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Immobilization of β-Galactosidase on an Insoluble Carrier with a Polyisocyanate Polymer. II. Kinetics and Stability
Immobilization of #-Galadosidase on an Insoluble Carrier with a Polyisocyana/e Polymer. II. Kinetics and Stability G. O. HUSTAD, T. RICHARDSON, and N. F. OLSON Department of Food Science University of Wisconsin, Madison 53706 Abstract
The kinetic properties of commercial fl-galactosidase (Escherichia coli K-12) bonded to .8 cm x 1.3 cm Teflon stirring bars via a polyisocyanate polymer were compared to those of the soluble enzyme Assays of immobilized fl-galactosidase were performed with test tubes placed in a 37 C water-jacketed container positioned on top of a stirring motor set at 80 -4"_5 rpm. The Michaelis-Menten constants for soluble and immobilized flgalactosidase at pH 6.5 were 13.1 mM and 22.1 raM. The pH optimum for immobilized fl-galactosidase shifted upward .8 pH units relative to the soluble enzyme. Activation energies for hydrolysis of lactose were 15,200 cal/mole and 12,500 cal/mole for soluble and immobilized /3-galactosidase. The immobilized fl-galactosidase exhibited good stability, retaining 65.6% of the original activity after intermittent use for 118 days. When not in use, the immobilized fl-galaetosidase was stored at 4 C in .1 M phosphate buffer (pH 6.5). Introduction
Enzymes have been immobilized by a variety of methods (9, 18), and the immobilized enzymes have been utilized in several different forms including particles, both for continuously stirred reactors (14) and packed bed reactors (12); porous cellulose sheets (10); membranes (1); and nylon tubes (6). Comparison of immobilized enzyme particles operating in continuous-feed stirred tanks and in packed bed reactors has shown the latter is more efficient (13, 14). Diffusion limitations, however, could be significant in either type of reaction form. Obtaining adequate flow rates with the present enzyme-support materials is a maior difficulty in operating packed bed reactors (5). Substrate solutions often contain Received January 9, 1973.
small amounts of suspended protein, polysaccharides, or lipids resulting in decreased flow rates (4, 5, 19). On the other hand, in continuously stirred reaction vessels not all the immobilized enzyme molecules are contributing to the reaction, even under the most turbulent conditions and highest substrate concentrations (14). This arises because of the slow diffusion of substrate ,through the external diffusional film and into the pores of the enzyme particles. According to Lilly and Sharp (14), diffusion into the particle pores probably could be eliminated ff the immobilized enzyme was located only on the outer surface of the insoluble polymer particles. A more accurate apparent Michaelis-Menten constant then could be realized. In the preceding paper (7), a method was described for immobilizing ~-galactosidase (E. coli K-12) to a polyisocyanate polymer coated oa magnetic stirring bars. With this form of immobilized enzyme, several assay measures were controlled easily. First, diffusion effects were fairly constant since the bound enzyme molecules were attached primarily to the outer surface of the insoluble support (7). Constant diffusion effects were maintained by controlling the stirring rate and the depth of immersion and general location of the bound enzyme in substrate. Reproducibility and accuracy of assays were enhanced because of the above controls. In this communication, the kinetic properties of fl-galaotosidase bound to stirring bars and soluble ~-galactosidase are compared, and stability studies d immobilized fI-ga/actosidase after both repeated use and long-term cold storage are presented.
Materials and Methods
The immobilization of fl-galactosidase (E. coli K-12) on stirring bars and the assay procedures for the immobilized and soluble enzymes were described (7). The fl-galactosidase stirring bars (.8 cm x 1.3 em) in these experiments were prepared in .1 M phosphate buffer at pH 6.5 and ranged in activity from
1118
1119
KINETICS OF IMMOBILIZED /~-GALACTOSIDASE
2.68 to 3.75 units per bar. A unit of /?-galaetosidase is the amount of enzyme which will liberate 1 /,mole of glucose from lactose in 15 min at 37 C (8). Assays of immobilized enzyme were performed with a test tube placed vertically in a 37 C water-jacketed container positioned on a stirring motor set at 80 -+- 5 rpm. Tests for possible losses in enzymic activity during use or between samples used two procedures. First, assays were duplicated or triplicated before and after all values for a kinetic parameter were obtained. Also, duplicate or triplicate determinations for one value of a kinetic parameter were assaved in a random sequence. Determination of pH profile, The pH-rate profile was determined with 2.0 ml of 2.5% lactose in .1 M potassium phosphate buffer adjusted to the pH of the assay with phosphoric acid. Assays were performed as described previously (7) except the enzyme bar was washed with deionized water and the bottom portion dried before insertion into the substrate.
IMMOBILIZED 13GALACTOSIDASE 7,
-
=.so k.-
SOLUBLE p-GALACTOSIOASE-~I
Determination of M ichaelis-Menten constant (Km). Values for Krn were determined by assaying soluble and immobilized fl-galactosidase at various substrate concentrations ranging from 7.5 to 60 raM. Immobilized fi-galactosidase was assayed both at pH 6.5 and pH 7.3 whereas soluble fl-galactosidase was assayed at pH 6.5 only.
Determination of apparent activation energies. Apparent activation energies for immobilized and soluble fi-galactosidase were calculated from Arrhenius plots of reaction rate and temperature data.
Determination of stability after repeated use and cold storage. Stability after intermittent use was determined for an enzyme stirring bar (BAR-M) during a period of 118 days and for BAR-21 during a period of 85 days. Cold storage stability was determined for" BAt/-19 and BAR-22 after 97 days at 4 C. The enzvme bars were stored in .1 M potassium phosphate buffer at pH 6.5 in all cases.
Results and Discussion pH profile. The pH profile curves for immobilized and soluble fi-galaetosidase reveal an upward shift in pH optimum of about .8 pH unit for immobilized (pH 7.3) relative to soluble (pH 6.5) fi-galaetosidase (Fig. 1). Presumably, this indicates that the matrix is negatively charged (16). The negative charge should not arise, from the polyisoeyanate polymer. If anything, this surface should be posi-
5.0
6.0
7.0
8.0
pH
Fro. I. pH profiles for soluble and immobilized ~-galaetosidase. Activity was plotted as percent of maximum activity at pH 6.5 and 7.a for soluble and immobilized fl-galactosidase. tive/y charged resulting from the protonaticn of primary amines (7). The negative charge might arise from denatured fi-galaetosidase and other protein impurities bound to the polymer surface since the immobilized speeit~e activity was only about 19% of the soluble specific aetivity (7). Based on an amino acid analysis by Craven et al. (3), each fi-galactosidase molecule was calculated to have a net charge greater than minus 600 Faradays at pH 6.5. Presumably, inactive fl-galaetosidase molecules also would carry a net negative charge at pH 6.5 thereby rendering the polymer surfaee and micro-environment of the bound enzyme molecules negative. Sinee the /?-galactosidase preparation was rather crude (68.9 units/rag under our assay conditions) (7), the effect of other impurities on the pH optimum is not known. Presumably, negative charges from signifieant quantities of bound protein impurities also could contribute to the upward shift of the pH optimum, tleeently, Line et al. JOURNAL OF DAIRY SCIENCE VOL. 56, NO. 9
1120
H U S T A D E T AL
6.C~iOL ~ 5 5' L. 4.00bE1 o.
E
particle depending on stirrer speed and substrate concerLtration. Thus, Lilly and Sharp (14) were unable to obtain single values for apparent Km because of nonlinearity of their Lineweaver-Burk plots. The apparent Km at the pH optimum of immobilized fl-galactosidase (pH 7.3) was 21.0 mM as compared to 13.1 mM at the pH 01o15mum of the soluble enzyme (pH 6.5). The apparent Km for immobilized fl-galactosidase at pH 6.5 was 22.1 mM. The variable effects on Kan values of several enzymes as a result of iramobilization on inorganic carriers were summarized recently (20). The apparent Krn values for immobilized enzymes ranged from .25 to 29 times ~the Km values for the respective soluble enzymes. In this study, immobilization of fl-galaetosidase increased apparent Km about 1.6 fold. Similarly, an increased apparent Km of about 1.9 fold was observed in a recent study where fl-galaetosidase was bound to porous cellulose sheets (17). The increased Km may be due to an effec-
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E
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,,~/ ~/
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~.
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8
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-
~,
¢/
x@~
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_G'~_~"
0~-~
,,¢~,v"/
3 2D if" ~,,Oy V , ¢~5"~i/ .j O [j,,~ t / ~_ 1.00~"~ ~"
tu
I I0
SOLUBLE
S5
p_GAL.A~rOSIDASEAT pH ,on:
T
40
I-
I
80 I/[S] OR
n
I
120
I
160
(M - I )
_
~
(15) reported that immobilized crude pepsin had a wider pH optimum range than another more active immobilized pepsin preparation. Michaelis-Menten constant (Km). The values of Km were obtained by plotting the reciprocal of substrate concentration (S) against the reciprocal of reaction velocity (v) at that substrate concentration (S). The data for immobilized fl-galaetosidase at pH 6.5 and pH 7.3 and for soluble fl-galactosidase at pH 6.5 are in Fig. 2. At the stirrer speed employed (80 ~ 5 rpm), the Lineweaver-Burk plots for immobilized fl-galactosidase (Fig. 2) are essential,ly linear permitting calculation of single values for Km. The linearity of the plots supports the assumption :that immobilized fl-galactosidase molecules are located primarily on the outer polymer surface. The diffusion of substrate molecules to and from the surface was thereby simplified compared to :the diffusion involved when enzymes are bound to porous supports. With chymotrypsin bound to earboxymethyl cellulose (14), the rate of reaction appeared to be controlled by diffusion of substrate through either the effective diffusional film around the particle or the pores within the JOURNAL OF DAIRY SCIENCE VOL. 56, NO. 9
III~( /
FIG. 2. Lineweaver-Burk plots for immobilized fl-galaetosidase at pH 6.5 and 7.3 and for soluble enzyme at pH 6.5.
SOLUBLE
4
> - m~
2-
I.O-
wILl
=.. :'3 °'8I
N o 0.6 w 00.4 w _j 0
-
~0.2
-
IMMOBILIZED/ " ( p-GALACTOSIDASE
L_J
0.1
3.O
I
I
3.2 3.4 103/T COK)-I
I 3.6
FIc. 3. Arrhenius plots for lactose hydrolysis by soluble and immobilized /~-galaetosidase. Log of rates as /anoles glueose/ml per 15 min for soluble fl-galactosidase or as /,moles glucose/bar per 15 min for immobilized fl-galaetosidase vs. reciprocal of absolute temperature.
KINETICS OF IMMOBILIZED fl-GALACTOSIDASE
five diffusional film around the stirring bar requiring higher concentrations of substrate to achieve half-maximal velocity. The presence of a diffusional film is supported by the lower activation energy for the immobilized enzyme (see below) which is indicative of diffusion limitation (17). Apparent activation energy. An Arrhenius plot (log k versus l / T ; k = zero order velocity constant, T = absolute temperature) for immobilized and soluble fl-galactosidase is in Fig. 3. With soluble fi-galactosidase, a deviation from Iinearity was observed beyond 37 C. With immobilized fi-galactosidase, however, no apparent deviation from linearity occurred up to 45 C indicating increased thermal stability. These assays were performed over 15-min time intervals. Thermal denaturation of immobilized fl-galactosidase occurs after 24 h at 42 C (7). Apparent activation energies (Ea) for immobilized and soluble fl-galactosidase were calculated from the slope of the linear portion of the plots in Fig. 3. The apparent activation energies for soluble fl-galaetosidase and the immobilized derivative were 15,200 eal/mole and 12,500 eal/mole, respectively. Sharp et al. (17) reported values of 17,500 eal/mole for soluble fl-galactosidase (E. cull ML-308) and 16,300 cal/mole for the immobilized derivative.
Stability after repeated use and after storage. An initial experiment designed to test the stability of immobilized B-galactosidase during intermittent use for 118 days indicated good stability. After the immobilized fl-galaetosidase was used for 82 assays, it still retained 65.6% of the original activity. Essentially no activity was lost in the last 105 days.
1121
The results of three other stability studies using BAR-19, BAR-21, and BAR-22 are in Table 1. BAR-21 retained 86.1% of the original activity after 85 days and 43 assays. In addit_ion, no loss in activity occurred in the last 56 days. The retention of enzyme activity of BAR-19 and BAR-21 was even greater after 97 days storage at 4 C. The enzyme-polymer surface was, in general, very durable and adhered quite well to the Teflon surface. This adherence is believed to result from hydropbobic bonding. During the hardening process, cross-linking reactions within the polymer continue to occur with subsequent sh6nking and strengthening of the polymer. As a result of shrinking, the polymer binds very tightly to the curved surface of the Teflon stirring bar. Recently, in this laboratory, /3-galactosidase was bonded to Lucite with the polyisocyanate polymer during preparation of a continuous-flow-stirred-tank-reactor. The results in this paper indicate very good stabilitv of the immobilized /3-galactosidase derivatives with intermittent use over long periods of time. /3-galactosidase stirring bars thus might be used routinely to determine lactose quantities at concentrations below .1 x Km ( < 2 . 1 raM). Glucose oxidase also could be bound to magnetic stirring bars resulting in an easier and more direct method of determining lactose concentrations. Acknowledgments
Research was supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison; by the Cooperative Research Service, U. S. Department of Agriculture; by Public Heatth Service Grant No. 1-ROl-l~D-00200; and by Dairy Research Inc. References
TABLE 1, Stability of three immobilized fl-galaetosidase preparations after repeated use and after cold storage at 4 C. Immobilized ~-galactosidase preparation BAR-21
BAR-19 BAR-22
Time ( days )
No. 15-rain assays at 37 C
Percent original activity retained
0 29 79 85
3 21 15 4
100 86.1 86.1 86.1
0
3
100
97
3
101
0
3
97
3
1O0 95.4
(1) Brown, G., E. Selegny, S. Arameus, and D. Thomas. 1969. Enzymatically active membranes: some properties of cellophane membranes supporting cross-/inked enzymes. Biochim. Biophys. Aeta 185:260. (2) Cohn, M., and J. Monod. 1951. Purification and properties of the fl-galactosidase (lactase) of E. coil. Biochim. Biophys. Acta 7:153. (3) Craven, G. R., E. Steers, Jr., and C. B. Anflnsen. 1965. Purification, composition, and Molecular weight of the fl-galactosidase of E. cull K-12. J. Biol. Chem. 240:2468. (4) Ferrier, L. K., T. Richardson, N. F. Olson, and C. L. Hicks. 1972. Characteristics of insoluble pepsin used in a continuous milkclotting system, J. Dairy Sci. 55:726. (5) Goldstein, L. 1969, Use of water-insoluble enzyme derivatives in synthesis and sepJOURNAL OF DAIRY SCIENCE VOL, 56, NO, 9
1122
(6)
(7)
(8) (9) (10)
(11)
12)
HUSTAD ET AL
aration. Pages 391-424 in Fermentation advances. D. Perlman, ed. Academic Press, Inc., New York, New York. Hornby, W. E., and H. Filippusson. 1970. The preparation of trypsin chemically attached to nylon tubes. Biochim. Biophys. Acta 220:343. Hustad, G. O., T. Richardson, and N. F. Olson. 1973. Immobilization of fi-galactosidase on an insoluble carrier with a polyisocyanate polymer. I. Preparation and properties. J. Dairy Sci. 56:1111. Jasewicz, L., and A. E. Wasserman. 1961. Quantitative determination of laetase. J. Dairy Sei. 44:393. Kay, G. 1968. Insolubilized enzymes. Process Biochem. 3:36. Kay, G., M. D. Lilly, A. K. Shaw, and R. J. H. Wilson. 1968. Preparation and use of porous sheets with enzyme action. Nature 217:641. Kuby, S. A., and H. A. Lardy. 1953. Purification and kinetics of ~-D-galactosidase from E. coli, strain K-12. J. Amer. Chem. Soc. 75:890. Lilly, M. D., W. E. Hornby, and E. M. Crook. 1966. The kinetics of carboxymethyl cellulose-ficin in packed beds. Biochem. J. 100:718.
JOURNAL OF DAIRY SCIENCE VOL. 56, No. 9
(13) Lilly, M. D., G. Kay, A. K. Sharp, and R. J. H. Wilson. 1968. The operation of biochemical reactors using fixed enzymes. Biochem. J. 107:5 P. (14) Lilly, M. D., and A. K. Sharp. 1968. The kinetics of enzymes attached to water-insoluble polymers. Chem. Eng. (London) No. 215, CE 12. (15) Line, W. F., A. Kwong, and H. H. Weetall. 1971. Pepsin insolubilized by covalent attachment to glass: preparation and characterization. Biochim. Biophys. Acta 242: 194. (16) Mosbach, K. 1971. Enzymes bound to artificial matrixes. Sci. Amer. 224:26. (17) Sharp, A. K., G. Kay, and M. D. Lilly. 1969. The kinetics of fi-galactosidase attached to porous cellulose sheets. Biotechnol. Bioeng. 11:363. (18) Silman, I. H., and E. Katchalski. 1966. Water-insoluble derivatives of enzymes, antigens and antibodies. Ann. Rev. Biochem. 35: 873. (19) Smiley, K. L. 1971. Continuous conversion of starch to glucose with immobilized glucoamylase. Biotechnol. Bioeng. 13:309. (20) Weetall, H. H. 1971. Enzymes immobilized on inorganic carriers. Res. Develop. 22:18.
The kinetic properties of commercial fl-galactosidase (Escherichia coli K-12) bonded to .8 cm x 1.3 cm Teflon stirring bars via a polyisocyanate polymer were compared to those of the soluble enzyme Assays of immobilized fl-galactosidase were performed with test tubes placed in a 37 C water-jacketed container positioned on top of a stirring motor set at 80 -4"_5 rpm. The Michaelis-Menten constants for soluble and immobilized flgalactosidase at pH 6.5 were 13.1 mM and 22.1 raM. The pH optimum for immobilized fl-galactosidase shifted upward .8 pH units relative to the soluble enzyme. Activation energies for hydrolysis of lactose were 15,200 cal/mole and 12,500 cal/mole for soluble and immobilized /3-galactosidase. The immobilized fl-galactosidase exhibited good stability, retaining 65.6% of the original activity after intermittent use for 118 days. When not in use, the immobilized fl-galaetosidase was stored at 4 C in .1 M phosphate buffer (pH 6.5). Introduction
Enzymes have been immobilized by a variety of methods (9, 18), and the immobilized enzymes have been utilized in several different forms including particles, both for continuously stirred reactors (14) and packed bed reactors (12); porous cellulose sheets (10); membranes (1); and nylon tubes (6). Comparison of immobilized enzyme particles operating in continuous-feed stirred tanks and in packed bed reactors has shown the latter is more efficient (13, 14). Diffusion limitations, however, could be significant in either type of reaction form. Obtaining adequate flow rates with the present enzyme-support materials is a maior difficulty in operating packed bed reactors (5). Substrate solutions often contain Received January 9, 1973.
small amounts of suspended protein, polysaccharides, or lipids resulting in decreased flow rates (4, 5, 19). On the other hand, in continuously stirred reaction vessels not all the immobilized enzyme molecules are contributing to the reaction, even under the most turbulent conditions and highest substrate concentrations (14). This arises because of the slow diffusion of substrate ,through the external diffusional film and into the pores of the enzyme particles. According to Lilly and Sharp (14), diffusion into the particle pores probably could be eliminated ff the immobilized enzyme was located only on the outer surface of the insoluble polymer particles. A more accurate apparent Michaelis-Menten constant then could be realized. In the preceding paper (7), a method was described for immobilizing ~-galactosidase (E. coli K-12) to a polyisocyanate polymer coated oa magnetic stirring bars. With this form of immobilized enzyme, several assay measures were controlled easily. First, diffusion effects were fairly constant since the bound enzyme molecules were attached primarily to the outer surface of the insoluble support (7). Constant diffusion effects were maintained by controlling the stirring rate and the depth of immersion and general location of the bound enzyme in substrate. Reproducibility and accuracy of assays were enhanced because of the above controls. In this communication, the kinetic properties of fl-galaotosidase bound to stirring bars and soluble ~-galactosidase are compared, and stability studies d immobilized fI-ga/actosidase after both repeated use and long-term cold storage are presented.
Materials and Methods
The immobilization of fl-galactosidase (E. coli K-12) on stirring bars and the assay procedures for the immobilized and soluble enzymes were described (7). The fl-galactosidase stirring bars (.8 cm x 1.3 em) in these experiments were prepared in .1 M phosphate buffer at pH 6.5 and ranged in activity from
1118
1119
KINETICS OF IMMOBILIZED /~-GALACTOSIDASE
2.68 to 3.75 units per bar. A unit of /?-galaetosidase is the amount of enzyme which will liberate 1 /,mole of glucose from lactose in 15 min at 37 C (8). Assays of immobilized enzyme were performed with a test tube placed vertically in a 37 C water-jacketed container positioned on a stirring motor set at 80 -+- 5 rpm. Tests for possible losses in enzymic activity during use or between samples used two procedures. First, assays were duplicated or triplicated before and after all values for a kinetic parameter were obtained. Also, duplicate or triplicate determinations for one value of a kinetic parameter were assaved in a random sequence. Determination of pH profile, The pH-rate profile was determined with 2.0 ml of 2.5% lactose in .1 M potassium phosphate buffer adjusted to the pH of the assay with phosphoric acid. Assays were performed as described previously (7) except the enzyme bar was washed with deionized water and the bottom portion dried before insertion into the substrate.
IMMOBILIZED 13GALACTOSIDASE 7,
-
=.so k.-
SOLUBLE p-GALACTOSIOASE-~I
Determination of M ichaelis-Menten constant (Km). Values for Krn were determined by assaying soluble and immobilized fl-galactosidase at various substrate concentrations ranging from 7.5 to 60 raM. Immobilized fi-galactosidase was assayed both at pH 6.5 and pH 7.3 whereas soluble fl-galactosidase was assayed at pH 6.5 only.
Determination of apparent activation energies. Apparent activation energies for immobilized and soluble fi-galactosidase were calculated from Arrhenius plots of reaction rate and temperature data.
Determination of stability after repeated use and cold storage. Stability after intermittent use was determined for an enzyme stirring bar (BAR-M) during a period of 118 days and for BAR-21 during a period of 85 days. Cold storage stability was determined for" BAt/-19 and BAR-22 after 97 days at 4 C. The enzvme bars were stored in .1 M potassium phosphate buffer at pH 6.5 in all cases.
Results and Discussion pH profile. The pH profile curves for immobilized and soluble fi-galaetosidase reveal an upward shift in pH optimum of about .8 pH unit for immobilized (pH 7.3) relative to soluble (pH 6.5) fi-galaetosidase (Fig. 1). Presumably, this indicates that the matrix is negatively charged (16). The negative charge should not arise, from the polyisoeyanate polymer. If anything, this surface should be posi-
5.0
6.0
7.0
8.0
pH
Fro. I. pH profiles for soluble and immobilized ~-galaetosidase. Activity was plotted as percent of maximum activity at pH 6.5 and 7.a for soluble and immobilized fl-galactosidase. tive/y charged resulting from the protonaticn of primary amines (7). The negative charge might arise from denatured fi-galaetosidase and other protein impurities bound to the polymer surface since the immobilized speeit~e activity was only about 19% of the soluble specific aetivity (7). Based on an amino acid analysis by Craven et al. (3), each fi-galactosidase molecule was calculated to have a net charge greater than minus 600 Faradays at pH 6.5. Presumably, inactive fl-galaetosidase molecules also would carry a net negative charge at pH 6.5 thereby rendering the polymer surfaee and micro-environment of the bound enzyme molecules negative. Sinee the /?-galactosidase preparation was rather crude (68.9 units/rag under our assay conditions) (7), the effect of other impurities on the pH optimum is not known. Presumably, negative charges from signifieant quantities of bound protein impurities also could contribute to the upward shift of the pH optimum, tleeently, Line et al. JOURNAL OF DAIRY SCIENCE VOL. 56, NO. 9
1120
H U S T A D E T AL
6.C~iOL ~ 5 5' L. 4.00bE1 o.
E
particle depending on stirrer speed and substrate concerLtration. Thus, Lilly and Sharp (14) were unable to obtain single values for apparent Km because of nonlinearity of their Lineweaver-Burk plots. The apparent Km at the pH optimum of immobilized fl-galactosidase (pH 7.3) was 21.0 mM as compared to 13.1 mM at the pH 01o15mum of the soluble enzyme (pH 6.5). The apparent Km for immobilized fl-galactosidase at pH 6.5 was 22.1 mM. The variable effects on Kan values of several enzymes as a result of iramobilization on inorganic carriers were summarized recently (20). The apparent Krn values for immobilized enzymes ranged from .25 to 29 times ~the Km values for the respective soluble enzymes. In this study, immobilization of fl-galaetosidase increased apparent Km about 1.6 fold. Similarly, an increased apparent Km of about 1.9 fold was observed in a recent study where fl-galaetosidase was bound to porous cellulose sheets (17). The increased Km may be due to an effec-
,~f~, _v~/
#
E
/ 3D0~-
,,~/ ~/
,X.~ , ~'~y
~.
/
8
/ /
~° ~ •~
-
~,
¢/
x@~
/ ,-~ /
_G'~_~"
0~-~
,,¢~,v"/
3 2D if" ~,,Oy V , ¢~5"~i/ .j O [j,,~ t / ~_ 1.00~"~ ~"
tu
I I0
SOLUBLE
S5
p_GAL.A~rOSIDASEAT pH ,on:
T
40
I-
I
80 I/[S] OR
n
I
120
I
160
(M - I )
_
~
(15) reported that immobilized crude pepsin had a wider pH optimum range than another more active immobilized pepsin preparation. Michaelis-Menten constant (Km). The values of Km were obtained by plotting the reciprocal of substrate concentration (S) against the reciprocal of reaction velocity (v) at that substrate concentration (S). The data for immobilized fl-galaetosidase at pH 6.5 and pH 7.3 and for soluble fl-galactosidase at pH 6.5 are in Fig. 2. At the stirrer speed employed (80 ~ 5 rpm), the Lineweaver-Burk plots for immobilized fl-galactosidase (Fig. 2) are essential,ly linear permitting calculation of single values for Km. The linearity of the plots supports the assumption :that immobilized fl-galactosidase molecules are located primarily on the outer polymer surface. The diffusion of substrate molecules to and from the surface was thereby simplified compared to :the diffusion involved when enzymes are bound to porous supports. With chymotrypsin bound to earboxymethyl cellulose (14), the rate of reaction appeared to be controlled by diffusion of substrate through either the effective diffusional film around the particle or the pores within the JOURNAL OF DAIRY SCIENCE VOL. 56, NO. 9
III~( /
FIG. 2. Lineweaver-Burk plots for immobilized fl-galaetosidase at pH 6.5 and 7.3 and for soluble enzyme at pH 6.5.
SOLUBLE
4
> - m~
2-
I.O-
wILl
=.. :'3 °'8I
N o 0.6 w 00.4 w _j 0
-
~0.2
-
IMMOBILIZED/ " ( p-GALACTOSIDASE
L_J
0.1
3.O
I
I
3.2 3.4 103/T COK)-I
I 3.6
FIc. 3. Arrhenius plots for lactose hydrolysis by soluble and immobilized /~-galaetosidase. Log of rates as /anoles glueose/ml per 15 min for soluble fl-galactosidase or as /,moles glucose/bar per 15 min for immobilized fl-galaetosidase vs. reciprocal of absolute temperature.
KINETICS OF IMMOBILIZED fl-GALACTOSIDASE
five diffusional film around the stirring bar requiring higher concentrations of substrate to achieve half-maximal velocity. The presence of a diffusional film is supported by the lower activation energy for the immobilized enzyme (see below) which is indicative of diffusion limitation (17). Apparent activation energy. An Arrhenius plot (log k versus l / T ; k = zero order velocity constant, T = absolute temperature) for immobilized and soluble fl-galactosidase is in Fig. 3. With soluble fi-galactosidase, a deviation from Iinearity was observed beyond 37 C. With immobilized fi-galactosidase, however, no apparent deviation from linearity occurred up to 45 C indicating increased thermal stability. These assays were performed over 15-min time intervals. Thermal denaturation of immobilized fl-galactosidase occurs after 24 h at 42 C (7). Apparent activation energies (Ea) for immobilized and soluble fl-galactosidase were calculated from the slope of the linear portion of the plots in Fig. 3. The apparent activation energies for soluble fl-galaetosidase and the immobilized derivative were 15,200 eal/mole and 12,500 eal/mole, respectively. Sharp et al. (17) reported values of 17,500 eal/mole for soluble fl-galactosidase (E. cull ML-308) and 16,300 cal/mole for the immobilized derivative.
Stability after repeated use and after storage. An initial experiment designed to test the stability of immobilized B-galactosidase during intermittent use for 118 days indicated good stability. After the immobilized fl-galaetosidase was used for 82 assays, it still retained 65.6% of the original activity. Essentially no activity was lost in the last 105 days.
1121
The results of three other stability studies using BAR-19, BAR-21, and BAR-22 are in Table 1. BAR-21 retained 86.1% of the original activity after 85 days and 43 assays. In addit_ion, no loss in activity occurred in the last 56 days. The retention of enzyme activity of BAR-19 and BAR-21 was even greater after 97 days storage at 4 C. The enzyme-polymer surface was, in general, very durable and adhered quite well to the Teflon surface. This adherence is believed to result from hydropbobic bonding. During the hardening process, cross-linking reactions within the polymer continue to occur with subsequent sh6nking and strengthening of the polymer. As a result of shrinking, the polymer binds very tightly to the curved surface of the Teflon stirring bar. Recently, in this laboratory, /3-galactosidase was bonded to Lucite with the polyisocyanate polymer during preparation of a continuous-flow-stirred-tank-reactor. The results in this paper indicate very good stabilitv of the immobilized /3-galactosidase derivatives with intermittent use over long periods of time. /3-galactosidase stirring bars thus might be used routinely to determine lactose quantities at concentrations below .1 x Km ( < 2 . 1 raM). Glucose oxidase also could be bound to magnetic stirring bars resulting in an easier and more direct method of determining lactose concentrations. Acknowledgments
Research was supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison; by the Cooperative Research Service, U. S. Department of Agriculture; by Public Heatth Service Grant No. 1-ROl-l~D-00200; and by Dairy Research Inc. References
TABLE 1, Stability of three immobilized fl-galaetosidase preparations after repeated use and after cold storage at 4 C. Immobilized ~-galactosidase preparation BAR-21
BAR-19 BAR-22
Time ( days )
No. 15-rain assays at 37 C
Percent original activity retained
0 29 79 85
3 21 15 4
100 86.1 86.1 86.1
0
3
100
97
3
101
0
3
97
3
1O0 95.4
(1) Brown, G., E. Selegny, S. Arameus, and D. Thomas. 1969. Enzymatically active membranes: some properties of cellophane membranes supporting cross-/inked enzymes. Biochim. Biophys. Aeta 185:260. (2) Cohn, M., and J. Monod. 1951. Purification and properties of the fl-galactosidase (lactase) of E. coil. Biochim. Biophys. Acta 7:153. (3) Craven, G. R., E. Steers, Jr., and C. B. Anflnsen. 1965. Purification, composition, and Molecular weight of the fl-galactosidase of E. cull K-12. J. Biol. Chem. 240:2468. (4) Ferrier, L. K., T. Richardson, N. F. Olson, and C. L. Hicks. 1972. Characteristics of insoluble pepsin used in a continuous milkclotting system, J. Dairy Sci. 55:726. (5) Goldstein, L. 1969, Use of water-insoluble enzyme derivatives in synthesis and sepJOURNAL OF DAIRY SCIENCE VOL, 56, NO, 9
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