelation mechanism from predominant hydrolysis/condensation to predominant direct polymerization of silicate precursors. The properties of sol-gel matrix, namely, transparency, large hydrophilic surface and good chemical and thermal stability, make it an ideal material for both biocatalysts and optical sensor devices. An example of a simple optical glucose sensor is demonstrated.
1. Introduction
2. Experimental procedures
We have recently demonstrated that enzymes can be entrapped within the matrix of a forming sol-gel, while retaining high catalytic activity in the xerogel [1-4]. Enzymes immobilized in silica xerogels obtained by sol-gel process do not leach out [2-4]. The lateral diffusion and conformational mobility of entrapped proteins are severely restricted, thus contributing to improved stability of enzymes [2-4]. It is shown here that an effective immobilization depends upon pH of the enzyme solution used in the sol-gel process. Possible relations between the biological activity of enzyme-containing xerogels, their physical properties and gelation process are discussed. The transparency of silicate xerogels makes them uniquely suitable for the development of fluorescent, luminescent and colourimetric enzymic sensors. An example of a simple optical glucose sensor is demonstrated.
Enzyme solutions (0.5 ml) in water or buffer were mixed at 4°C with tetra-methyl orthosilicate (TMOS, 98%, Aldrich, 0.5 ml). No addition of alcohol was needed [3-5]. The reaction mixture was gently shaken for about 15 min or until mixtures became homogeneous, and allowed to gel at room temperature. The gels were air-dried for a week at ambient temperature. Additional experimental details are given in figure captions. The time elapsing between the addition of TMOS to the water solution and the loss of flow in the tilted test tube was taken as the gelation time. The specific surface areas was obtained from the BET equation. Nitrogen adsorption isotherms were determined using Micrometrics ASAP 2000. The pore size distribution was calculated from an adsorption-desorption isotherm using the Kelvin equation. The glass sample was washed overnight,
0022-3093/92/$05.00 © 1992 - Elsevier Science Publishers B.V. All rights reserved
740
S. Braun et aL / Biocatalysis by sol-gel entrapped enzymes
dried in vacuum at about 0.05 mbar for 48 h, and then degassed for 12 h at ambient temperature and 0.01 mbar prior to the measurement of nitrogen adsorption. Prior to determination of enzyme activity, the dry glassy xerogels containing either trypsin or acetylated trypsin were ground to a size of 60-100 mesh and washed overnight with running water. The assay mixture (1 ml) containing the substrate N-benzoyl-L-arginine-4-nitroanilide (L-BAPNA, 1 mM) in TRIS- HC1 buffer (40 raM, pH 8.0) was added to an aliquot (3-5 rag) of glass powder and vigorously stirred for a suitable period of time at 25°C [4]. Activity of an immobilized enzyme was calculated as a percentage of the activity used for the preparation of xerogel. Glucose oxidase (from A. niger, Sigma, 203 U/mg, 10 Units) and peroxidase (from horseradish, Sigma, 157 U/rag, 20 units) were dissolved in water (0.5 ml). The solution was mixed and then gently shaken with TMOS (0.5 ml) containing 0.5% polydimetylsiloxane trimethylsiloxyterminated (molecular weight: 162, Petrarch Systems Inc.). The resulting gel was allowed to dry at ambient temperature for 2 weeks. The transparent glassy xerogel was impregnated with a chromogenic mixture [6] solution containing 3.3 mM 3-(dimethylamino) benzoic acid (DMAB) and 2 mM 3-methyl-benzothiazolinone hydrazone hydrochloride (MBTH) in 0.1 M phosphate buffer, pH 6.5. The xerogel was then air-dried. The formation of dye in the glassy xerogel in the presence of glucose (fig. 1) was recorded with a densitometer (SLTRFF, Biomedical Instruments Ltd., CA, USA) filtered at 595 nm.
3. Results
Xerogels prepared in the absence or at low concentration of buffer were monolith transparent glasses of considerable mechanical strength (fig. 1). Most proteins did not aggregate during xerogel preparations even at concentrations up to 20 m g / g glass [4]. Repeated hydration and drying of glasses resulted in fractures in large (1-2 cm in diameter) blocks. Xerogels prepared with addition of polydimetylsiloxane resulted in improved
Fig. 1. Transparent
sol-gel disks of protein-containing xerogels.
stability of glasses to cycles of drying and hydration. To study the effect of pH upon the properties of xerogels and the effectiveness of enzyme immobilization, sol-gel glasses were prepared with the addition of Teorell-Stenhagen [7] citratephosphate-borate buffer (CPB). CPB buffer has essentially constant conductivity of 6-7 mS at all pH values. Proteins at 2 m g / g xerogel had no effect on the xerogel N2-BET surface area. Lower pH resulted in larger BET surface areas (650-700 m2/g). With the increase in pH, surface areas gradually decreased, measuring 500-550 m2/g at pH 12. A concomitant increase in pore volume was observed (0.3-0.4 m l / g at pH 2 versus 0.7-0.9 m l / g at pH 12). All xerogels were characterized by a very narrow pore size distribution. The average pore diameter was 3-4 nm at pH 2, and increased to 7-10 nm at pH 7 with slight variation above this pH. Two forms of trypsin have been used to probe interactions between the silicate matrix and encapsulated protein molecules. Native trypsin is a relatively basic protein (isoelectric point pI = 10.5). Trypsin may be subjected to full acetylation of available side-chain amino groups, which results in the shift of p I < 3 to the pI of silica particles. Acetylated trypsin retains full catalytic activity of the native enzyme. The' catalytic properties, size and form of both protein molecules
741
S. Braun et al. / Biocatalysis by sol-gel entrapped enzymes 40"
in a manner similar to that of acetylated trypsin.
35
A t 5 < p H < 9, however, trypsin activity was lower
and almost independent of pH. Below pH 4, no
mm
30
[]
trypsin activity was recovered. It is i n t e r e s t i n g to
[] >
25'
,<
20
.E
15
note that border conditions tween the three pH domains tive trypsin coincide with the tein carboxy ( p K a = 4 ) and
rl
g
[] D D
1.-
n
[]
•
10
groups.
5 O ,
2
4
6
•
8
,
•
10
,
14
12
pH 10 3
.=_ E 10 2
E l-
g 101
•
• •
10 0
for transition beof immobilized napK a values of proamino ( p K a ~ 11)
•
,
2
.
,
4
.
,
.
6
,
8
.
,
10
•
•
,
12
.
14
The sol-gel immobilization procedure in its various modifications is a general immobilization technique: we have efficiently immobilized numerous enzymes belonging to various enzyme groups and classes [1-4], including coupled enzyme systems. The transparency of sol-gel glass is a unique property, allowing the building of optical enzyme sensors. These sensors may be built on the basis of differences in absorption spectra, luminescence or fluorescence of various enzyme-substrate, enzyme-inhibitor or enzyme-antibody complexes. A simple irreversible prototype described below demonstrates the feasibility of the sol-gel technique for this type of sensor.
pH
Fig. 2. Effect of pH upon the gelation time and upon the recovery of trypsin activity in enzyme-containing xerogels. Trypsin (0.5 rag) or acetylated trypsin (0.5 mg) were dissolved in Teorell-Stenhagen citrate-phosphate-borate (CPB) buffer [7] containing 5 mM CaC12 and prepared at pH values indicated in the figure. TMOS (0.25 ml) was added to the solution at 22°C under stirring. Gelation time (lower figure) was determined and the gels were processed as described in section 2. Trypsin ([]) and acetylated trypsin ( • ) activities (upper figure) were determined at 25°C using L-BAPNA (1 mM) as the substrate.
140 •
E 120 •
c
100'
100 m
/
80
60 3 0 rnM O
40"
,.O '~
20'
2
10 m M 0
, 0
are closely similar. Thus, c o n s i d e r a b l e variations (fig. 2) in the recovery of trypsin activity in xerogels, could b e i n t e r p r e t e d in terms of charge interactions. Xerogels p r e p a r e d at p H < 7 were practically devoid of acetylated trypsin activity, while at p H > 7 t h e activity i n c r e a s e d with pH. Thus, the recovery of acetylated trypsin activity falls into the same two p H d o m a i n s i n d i c a t e d for the gelation process. T h e activity of native trypsin i m m o bilized at p H > pI value d e p e n d e d u p o n the p H
'
, 1
-
, 2
•
, 3
-
, 4
•
, 5
•
, 6
-
, 7
•
, 8
•
, 9
•
, 10
.
, 11
, 2
Time, min
Fig. 3. Sol-gel optical glucose sensor. Xerogel disk (8 mm in diameter and 2 mm thick) containing glucose oxidase, peroxidase and a chromogenic assay for peroxidase (see final paragraph of section 2) was placed in the optical pathway of the densitometer in a vial containing 3 ml water. At time 0, a concentrated solution of glucose was added, bringing glucose concentration to t0 mM. Enzymatic oxidation of glucose produced H202, which in the presence of peroxidase and of chromogenic assay components resulted in dye formation [6]. Dye accumulation in the sol-gel disk was recorded in arbitrary optical-density units. More glucose was added at 6, 8 and 10 min. Glucose concentrations are shown at the arrows.
742
S. Braun et al. / Biocatalysis by sol-gel entrapped enzymes
Glucose oxidase and peroxidase were co-immobilized in a sol-gel xerogel. The two enzymes catalyze following reactions: (a) Glucose oxidase: glucose + 02 = glucon01actone + H202; (b) Peroxidase: donor + H 2 0 2 = oxidized donor + H 2 0 . M T B H was used as the electron donor in reaction (b), and the oxidized donor reacted with DMAB forming 1 mol of indamine dye per mole glucose [6]. The kinetics of dye formation at various glucose concentrations (fig. 3) demonstrate that the device was glucose-sensitive in the range between 0 and 100 mM.
4. Discussion
As indicated by a discontinuity in gelation time curve plotted as a function of p H (fig. 2), gelation mechanism varied between the two pH domains: pH 2 - 7 and pH > 7. These domains have been well characterized earlier [8] in terms of combination of processes of hydrolysis, condensation and particle growth. In short, one expects formation of small rough silica particles which condense at 2 _< pH < 7, while large smooth particles form at pH > 7 by continuous polymerization. Although it is not clear whether the p H of the buffer is strictly kept in the gel, the p H dependence of xerogel surface characteristics is consistent with the expected change in form and size of gel particles at different pH values of CPB buffer. In has been shown previously [4] that, at pore sizes obtained throughout the pH range reported here, diffusion does not limit the enzymatic reaction when such relatively poor substrate as Nbenzoyl-L-arginine-4-nitroanilide (L-BAPNA) is used. Thus, the recovery of trypsin activity may be affected by (a) availability of the catalytic center of the protein to the substrate, or (b) loss of enzyme activity through denaturation. The former is more probable. It has been demonstrated [1-4] that no protein is lost by leaching, and that the binding of dye to sol-gel immobilized protein correlates well with enzyme activity. Denatured but accessible protein would bind the dye to the
same extent as the active non-denatured species. Thus, the loss of enzyme activity is probably a result of embedding the catalytic center below the surface in the bulk of a xerogel. It follows that the accessibility and, therefore, activity of sol-gel immobilized enzymes should depend upon both gel formation mechanism and protein molecule charge. Above p H 7, silica particles grow primarily by a continuous polymerization mechanism, forming particles which are likely to carry more negative charge than the oligomeric precursors [8]. Negatively charged protein molecules (at pH < pI) will be well dispersed by electrostatic repulsion among large elementary silica sol particles. Thus, after gelation and drying, most enzyme molecules will be trapped with access to the pores of the xerogel. Below pH 7, the growth occurs mainly by particle aggregation [8]. It is obvious that adsorption of protein to small elementary particles would contribute to the loss of activity to species embedded without the access to the surface, as a result of aggregate formation. Since adsorption of proteins to most surfaces, includiflg silica, increases with the increase of protein molecule hydrophobicity [9-11], acetylated and, generally, acylated proteins absorb at interfaces much faster and to a larger extent than native proteins [12-15]. Thus, at 5 < pH < 7, relatively hydrophobic acetylated trypsin tends to become embedded more than the native trypsin. One may formulate the following 'rule of thumb': an efficient sol-gel immobilization of enzyme is expected at pH values above 8 and above the pI value of the enzyme.
5. Conclusion
Transparent xerogels containing various enzymes were obtained by mixing a solution of an enzyme with tetra-methoxy orthosigcate (TMOS) at room temperature followed by gelation and drying. Various procedures for this entrapment were developed. Recovery of enzyme activity depended upon gelation mechanism and on protein molecule charge. A feasibility of an optical enzyme sensor based on sol-gel immobilized en-
S. Braun et al. / Biocatalysis by sol-gel entrapped enzymes
zymes was demonstrated. A worldwide patent application by the Yissum R & D Co of the Hebrew University is pending.
This work is supported by the Harry Kay (Minneapolis) Foundation for Applied Research of the Hebrew University.
References [1] S. Braun, S. Rappoport, R. Zusman, D. Avnir and M. L Ottolenghi, Mater. Lett. 10 (1990) 1. [2] S. Braun, S. Rappoport, R. Zusman, S. Shtelzer, S. Druckman, D. Avnir and M. Ottolenghi, in: Biotechnology: Bridging Research and Applications, ed. D. Kamely, A, Chakrabarty and S.E. Kornguth (Kluwer Academic, Boston, 1990) p. 205. [3] D. Avnir, S. Braun and M. Ottolenghi, in: Supermolecular Architecture in Two and Three Dimensions, ed. T. Bein, ACS Symp. Series, Vol. 499 (American Chemical Society, New York 1992).
743
[4] S. Shtelzer, S. Rappoport, D. Avnir, M. Ottolenghi and S. Braun, Biotechnol. Appl. Biochem., 15 (1992) 227. [5] D. Avnir and V. Kaufman, J. Non-Cryst. Solids 192 (1987) 177. [6] T.T. Ngo and H.M. Lenhoff, Anal. Biochem. 105 (1980) 389. [7] H.A. Sober, ed., Handbook of Biochemistry and Molecular Biology, 2nd Ed. (CRC, Cleveland, OH, 1970). [8] C.J. Brinker and G.W. Scherer, Sol-Gel Science (Academic Press, San Diego, 1990). [9] W. Norde and J. Lykelema, J. Colloid Interf. Sci. 71 (1979) 350. [10] J. Lykelema, J. Colloid Surf. 10 (1984) 33. [11] A.L. Kamyshnyi, J. Phys. Chem. (Russ.) 55 (1981) 319. [12] R.A. Barford and B.J. Sliwinski, J. Disp. Sci. Technol. 6 (1985) 1. [13] R.E. Feeny, G. Blackhorn and H.B.F. Dixon, Adv. Protein Chem. 29 (1975) 135. [14] M.T.A. Evans, J. Mitchell, P.R. Mussewhite and L. Irons, Adv. Exp. Med. Biol. 7 (1970) 1. [15] S. Magdassi, D. Leibler and S. Braun, Langmuir 6 (1990) 376.
Journal of Non-Crystalline Solids 147&148 (1992) 739-743 North-Holland
L OF
NON-CRYSTALLINE SOLIDS
Biocatalysis by sol-gel entrapped enzymes Sergei B r a u n a, S a r a h S h t e l z e r a, Sara R a p p o p o r t a, D a v i d A v n i r b and M i c h a e l O t t o l e n g h i b a A. Silberman Institute of Life Sciences, The Hebrew University, Jerusalem 91904, Israel b Institute of Chemistry, The Hebrew University, Jerusalem 91904, Israel
Attachment of enzymes to insoluble matrix is an essential step in the development of biocatalysts. Transparent xerogels containing various enzymes were obtained by mixing a solution of an enzyme with tetra-methoxy orthosilicate (TMOS) at room temperature followed by gelation and drying. Effective immobilization was usually obtained at initial pH values > 7, where there is a change in the gelation mechanism from predominant hydrolysis/condensation to predominant direct polymerization of silicate precursors. The properties of sol-gel matrix, namely, transparency, large hydrophilic surface and good chemical and thermal stability, make it an ideal material for both biocatalysts and optical sensor devices. An example of a simple optical glucose sensor is demonstrated.
1. Introduction
2. Experimental procedures
We have recently demonstrated that enzymes can be entrapped within the matrix of a forming sol-gel, while retaining high catalytic activity in the xerogel [1-4]. Enzymes immobilized in silica xerogels obtained by sol-gel process do not leach out [2-4]. The lateral diffusion and conformational mobility of entrapped proteins are severely restricted, thus contributing to improved stability of enzymes [2-4]. It is shown here that an effective immobilization depends upon pH of the enzyme solution used in the sol-gel process. Possible relations between the biological activity of enzyme-containing xerogels, their physical properties and gelation process are discussed. The transparency of silicate xerogels makes them uniquely suitable for the development of fluorescent, luminescent and colourimetric enzymic sensors. An example of a simple optical glucose sensor is demonstrated.
Enzyme solutions (0.5 ml) in water or buffer were mixed at 4°C with tetra-methyl orthosilicate (TMOS, 98%, Aldrich, 0.5 ml). No addition of alcohol was needed [3-5]. The reaction mixture was gently shaken for about 15 min or until mixtures became homogeneous, and allowed to gel at room temperature. The gels were air-dried for a week at ambient temperature. Additional experimental details are given in figure captions. The time elapsing between the addition of TMOS to the water solution and the loss of flow in the tilted test tube was taken as the gelation time. The specific surface areas was obtained from the BET equation. Nitrogen adsorption isotherms were determined using Micrometrics ASAP 2000. The pore size distribution was calculated from an adsorption-desorption isotherm using the Kelvin equation. The glass sample was washed overnight,
0022-3093/92/$05.00 © 1992 - Elsevier Science Publishers B.V. All rights reserved
740
S. Braun et aL / Biocatalysis by sol-gel entrapped enzymes
dried in vacuum at about 0.05 mbar for 48 h, and then degassed for 12 h at ambient temperature and 0.01 mbar prior to the measurement of nitrogen adsorption. Prior to determination of enzyme activity, the dry glassy xerogels containing either trypsin or acetylated trypsin were ground to a size of 60-100 mesh and washed overnight with running water. The assay mixture (1 ml) containing the substrate N-benzoyl-L-arginine-4-nitroanilide (L-BAPNA, 1 mM) in TRIS- HC1 buffer (40 raM, pH 8.0) was added to an aliquot (3-5 rag) of glass powder and vigorously stirred for a suitable period of time at 25°C [4]. Activity of an immobilized enzyme was calculated as a percentage of the activity used for the preparation of xerogel. Glucose oxidase (from A. niger, Sigma, 203 U/mg, 10 Units) and peroxidase (from horseradish, Sigma, 157 U/rag, 20 units) were dissolved in water (0.5 ml). The solution was mixed and then gently shaken with TMOS (0.5 ml) containing 0.5% polydimetylsiloxane trimethylsiloxyterminated (molecular weight: 162, Petrarch Systems Inc.). The resulting gel was allowed to dry at ambient temperature for 2 weeks. The transparent glassy xerogel was impregnated with a chromogenic mixture [6] solution containing 3.3 mM 3-(dimethylamino) benzoic acid (DMAB) and 2 mM 3-methyl-benzothiazolinone hydrazone hydrochloride (MBTH) in 0.1 M phosphate buffer, pH 6.5. The xerogel was then air-dried. The formation of dye in the glassy xerogel in the presence of glucose (fig. 1) was recorded with a densitometer (SLTRFF, Biomedical Instruments Ltd., CA, USA) filtered at 595 nm.
3. Results
Xerogels prepared in the absence or at low concentration of buffer were monolith transparent glasses of considerable mechanical strength (fig. 1). Most proteins did not aggregate during xerogel preparations even at concentrations up to 20 m g / g glass [4]. Repeated hydration and drying of glasses resulted in fractures in large (1-2 cm in diameter) blocks. Xerogels prepared with addition of polydimetylsiloxane resulted in improved
Fig. 1. Transparent
sol-gel disks of protein-containing xerogels.
stability of glasses to cycles of drying and hydration. To study the effect of pH upon the properties of xerogels and the effectiveness of enzyme immobilization, sol-gel glasses were prepared with the addition of Teorell-Stenhagen [7] citratephosphate-borate buffer (CPB). CPB buffer has essentially constant conductivity of 6-7 mS at all pH values. Proteins at 2 m g / g xerogel had no effect on the xerogel N2-BET surface area. Lower pH resulted in larger BET surface areas (650-700 m2/g). With the increase in pH, surface areas gradually decreased, measuring 500-550 m2/g at pH 12. A concomitant increase in pore volume was observed (0.3-0.4 m l / g at pH 2 versus 0.7-0.9 m l / g at pH 12). All xerogels were characterized by a very narrow pore size distribution. The average pore diameter was 3-4 nm at pH 2, and increased to 7-10 nm at pH 7 with slight variation above this pH. Two forms of trypsin have been used to probe interactions between the silicate matrix and encapsulated protein molecules. Native trypsin is a relatively basic protein (isoelectric point pI = 10.5). Trypsin may be subjected to full acetylation of available side-chain amino groups, which results in the shift of p I < 3 to the pI of silica particles. Acetylated trypsin retains full catalytic activity of the native enzyme. The' catalytic properties, size and form of both protein molecules
741
S. Braun et al. / Biocatalysis by sol-gel entrapped enzymes 40"
in a manner similar to that of acetylated trypsin.
35
A t 5 < p H < 9, however, trypsin activity was lower
and almost independent of pH. Below pH 4, no
mm
30
[]
trypsin activity was recovered. It is i n t e r e s t i n g to
[] >
25'
,<
20
.E
15
note that border conditions tween the three pH domains tive trypsin coincide with the tein carboxy ( p K a = 4 ) and
rl
g
[] D D
1.-
n
[]
•
10
groups.
5 O ,
2
4
6
•
8
,
•
10
,
14
12
pH 10 3
.=_ E 10 2
E l-
g 101
•
• •
10 0
for transition beof immobilized napK a values of proamino ( p K a ~ 11)
•
,
2
.
,
4
.
,
.
6
,
8
.
,
10
•
•
,
12
.
14
The sol-gel immobilization procedure in its various modifications is a general immobilization technique: we have efficiently immobilized numerous enzymes belonging to various enzyme groups and classes [1-4], including coupled enzyme systems. The transparency of sol-gel glass is a unique property, allowing the building of optical enzyme sensors. These sensors may be built on the basis of differences in absorption spectra, luminescence or fluorescence of various enzyme-substrate, enzyme-inhibitor or enzyme-antibody complexes. A simple irreversible prototype described below demonstrates the feasibility of the sol-gel technique for this type of sensor.
pH
Fig. 2. Effect of pH upon the gelation time and upon the recovery of trypsin activity in enzyme-containing xerogels. Trypsin (0.5 rag) or acetylated trypsin (0.5 mg) were dissolved in Teorell-Stenhagen citrate-phosphate-borate (CPB) buffer [7] containing 5 mM CaC12 and prepared at pH values indicated in the figure. TMOS (0.25 ml) was added to the solution at 22°C under stirring. Gelation time (lower figure) was determined and the gels were processed as described in section 2. Trypsin ([]) and acetylated trypsin ( • ) activities (upper figure) were determined at 25°C using L-BAPNA (1 mM) as the substrate.
140 •
E 120 •
c
100'
100 m
/
80
60 3 0 rnM O
40"
,.O '~
20'
2
10 m M 0
, 0
are closely similar. Thus, c o n s i d e r a b l e variations (fig. 2) in the recovery of trypsin activity in xerogels, could b e i n t e r p r e t e d in terms of charge interactions. Xerogels p r e p a r e d at p H < 7 were practically devoid of acetylated trypsin activity, while at p H > 7 t h e activity i n c r e a s e d with pH. Thus, the recovery of acetylated trypsin activity falls into the same two p H d o m a i n s i n d i c a t e d for the gelation process. T h e activity of native trypsin i m m o bilized at p H > pI value d e p e n d e d u p o n the p H
'
, 1
-
, 2
•
, 3
-
, 4
•
, 5
•
, 6
-
, 7
•
, 8
•
, 9
•
, 10
.
, 11
, 2
Time, min
Fig. 3. Sol-gel optical glucose sensor. Xerogel disk (8 mm in diameter and 2 mm thick) containing glucose oxidase, peroxidase and a chromogenic assay for peroxidase (see final paragraph of section 2) was placed in the optical pathway of the densitometer in a vial containing 3 ml water. At time 0, a concentrated solution of glucose was added, bringing glucose concentration to t0 mM. Enzymatic oxidation of glucose produced H202, which in the presence of peroxidase and of chromogenic assay components resulted in dye formation [6]. Dye accumulation in the sol-gel disk was recorded in arbitrary optical-density units. More glucose was added at 6, 8 and 10 min. Glucose concentrations are shown at the arrows.
742
S. Braun et al. / Biocatalysis by sol-gel entrapped enzymes
Glucose oxidase and peroxidase were co-immobilized in a sol-gel xerogel. The two enzymes catalyze following reactions: (a) Glucose oxidase: glucose + 02 = glucon01actone + H202; (b) Peroxidase: donor + H 2 0 2 = oxidized donor + H 2 0 . M T B H was used as the electron donor in reaction (b), and the oxidized donor reacted with DMAB forming 1 mol of indamine dye per mole glucose [6]. The kinetics of dye formation at various glucose concentrations (fig. 3) demonstrate that the device was glucose-sensitive in the range between 0 and 100 mM.
4. Discussion
As indicated by a discontinuity in gelation time curve plotted as a function of p H (fig. 2), gelation mechanism varied between the two pH domains: pH 2 - 7 and pH > 7. These domains have been well characterized earlier [8] in terms of combination of processes of hydrolysis, condensation and particle growth. In short, one expects formation of small rough silica particles which condense at 2 _< pH < 7, while large smooth particles form at pH > 7 by continuous polymerization. Although it is not clear whether the p H of the buffer is strictly kept in the gel, the p H dependence of xerogel surface characteristics is consistent with the expected change in form and size of gel particles at different pH values of CPB buffer. In has been shown previously [4] that, at pore sizes obtained throughout the pH range reported here, diffusion does not limit the enzymatic reaction when such relatively poor substrate as Nbenzoyl-L-arginine-4-nitroanilide (L-BAPNA) is used. Thus, the recovery of trypsin activity may be affected by (a) availability of the catalytic center of the protein to the substrate, or (b) loss of enzyme activity through denaturation. The former is more probable. It has been demonstrated [1-4] that no protein is lost by leaching, and that the binding of dye to sol-gel immobilized protein correlates well with enzyme activity. Denatured but accessible protein would bind the dye to the
same extent as the active non-denatured species. Thus, the loss of enzyme activity is probably a result of embedding the catalytic center below the surface in the bulk of a xerogel. It follows that the accessibility and, therefore, activity of sol-gel immobilized enzymes should depend upon both gel formation mechanism and protein molecule charge. Above p H 7, silica particles grow primarily by a continuous polymerization mechanism, forming particles which are likely to carry more negative charge than the oligomeric precursors [8]. Negatively charged protein molecules (at pH < pI) will be well dispersed by electrostatic repulsion among large elementary silica sol particles. Thus, after gelation and drying, most enzyme molecules will be trapped with access to the pores of the xerogel. Below pH 7, the growth occurs mainly by particle aggregation [8]. It is obvious that adsorption of protein to small elementary particles would contribute to the loss of activity to species embedded without the access to the surface, as a result of aggregate formation. Since adsorption of proteins to most surfaces, includiflg silica, increases with the increase of protein molecule hydrophobicity [9-11], acetylated and, generally, acylated proteins absorb at interfaces much faster and to a larger extent than native proteins [12-15]. Thus, at 5 < pH < 7, relatively hydrophobic acetylated trypsin tends to become embedded more than the native trypsin. One may formulate the following 'rule of thumb': an efficient sol-gel immobilization of enzyme is expected at pH values above 8 and above the pI value of the enzyme.
5. Conclusion
Transparent xerogels containing various enzymes were obtained by mixing a solution of an enzyme with tetra-methoxy orthosigcate (TMOS) at room temperature followed by gelation and drying. Various procedures for this entrapment were developed. Recovery of enzyme activity depended upon gelation mechanism and on protein molecule charge. A feasibility of an optical enzyme sensor based on sol-gel immobilized en-
S. Braun et al. / Biocatalysis by sol-gel entrapped enzymes
zymes was demonstrated. A worldwide patent application by the Yissum R & D Co of the Hebrew University is pending.
This work is supported by the Harry Kay (Minneapolis) Foundation for Applied Research of the Hebrew University.
References [1] S. Braun, S. Rappoport, R. Zusman, D. Avnir and M. L Ottolenghi, Mater. Lett. 10 (1990) 1. [2] S. Braun, S. Rappoport, R. Zusman, S. Shtelzer, S. Druckman, D. Avnir and M. Ottolenghi, in: Biotechnology: Bridging Research and Applications, ed. D. Kamely, A, Chakrabarty and S.E. Kornguth (Kluwer Academic, Boston, 1990) p. 205. [3] D. Avnir, S. Braun and M. Ottolenghi, in: Supermolecular Architecture in Two and Three Dimensions, ed. T. Bein, ACS Symp. Series, Vol. 499 (American Chemical Society, New York 1992).
743
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