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Kinetic parameters of immobilized urease
0038-0717/92$5.00+ 0.00 Copyright G 1992 Pergamon Press plc
Soil Bid. Biochcm. Vol. 24, No. 3, pp. 225-228, 1992
Printed in Great Britain. AISrights reserved
KINETIC PARAMETERS C. M.
LAI*
OF IMM~~I~IZE~
and M, A.
UREASE
TABATABAI~
Department of Agronomy, Iowa State University, Ames, IA 50011, U.S.A.
Summary-Jackbean urease was immobilized on two clay minerals (kaolinite and montmorillonite) and the mineral constituents of tsvo Iowa surface soils after s~lanizat~on of the support surfaces by 3-am~nopropyltriethoxysiiane and derivitat~on with glutaraldehyde. Results showed that the amounts of urease immobilized decreased in the order: montmorillonite > kaolinite > Tama soil r Lester soil. The amounts of urease required for maximum immobilization were 70, 90, 30 and 70mg g-’ of kaolin&e, montmorillonite, Tama soil and Lester soil, respectively. The optimal pH value of immobilized urease, except that on montmorillonite (8.5), was similar to that of free unease (7.0) and it was lower than those of native soil urease. The Km values of immobilized urease (2.5.14tX8 mM) were in the same order of magnitude as that of free urease (29.4 mM) but one order of magnitude higher than those of soil urease (1.77-2.90 mM). Optimal temperature of immobilized urease (60°C) was the same as that for free urease but was lower than those of soil urease (70°C). Ea and AH, values of urease immobilized on kaotinite and on soil mineral constituents were similar to those of the free enzyme but were lower than those of native soil urease. Immobilization of urease on clay surfaces lead to increases in the kinetic constants.
but Albert and Harter (1973) reported that adsorption of lysozyme and ~valbumin by Na-clay minerals Soil as a system of humus and minerals contains both resulted in an increase in Na’ concentration of the immobilized enzymes, stabilized by a three-dimenclay-protein suspensions; they interpreted this as evisional network of micromolecules, and occluded dence of a cation-exchange adsorption mechanism. microbial cells. Several theories have been proposed More recently, adsorption of urease on hexadeto explain the protective influence of soil on extracyltrimethyl-ammonium-smectite by hydrophobic cellular enzymatic activity. Ladd and Butler (197.5) bonding has been demonstrated (Boyd and Mortsuggested that the enzyme partion of a humusland, 198.5). enzyme complexes to soil humus by hydrogen, ionic Stabilization of enzymes in the soil environment by or covalent bonding. The extent that enzymes are soil organic matter rather than by soil inorganic bound by each of these mechanisms is difficult to determine. Studies by Simonart et al. (1967) components has also been suggested. Much of the information dealing with this hypothesis has been suggested that hydrogen bonding is a minor factor in obtained by studies involving synthetic polymerenzyme stabiIization in soils. By using phenol as a enzyme complexes (Ladd and Butler, 1975). Studies hydrogen-bond-breaking solvent, they were able to by Sarkar and Burns (1983, 1984) showed that desorb only a small amount of proteinaceous mafi-D-glucosidase can be fixed on phenolic copolymers terial. The dominant mechanisms of enzyme immobilization and stabilization have been summarized by containing L-tyrosine, pyrogallol and resorcinol The enzyme activity of the copofymers showed various Weetall (1975). These include microencapsulation, degrees of resistance to proteolysis, organic solvents cross-linking, copolymer formation, adsorption, enand storage at high temperatures. The immobilized trapment, ion exchange, adsorption and cross-linking enzyme showed increased K,, values and decreased and covalent attachment. Perhaps all these mechanV,,, values in comparison with soluble f(-u-glucosiisms are involved in stabilization of enzymes in soils. Early work by Ensminger and Gieseking (1942) dase. Other studies have shown that the stability of this and other enzymes (lactase, acid phosphatase provided evidence that protein adsorbed on montmoand tyrosinase) can be improved by adsorption on rillonite was stabilized against microbial attack. Since clay and soils (Leonowicz er al., 1988; Sarkar et al., then, the adsorption of enzyme proteins by clay 1989). minerals has been reported by several workers Information on kinetic parameters of immobilized (McLaren, 1954; Armstrong and Chesters, 1964; enzymes is needed because it should be possible to use Burns, 1986). The exact mechanisms by which clay enzyme technology in degradation of xenobiotics in minerals bind proteins has not yet been determined, groundwater and in the subsurface soil environment. Because of the importance of urease in soils and because of the potential of using immobilized en*Present address: Department of Agricultural Chemistry, zymes for in situ remediation, in this work, we used National Taiwan University, Taipei, Taiwan. tAuthor for correspondence. urease as a model enzyme and studied the changes in
225
226
C. M. LAI and M. A. TABATABAI
the kinetic parameters of this enzyme when immobilized on clays and on soil mineral constituents and compared these parameters with those of free and native soil urease.
MATERIALS
AND METHODS
e
3
The jackbean Sigma Chemical
urease used was obtained Co., St Louis, MO.
from
1 t
01 6.0
,._y--=-~
m-m-m-m 1
6.5
’
7.0
7.5
3
8.0
j
8.5
9.0
’
’
9.5 10.0
pH of buffer
Supports
The supports used included kaolinite (< 150 pm, pH 6.5, CEC 3.2 meq 100 g-l, Peerless No. 2 Kaolin from South Carolina), montmorillonite (< 150 pm, pH9.8, CEC 70.3 meq lOOg_‘, Volclay Wyoming bentonite from American Colloid Co.), Tama surface soil (< 180 pm, pH 5.4, Organic C 2.2%, clay 23%, sand 5%) and Lester surface soil (< 180 pm, pH 6.6, Organic C 3.4%, clay 16%, sand 33%). U-ease
immobilization
The urease was immobilized on clays and soil minerals as described by Robinson et al. (1971) and Sarkar et al. (1989). The procedures involved boiling the support in cont. HNO,, centrifuging at 14,000g and washing the residue free of acidity. The support was then activated by mixing with 2% solution of 3-aminopropyl-triethoxysilane in acetone. The activated supports were then treated with 5% glutaraldehyde prepared in 100 mM phosphate buffer (pH 7.0). After the removal of excess glutaraldehyde, the support was treated with urease solution at 4°C for 24 h, centrifuged and washed several times to remove the free urease. The reactions involved are reported by Weetall (1988). Urease
assay
Urease activity was assayed by determining the amount of ammonium-N released in the presence of Tris(hydroxymethyl)aminomethane (THAM) buffer (Tabatabai and Bremner, 1972).
ii-
I
6 t
01I
d
0
10
I I
30 Ureose added
50 (mg g-’
70
80
90
100
of support)
Fig. I. Immobilization of urease activity on clays and soil minerals as affected by the amount of enzyme applied. Urease activity is expressed in pg of ammonium N released gg’ of support h-’ at pH 7.0 and 37°C. Urease immobilized on kaolinite (O), on montmorillonite (a), on Tama soil (A), and on Lester soil (A).
Fig. 2. Effect of buffer pH on free, immobilized and soil urease activity. Urease activity is expressed in pg of ammonium N released 5~g~’ of urease, 1 mgg’ of support containing immobilized urease, or 5 gg’ of soil h-‘. Urease immobilized on kaolinite (0) on montmorillonite (a), on Tama soil (a) and on Lester soil (A). Free enzyme (0). Native urease in Tama soil (w) and in Lester soil (V).
RESULTS AND DISCUSSION
Immobilization of urease on the supports studied was affected by the amount of enzyme added (Fig. 1). Optimal urease immobilization was obtained at 50 mg of enzyme protein g-’ support. Increasing the amount of urease above this concentration did not markedly increase its immobilization on the supports. At urease concentration ~50 mg gg’ support, the amounts of urease activity immobilized varied with the four types of supports used. The buffer pH significantly affected the activity of free, immobilized and native urease (Fig. 2). Unlike native soil urease activity, which showed optimum activity at THAM buffer pH 9.0, the optimal activity of free urease was at pH 7.0. A similar pH optimum was reported by Boyd and Mortland (1985) for free and immobilized urease on hexadecyltrimethyll ammonium-smectite. Although the optimal pH of urease activity immobilized on the four supports varied somewhat, the urease activity immobilized on the same support at pH 7.0 and those at slightly lower or higher pH values were similar. Thus, comparison of the kinetic parameters of free, immobilized and native urease was done at their optimal pH values. The results obtained in studies of the effect of urea concentration on urease activity expressed in Lineweaver-Burk plots are shown in Fig. 3. In general, the data fitted the Michaelis-Menten equation; the lines shown in Fig. 3 are those obtained by linear regression equations. The K,,, value of free urease (29.4m~) was lower than those of urease immobilized on kaolinite, montmorillonite and Lester soil mineral constituents (Table 1). The K, values of native urease in Tama and Lester soils were much lower than those of free and immobilized urease. Immobilization of urease on clay and soil mineral constituents significantly altered its K, value. The difference between the K,,, values of native soil urease and those of free and immobilized urease is not due to the source of the enzyme. We used jackbean urease
Kinetic
parameters
of immobilized
227
urease
1 0 -0.2
0.0
0.2
0.4
0
0.8
10
20
30
40
50
60
70
80
Incubation temperature PC)
l/S
Fig. 3. Lineweaver-Burk plots for free, immobilized and soil urease activity. The substrate (S) is in mM, velocity (V) is expressed in pg of ammonium N released 0.05 mg-’ of jack bean urease (PH 7), 10 mgg’ of support containing immobilized urease (PH 7), or 1 g-’ of soil @H 9) h-’ at 37°C. Urease immobilized on kaolinite (O), on montmorillonite (a), on Tama soil (A) and on Lester soil (A). Free enzyme (0). Native urease in Tama soil (m) and in lester soil (V).
Fig. 4. Effect of incubation temperature on free, immobilized and soil urease activity. Urease activity is expressed in pg of ammonium N released 5 pg-’ of jack bean urease (pH 7), 1 mg-’ of support containing immobilized urease (pH 7), 5 g-l of Tama soil or 1.25 g-’ of Lester soil (pH 9) h-l. Urease immobilized on kaolinite (O), on montmorillonite (a), on Tama soil (A) and on Lester soil (A). Free enzyme (0). Native urease in Tama soil (W) and in Lester soil (V).
and, although plant materials added to soils contain urease activity (Frankenberger and Tabatabai, 1982), it is likely that soil urease is mainly derived from microbial sources. From the Km values obtained, it is clear that soil urease has greater affinity for urea than the immobilized urease. The P’,,,,, values of urease immobilized on the two clay minerals were in the same order of magnitude. Although the V,,,,, value of urease immobilized on Lester soil mineral constituents was somewhat greater than that immobilized on Tama soil, both values were within the same order of magnitude. Temperature of inactivation of native soil urease and that of free and immobilized urease occurred at 60°C (Fig. 4). Previous work showed that plant urease was inactivated at 70°C (Frankenberger and Tabatabai, 1982). The temperature dependence of the rate constant at a temperature below inactivation can be described by the Arrhenius equation:
intercept and slope, respectively, of the linear plot of the dependence of log k on l/T. The Arrhenius plot for the activities of native soil urease and for free and immobilized urease were linear from 20 to 60°C. The slopes of the lines and, thus, the energies of activation of the three states of urease were similar. The energies of activation of the reaction catalyzed by urease, expressed in kJ mol-‘, ranged from 13.5 for urease immobilized on montmorillonite to 39.1 for native urease in Lester soil (Table 2). The activation energy values obtained in this study are within the range reported for this enzyme in plants (41-51 kJ mol-‘) (Frankenberger and Tabatabai, 1982; Lynn and Yankwich, 1962). Jespersen (1975) reported that determination of urease activation energies by using THAM and phosphate buffer differs significantly and that, with THAM buffer, the energy of activation increased with increasing urea concentration, whereas the phosphate buffer tends to decrease the energy of activation of urease. Therefore, activation energies should be compared only under similar conditions such as optimal pH, choice of buffer, substrate concentration and temperature of incubation. All these conditions were observed in this work. Transition-state theory uses thermodynamic concepts to describe chemical- and enzyme-catalyzed reactions. A large enthalpy of activation (AH,) indicates that a large amount of stretching, squeezing or even breaking of chemical bonds is necessary for the
k = A exp( - Ea/RT) where A is the pre-exponential factor, Ea is the energy of activation, R is the gas constant, and T is the temperature in “K. The Arrhenius equation also can be expressed in the log form: log k = (- Ea/2.30RT) where
log A and
+ log A,
Ea can be determined
from
the
Table I. K, and V,,, values of free, immobilized and soil urease calculated from the Lineweaver-Burk plots of the Michaelis-Menten equation Urease Jack bean Immobilized Immobilized Immobilized Immobilized Tama soil Lester soil
on on on on
kaolinite montmorillonite Tama soil Lester soil
Km* 29.4 60.8 45.0 25. I 40.8 I .77 2.90
vlll,,t 7.62 x IO1 1.24 x IO4 I.13 x I04 5.32 x IO’ 8.86 x IO’ 1.60 x IO 8.56 x IO
*K,,, is expressed in rn~. tvtn,, is expressed in fig of ammonium N released 0.05 mg-’ ofjack bean urease, IO mg-’ of support containing immobilized urease, or I g’ of soil h-’ at 37°C.
Table 2. Energy of activation (E,) and enthalpy of activation (AH,) for free, immobilized and soil urease
Urease Jack bean Immobilized Immobilized Immobilized Immobilized Tama soil Lester soil
on on on on
kaolinite montmorillonite Tama soil Lester soil
AHa
(kJ mol-‘)
E,
(kJ mol-‘) at 25°C
19.1 25.8 13.5 24.1 25.2 38.9 39. I
16.7 23.3 II.0 21.6 22.8 36.4 36.6
228
C. M. LAI and M. A. TABATABAI
formation of transition state 1979). AH, is always less than following relation:
(Cornish-Bowden, Ea because of the
AH, = Ea - Rt. The results obtained for AH, are always
Acknowledgement-The Ministry of Education,
senior author was supported by the Taipei, Taiwan, Republic of China.
REFERENCES Albert J. T. and Harter R. D. (1973) Adsorption of lysozyme and ovalbumin by clay: effect of clay-suspension pH and clay mineral type. Soil Science 115, 130-136. Armstrong D. E. and Chesters G. (1964) Properties of protein-bentonite complexes as influenced by equilibrium conditions. Soil Science 98, 39-52. Boyd S. A. and Mortland M. M. (1985) Urease activity on a clay+rganic complex. Soil Science Society of America Journal 49, 617-622. Burns R. G. (1986) Interaction of enzymes with soil minerals and organic colloids. In Interactions of Soil Minerals with Natural Organics and Microbes (P. M. Huang and M. Schnitzer, Eds), pp. 4299451. Soil Science Society of America Special Publication Number 17, Madison. Cornish-Bowden A. (1979) Fundamentals of Enzyme Kinetics. Butterworths, London. Ensminger L. E. and Gieseking J. E. (1942) Resistance of clay-adsorbed proteins to proteolytic hydrolysis. Soil Science 53, 2055209.
Frankenberger W. T. Jr and Tabatabai M. A. (1982) Amidase and urease activities in plants. Plant and Soil 64, 153-166. Jespersen N. D. (1975) A thermochemical study of the hydrolysis of urea by urease. Journal of American Chemical Society 97, 1662-1667. Ladd J. N. and Butler J. H. A. (1975) Humus-enzyme systems and synthetic, organic polymer-enzyme analogs. In Soil Biochemistry (E. P. Paul and A. D. McLaren, Eds), Vol. 4, pp. 143-194. Dekker, New York. Leonowicz A., Sarker J. M. and Bollag J.-M. (1988) Improvement in stability of an immobilized fungal lactase. Applied Microbiology and Biotechnology 29, 1299135. Lynn K. R. and Yankwich P. E. (1962) “C kinetic isotope effects in the urease-catalyzed hydrolysis of urea. I. Temperature dependence. Biochimica et Biophysics Acta 56, 512-530. McLaren A. D. (1954) The adsorption and reaction of enzymes and proteins on kaolinite. II. The action of chymotrypsin on lysozyme. Soil Science Society of America Proceedings 18, 170-I 74. Robinson P. J., Dunnill P. and Lilly M. D. (1971) Porous glass as a solid support for the immobilization or affinity chromatography of enzymes. Biochimica et Biophysics Acta 242, 6599661. Sarker J. M. and Burns R. G. (1983) Immobilized of D-ghicosidase and o-glucosidase-polyphenolic complexes. Biotechnology Letters 5, 619-624. Sarker J. M. and Burns R. G. (1984) Synthesis and properties of o-glucosidase-phenohc copolymers as analogues of soil humic-enzyme complexes. Soil Biology & Biochemistry 16, 619-625. Sarker J. M., Leonowicz A. and Bollag J.-M. (1989) Immobilization of enzymes on clays and soils. Soil Biology & Biochemistry 21, 2233230. Simonart P., Batistic L. and Mayaudon J. (1967) Isolation of protein from humic acid extracted from soil. PIant and Soil 27, 1533161. Tabatabai M. A. and Bremner J. M. (1972) Assay of urease activity in soils. Soil Biology & Biochemistry 4, 4799487. Weetall H. H. (1975) Immobilized enzymes and their application in the food and beverage industry. Process Biochemistry 10, 3-24. Weetall H. H. (1988) Enzymes immobilized on inorganic supports. In Analytical Uses of Immobilized Biological Compounds for Detection, Medical and Industrial Uses (G. G. Guilbault and M. Mascini. Eds), pp. I -15.Reidel. Boston. Wells K. L. and Riecken F. F. (1969) Regional distribution of potassium in the B horizon clay of some prairie loess soils of the midwest. Soil Science Society of America Proceedings 33, 582-587.
Soil Bid. Biochcm. Vol. 24, No. 3, pp. 225-228, 1992
Printed in Great Britain. AISrights reserved
KINETIC PARAMETERS C. M.
LAI*
OF IMM~~I~IZE~
and M, A.
UREASE
TABATABAI~
Department of Agronomy, Iowa State University, Ames, IA 50011, U.S.A.
Summary-Jackbean urease was immobilized on two clay minerals (kaolinite and montmorillonite) and the mineral constituents of tsvo Iowa surface soils after s~lanizat~on of the support surfaces by 3-am~nopropyltriethoxysiiane and derivitat~on with glutaraldehyde. Results showed that the amounts of urease immobilized decreased in the order: montmorillonite > kaolinite > Tama soil r Lester soil. The amounts of urease required for maximum immobilization were 70, 90, 30 and 70mg g-’ of kaolin&e, montmorillonite, Tama soil and Lester soil, respectively. The optimal pH value of immobilized urease, except that on montmorillonite (8.5), was similar to that of free unease (7.0) and it was lower than those of native soil urease. The Km values of immobilized urease (2.5.14tX8 mM) were in the same order of magnitude as that of free urease (29.4 mM) but one order of magnitude higher than those of soil urease (1.77-2.90 mM). Optimal temperature of immobilized urease (60°C) was the same as that for free urease but was lower than those of soil urease (70°C). Ea and AH, values of urease immobilized on kaotinite and on soil mineral constituents were similar to those of the free enzyme but were lower than those of native soil urease. Immobilization of urease on clay surfaces lead to increases in the kinetic constants.
but Albert and Harter (1973) reported that adsorption of lysozyme and ~valbumin by Na-clay minerals Soil as a system of humus and minerals contains both resulted in an increase in Na’ concentration of the immobilized enzymes, stabilized by a three-dimenclay-protein suspensions; they interpreted this as evisional network of micromolecules, and occluded dence of a cation-exchange adsorption mechanism. microbial cells. Several theories have been proposed More recently, adsorption of urease on hexadeto explain the protective influence of soil on extracyltrimethyl-ammonium-smectite by hydrophobic cellular enzymatic activity. Ladd and Butler (197.5) bonding has been demonstrated (Boyd and Mortsuggested that the enzyme partion of a humusland, 198.5). enzyme complexes to soil humus by hydrogen, ionic Stabilization of enzymes in the soil environment by or covalent bonding. The extent that enzymes are soil organic matter rather than by soil inorganic bound by each of these mechanisms is difficult to determine. Studies by Simonart et al. (1967) components has also been suggested. Much of the information dealing with this hypothesis has been suggested that hydrogen bonding is a minor factor in obtained by studies involving synthetic polymerenzyme stabiIization in soils. By using phenol as a enzyme complexes (Ladd and Butler, 1975). Studies hydrogen-bond-breaking solvent, they were able to by Sarkar and Burns (1983, 1984) showed that desorb only a small amount of proteinaceous mafi-D-glucosidase can be fixed on phenolic copolymers terial. The dominant mechanisms of enzyme immobilization and stabilization have been summarized by containing L-tyrosine, pyrogallol and resorcinol The enzyme activity of the copofymers showed various Weetall (1975). These include microencapsulation, degrees of resistance to proteolysis, organic solvents cross-linking, copolymer formation, adsorption, enand storage at high temperatures. The immobilized trapment, ion exchange, adsorption and cross-linking enzyme showed increased K,, values and decreased and covalent attachment. Perhaps all these mechanV,,, values in comparison with soluble f(-u-glucosiisms are involved in stabilization of enzymes in soils. Early work by Ensminger and Gieseking (1942) dase. Other studies have shown that the stability of this and other enzymes (lactase, acid phosphatase provided evidence that protein adsorbed on montmoand tyrosinase) can be improved by adsorption on rillonite was stabilized against microbial attack. Since clay and soils (Leonowicz er al., 1988; Sarkar et al., then, the adsorption of enzyme proteins by clay 1989). minerals has been reported by several workers Information on kinetic parameters of immobilized (McLaren, 1954; Armstrong and Chesters, 1964; enzymes is needed because it should be possible to use Burns, 1986). The exact mechanisms by which clay enzyme technology in degradation of xenobiotics in minerals bind proteins has not yet been determined, groundwater and in the subsurface soil environment. Because of the importance of urease in soils and because of the potential of using immobilized en*Present address: Department of Agricultural Chemistry, zymes for in situ remediation, in this work, we used National Taiwan University, Taipei, Taiwan. tAuthor for correspondence. urease as a model enzyme and studied the changes in
225
226
C. M. LAI and M. A. TABATABAI
the kinetic parameters of this enzyme when immobilized on clays and on soil mineral constituents and compared these parameters with those of free and native soil urease.
MATERIALS
AND METHODS
e
3
The jackbean Sigma Chemical
urease used was obtained Co., St Louis, MO.
from
1 t
01 6.0
,._y--=-~
m-m-m-m 1
6.5
’
7.0
7.5
3
8.0
j
8.5
9.0
’
’
9.5 10.0
pH of buffer
Supports
The supports used included kaolinite (< 150 pm, pH 6.5, CEC 3.2 meq 100 g-l, Peerless No. 2 Kaolin from South Carolina), montmorillonite (< 150 pm, pH9.8, CEC 70.3 meq lOOg_‘, Volclay Wyoming bentonite from American Colloid Co.), Tama surface soil (< 180 pm, pH 5.4, Organic C 2.2%, clay 23%, sand 5%) and Lester surface soil (< 180 pm, pH 6.6, Organic C 3.4%, clay 16%, sand 33%). U-ease
immobilization
The urease was immobilized on clays and soil minerals as described by Robinson et al. (1971) and Sarkar et al. (1989). The procedures involved boiling the support in cont. HNO,, centrifuging at 14,000g and washing the residue free of acidity. The support was then activated by mixing with 2% solution of 3-aminopropyl-triethoxysilane in acetone. The activated supports were then treated with 5% glutaraldehyde prepared in 100 mM phosphate buffer (pH 7.0). After the removal of excess glutaraldehyde, the support was treated with urease solution at 4°C for 24 h, centrifuged and washed several times to remove the free urease. The reactions involved are reported by Weetall (1988). Urease
assay
Urease activity was assayed by determining the amount of ammonium-N released in the presence of Tris(hydroxymethyl)aminomethane (THAM) buffer (Tabatabai and Bremner, 1972).
ii-
I
6 t
01I
d
0
10
I I
30 Ureose added
50 (mg g-’
70
80
90
100
of support)
Fig. I. Immobilization of urease activity on clays and soil minerals as affected by the amount of enzyme applied. Urease activity is expressed in pg of ammonium N released gg’ of support h-’ at pH 7.0 and 37°C. Urease immobilized on kaolinite (O), on montmorillonite (a), on Tama soil (A), and on Lester soil (A).
Fig. 2. Effect of buffer pH on free, immobilized and soil urease activity. Urease activity is expressed in pg of ammonium N released 5~g~’ of urease, 1 mgg’ of support containing immobilized urease, or 5 gg’ of soil h-‘. Urease immobilized on kaolinite (0) on montmorillonite (a), on Tama soil (a) and on Lester soil (A). Free enzyme (0). Native urease in Tama soil (w) and in Lester soil (V).
RESULTS AND DISCUSSION
Immobilization of urease on the supports studied was affected by the amount of enzyme added (Fig. 1). Optimal urease immobilization was obtained at 50 mg of enzyme protein g-’ support. Increasing the amount of urease above this concentration did not markedly increase its immobilization on the supports. At urease concentration ~50 mg gg’ support, the amounts of urease activity immobilized varied with the four types of supports used. The buffer pH significantly affected the activity of free, immobilized and native urease (Fig. 2). Unlike native soil urease activity, which showed optimum activity at THAM buffer pH 9.0, the optimal activity of free urease was at pH 7.0. A similar pH optimum was reported by Boyd and Mortland (1985) for free and immobilized urease on hexadecyltrimethyll ammonium-smectite. Although the optimal pH of urease activity immobilized on the four supports varied somewhat, the urease activity immobilized on the same support at pH 7.0 and those at slightly lower or higher pH values were similar. Thus, comparison of the kinetic parameters of free, immobilized and native urease was done at their optimal pH values. The results obtained in studies of the effect of urea concentration on urease activity expressed in Lineweaver-Burk plots are shown in Fig. 3. In general, the data fitted the Michaelis-Menten equation; the lines shown in Fig. 3 are those obtained by linear regression equations. The K,,, value of free urease (29.4m~) was lower than those of urease immobilized on kaolinite, montmorillonite and Lester soil mineral constituents (Table 1). The K, values of native urease in Tama and Lester soils were much lower than those of free and immobilized urease. Immobilization of urease on clay and soil mineral constituents significantly altered its K, value. The difference between the K,,, values of native soil urease and those of free and immobilized urease is not due to the source of the enzyme. We used jackbean urease
Kinetic
parameters
of immobilized
227
urease
1 0 -0.2
0.0
0.2
0.4
0
0.8
10
20
30
40
50
60
70
80
Incubation temperature PC)
l/S
Fig. 3. Lineweaver-Burk plots for free, immobilized and soil urease activity. The substrate (S) is in mM, velocity (V) is expressed in pg of ammonium N released 0.05 mg-’ of jack bean urease (PH 7), 10 mgg’ of support containing immobilized urease (PH 7), or 1 g-’ of soil @H 9) h-’ at 37°C. Urease immobilized on kaolinite (O), on montmorillonite (a), on Tama soil (A) and on Lester soil (A). Free enzyme (0). Native urease in Tama soil (m) and in lester soil (V).
Fig. 4. Effect of incubation temperature on free, immobilized and soil urease activity. Urease activity is expressed in pg of ammonium N released 5 pg-’ of jack bean urease (pH 7), 1 mg-’ of support containing immobilized urease (pH 7), 5 g-l of Tama soil or 1.25 g-’ of Lester soil (pH 9) h-l. Urease immobilized on kaolinite (O), on montmorillonite (a), on Tama soil (A) and on Lester soil (A). Free enzyme (0). Native urease in Tama soil (W) and in Lester soil (V).
and, although plant materials added to soils contain urease activity (Frankenberger and Tabatabai, 1982), it is likely that soil urease is mainly derived from microbial sources. From the Km values obtained, it is clear that soil urease has greater affinity for urea than the immobilized urease. The P’,,,,, values of urease immobilized on the two clay minerals were in the same order of magnitude. Although the V,,,,, value of urease immobilized on Lester soil mineral constituents was somewhat greater than that immobilized on Tama soil, both values were within the same order of magnitude. Temperature of inactivation of native soil urease and that of free and immobilized urease occurred at 60°C (Fig. 4). Previous work showed that plant urease was inactivated at 70°C (Frankenberger and Tabatabai, 1982). The temperature dependence of the rate constant at a temperature below inactivation can be described by the Arrhenius equation:
intercept and slope, respectively, of the linear plot of the dependence of log k on l/T. The Arrhenius plot for the activities of native soil urease and for free and immobilized urease were linear from 20 to 60°C. The slopes of the lines and, thus, the energies of activation of the three states of urease were similar. The energies of activation of the reaction catalyzed by urease, expressed in kJ mol-‘, ranged from 13.5 for urease immobilized on montmorillonite to 39.1 for native urease in Lester soil (Table 2). The activation energy values obtained in this study are within the range reported for this enzyme in plants (41-51 kJ mol-‘) (Frankenberger and Tabatabai, 1982; Lynn and Yankwich, 1962). Jespersen (1975) reported that determination of urease activation energies by using THAM and phosphate buffer differs significantly and that, with THAM buffer, the energy of activation increased with increasing urea concentration, whereas the phosphate buffer tends to decrease the energy of activation of urease. Therefore, activation energies should be compared only under similar conditions such as optimal pH, choice of buffer, substrate concentration and temperature of incubation. All these conditions were observed in this work. Transition-state theory uses thermodynamic concepts to describe chemical- and enzyme-catalyzed reactions. A large enthalpy of activation (AH,) indicates that a large amount of stretching, squeezing or even breaking of chemical bonds is necessary for the
k = A exp( - Ea/RT) where A is the pre-exponential factor, Ea is the energy of activation, R is the gas constant, and T is the temperature in “K. The Arrhenius equation also can be expressed in the log form: log k = (- Ea/2.30RT) where
log A and
+ log A,
Ea can be determined
from
the
Table I. K, and V,,, values of free, immobilized and soil urease calculated from the Lineweaver-Burk plots of the Michaelis-Menten equation Urease Jack bean Immobilized Immobilized Immobilized Immobilized Tama soil Lester soil
on on on on
kaolinite montmorillonite Tama soil Lester soil
Km* 29.4 60.8 45.0 25. I 40.8 I .77 2.90
vlll,,t 7.62 x IO1 1.24 x IO4 I.13 x I04 5.32 x IO’ 8.86 x IO’ 1.60 x IO 8.56 x IO
*K,,, is expressed in rn~. tvtn,, is expressed in fig of ammonium N released 0.05 mg-’ ofjack bean urease, IO mg-’ of support containing immobilized urease, or I g’ of soil h-’ at 37°C.
Table 2. Energy of activation (E,) and enthalpy of activation (AH,) for free, immobilized and soil urease
Urease Jack bean Immobilized Immobilized Immobilized Immobilized Tama soil Lester soil
on on on on
kaolinite montmorillonite Tama soil Lester soil
AHa
(kJ mol-‘)
E,
(kJ mol-‘) at 25°C
19.1 25.8 13.5 24.1 25.2 38.9 39. I
16.7 23.3 II.0 21.6 22.8 36.4 36.6
228
C. M. LAI and M. A. TABATABAI
formation of transition state 1979). AH, is always less than following relation:
(Cornish-Bowden, Ea because of the
AH, = Ea - Rt. The results obtained for AH, are always
Acknowledgement-The Ministry of Education,
senior author was supported by the Taipei, Taiwan, Republic of China.
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