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Characterization of molecular-sieve-bound inulinase
[J. Ferment. Technol., Vol. 65, No. 2, 239-242.
19871
Note
Characterization
of Molecular-Sieve-Bound
PRATIMA BAJPAI*
and
Inulinase
ARGYRIOS MARGARITIS
DepmtmGnt of Chemical and Biochemical Engineering, The University of Western Ontario, London, Ontario, Canada N6G 5B9
The enzyme inulinase (2,1-p-n-fructan
fructanohydrolase,
EC 3.2.1.7),
prepared
from Kluyveromycesmarxianus has been immobilized using an inorganic solid support, molecular sieve 4A via the metal link method. The immobilized enzyme had around 22 units of inulinase activity per g of the support with retention of 72% of the original activity.
The optimum protein to molecular sieve ratio for the maximum retention of
inulinase activity
was 9 mg/g molecular sieve. The properties of soluble and immobilized enzyme differed in many respects. The optimum pH of the enzyme shifted from 6 to 5 and the optimum temperature of enzyme activity changed from 50 to 55°C. Km values were 6.7 mM for soluble enzyme and 10 mM for immobilized enzyme. heat stability of the enzyme was improved by immobilization.
The
Immobilized enzyme
retained about 76% of the original activity after 40 days of storage at room temperature (30&2’C).
Both inorganic and organic materials have been used for the preparation of immobilized enzymes but inorganic supports are more suitable as they are not vulnerable to microbial attack and are available in a wide range of particle sizes and porosity. Porous glass has been used extensively for immobilizing many enzymes but it is expensive. Molecular sieves, alkali salts of alumino silicates, are manufactured commercially and are used to separate hydrocarbons. They are less expensive. Very little published information is available on inulinase immobilization.l-8) Bajpai and MargariW-6) have recently reported the immobilization of Kluyveromyces marxianus cells having inulinase activity. In this investigation partially purified inulinase was immobilized on a molecular sieve in order to hydrolyze inulin sugars present in Jerusalem artichoke tubers to fructose. Materials Materials
and Methods
Molecular
sieve was obtained from
* Correspondence at: Thapar Centre, Patiala 147001, India
Corporate
R
&
the Fisher Scientific Company,
U.S.A.
The material
was supplied in the form of beads (812 mesh). and inulin (from chicory root, moLwt.=5000)
TiCls were
obtained from the Sigma Chemical Company (U.S.A.). All other chemicals used were of analytical grade. Support activation The molecular sieve was treated with 5% nitric acid at 80°C for about an hour. It was washed repeatedly with distilled water and dried at 100°C for 24 h.
Titanous
chloride was oxidized
by the dropwise addition of 70% perchloric
acid or
30% HsOs till the solution became colourless. Ten grams of molecular sieve was suspended in 20 ml of titanous chloride (TiCls or oxidized form) and left at 40°C overnight. Excess titanium ions were removed washing with distilled water
and the
molecular sieve was dried. Enzyme preparation and immobilization
by repeated
An
inulinase of KruyuGromyus marxianus was partially purified by tan& acid precipitation to 14 units/mg proteins)
Activated
molecular
sieve was suspended
in inulinase solution and left overnight at 4°C. The immobilized enzyme was washed with distilled water, 0.5 M NaCl, and distilled water successively. The activity was measured by the sugars released from inulin by dinitrosalicylic acid.‘)
Results
and Discussion
D
Titanium
chloride
was
used
to activate
[J. Ferment. Technol.,
BAJPAIand MARGARITIS
240
It was observed that storing molecular sieve in a humid atmosphere over a long period resulted in the non-reactivity of the This molecular sieve for binding enzymes. may be due to the masking of groups on the surface of molecular sieve by water molecules, because reactivation is possible by drying at 105°C for 3 to 4 days. Molecular sieve activated with titanium chloride can be stored in a dry state at a room temperature for a month. The optimum pH for binding of enzyme to the molecular sieve was studied in the presence of citrate phosphate buffer and the maximum activity was observed at pH 3.0. The activity decreased rather sharply when the pH was increased to 6.0. However, with acetate buffer, the activity was found to be more or less constant in the range of pH 3.5 to 5.5. The maximum value was almost the same as that without buffer. Hence in all further experiments, buffer was not used during immobilization. Optimum molecular sieve enzyme Inulinase was immobilized with ratio the molecular sieve in the presence of varying initial protein concentrations to establish the optimum ratio (Table 2). The bound inulinase activity increased as the protein loading was increased. However, increasing the amount of protein beyond 9 mg did not cause any significant increase in the retention of inulinase activity. The percent activity
Table 1. Effects of pretreatment on the retention of inulinase on molecular sieve.
acid.
Umts of mulmase retained/g of molecular sieve
Treatment Untreated molecular sieve
5.2
Perchloric acid treated molecular sieve
10.8
Titanium trichloride treated molecular sieve
18.0
Titanium trichloride oxidized with HsOs
35. 1
Titanium trichloride oxidized with perchloric acid
60.2
One gram of molecular sieve was suspended in a total volume of 6 ml of enzyme containing a total of 8 mg of protein. After 24 h of contact, the support was washed with distilled water, 0.5 M NaCl, and diitilled water successively.
molecular sieves as described by Emery et al.8) As acidic conditions were reported to induce loss in the crystalline structure of molecular sieves,91 we tried perchloric acid The results presented in Table 1 treatment. clearly indicated that untreated molecular sieve and perchloric acid treated molecular sieve retained only a limited amount of protein. However, titanium chloride increased the retention of inulinase activity. Activation of molecular sieve with Tic18 and perchloric acid increased the activity J-fold as compared to molecular sieve treated with TiCls. Hence in all further experiments Tic18 was oxidized with perchloric Table 2. Protein added*
Measurement of optimum molecular .sieve/inulinase ratio. Protein** removed
W/g of molecular sieve)
Theoretical immobilized activity O-mWg molecular sieve) ~__~~ (A) ~.
Actsvny of immobilized enzyme Wits/g molecular sieve) (B) __
Retention (%) B/Ax
100
_.
~~_
2.8
2. 1
30
22.1
74.6
6.02
5.6
80
48. 1
60.5
9.2
8.5
120
59. 1
49. 1
13. 1
11.2
158
60.2
38. 1
18.4
14. 1
199
61. 2
30.8
* Specific activity of inulinase was 14.1 units/mg protein. ** Protein removed is calculated by taking the difference between protein added and protein present in the supematant.
Vol. 65, 19871 retained loading
was
Immobilized Inulinase dependent
on
the
protein
Effects of pH Soluble inulinase had an optimum pH around 6.0 while the maximum activity for immobilized inulinase was at pH 5.0. The shift of the pH optimum of immobilized inulinase to the acidic side was also noted when DEAE cellulose and tygon tubing
100
and varied from 74 to 30%.
were used as solid supports.112)
80 g z
5
60
I
::
40 : 5 g
20
0
Effects of temperature With the immobilized inulinase, the highest activity for inulin hydrolysis was obtained at 55°C while with the free inulinase, maximum activity occurred at 50°C. The activation energies for the free and immobilized inulinase were estimated to be 7.8 kcal/mol and 3.62 kcal/mol respectively, from the activity at 30 to 65°C. Kim et d.1) found that the optimum temperature of aminoethyl-celluloseimmobilized inulinase was lower than that for the soluble inulinase. Effects of substrate concentration Molecular-sieve-bound and soluble inulinases were assayed at various initial inulin concentrations (2 to 32 mm). The michaelis constants calculated from Lineweaver-Burk plots were 6.66 mM for free enzyme and 13.3 mM for the bound enzyme. Kim et aZ.1) have reported K, values for soluble inulinase from Kluyveromyces fragilis as 6.7 mM and immobilized inulinase on tygon tubing and aminoethyl cellulose as 10 mM and 7.7 mM, respectively. Thermal stability Soluble and bound enzymes were exposed to temperatures from 30 to 65°C at pH 6 in 0.1 M acetate buffer for various lengths of time to 140 mm. Although no inulinase activity of the soluble enzyme was lost at 30 and 40°C for 140 min, the activity decreased rapidly over 50°C. At 60 and 65”C, about 75 and 90% of the activities were lost in 30 min, respectively (Fig. 1A). The activity of immobilized enzyme after 140 min remained fairly stable up to 55°C but decreased beyond that (Fig. 1B). At 60°C about 30% activity was lost in 140 min and at 65°C about 70% activity was lost in 30 min. Thus immobilization brings about an increase in the thermal
Fig. 1. Thermal stability of free enzyme (A) and immobilized enzyme (B) after preincubation at different temperatures: (0) 80%; (A)40°C; (0) 50°C; (0) 55°C; (A) 60%; (0) 65°C. stability. Storage stability Storage stability of molecular-sieve-bound inulinase was measured at room temperature and the activity losses were found to be only 24% for 40 days. Operational stability Operational stability of molecular-sieve-bound inulinase was measured by passing 5% inulin solution at a flow rate of 105 ml/h for 25 days through a column where 43 g of molecular sieve was placed in a water jacketed glass column (42 x 28 cm). Reducing sugar formed were measured at regular interval. The half life was found to be about 30 days. References 1) Kim, W. Y., Byun, S. M., Nahm, B. H.: J. Food Sci. Technol., 11, 238 (1979). 2) Kim, W. Y., Byun, S. M., Uhm, T. B.: Enz. ML-rob. Technol., 4, 239 (1982). 3) Guiraud, J. P., Dcmeulle, S., Galzy, P.: technol. Z.&t., 3, 683 (1981). 4) Bajpai, P., Margaritis, A.:
Em
Bio-
Microb. Tcchnol.,
BAJPAIand MAFCGARITIS
242 7, 373 (1985). 5) Bajpai, P., Margaritis,
8) Emery, A. N., Hough, J. S., Novais, J. M., Lyons, A.:
Microbial., 31, 297 (1985). 6) Bajpai, P., Margaritis, A.: tation, 11, p. Rickmansworth, 7) Miller,
G. L.:
J.
Gen.
&
A@.
Advances in Fermen-
103, Turret Wheatland England (1985). Anal. Chem., 31, 426 (1959).
Ltd.,
Chem. Eng., 258, 71 (1982). T. P.: 9) Lee, M. N. Y.: Recent Developments in Separation Science, Vol. 1,p. 75, CRC Press, Cleaveland (1972). (Received August 13, 1986)
19871
Note
Characterization
of Molecular-Sieve-Bound
PRATIMA BAJPAI*
and
Inulinase
ARGYRIOS MARGARITIS
DepmtmGnt of Chemical and Biochemical Engineering, The University of Western Ontario, London, Ontario, Canada N6G 5B9
The enzyme inulinase (2,1-p-n-fructan
fructanohydrolase,
EC 3.2.1.7),
prepared
from Kluyveromycesmarxianus has been immobilized using an inorganic solid support, molecular sieve 4A via the metal link method. The immobilized enzyme had around 22 units of inulinase activity per g of the support with retention of 72% of the original activity.
The optimum protein to molecular sieve ratio for the maximum retention of
inulinase activity
was 9 mg/g molecular sieve. The properties of soluble and immobilized enzyme differed in many respects. The optimum pH of the enzyme shifted from 6 to 5 and the optimum temperature of enzyme activity changed from 50 to 55°C. Km values were 6.7 mM for soluble enzyme and 10 mM for immobilized enzyme. heat stability of the enzyme was improved by immobilization.
The
Immobilized enzyme
retained about 76% of the original activity after 40 days of storage at room temperature (30&2’C).
Both inorganic and organic materials have been used for the preparation of immobilized enzymes but inorganic supports are more suitable as they are not vulnerable to microbial attack and are available in a wide range of particle sizes and porosity. Porous glass has been used extensively for immobilizing many enzymes but it is expensive. Molecular sieves, alkali salts of alumino silicates, are manufactured commercially and are used to separate hydrocarbons. They are less expensive. Very little published information is available on inulinase immobilization.l-8) Bajpai and MargariW-6) have recently reported the immobilization of Kluyveromyces marxianus cells having inulinase activity. In this investigation partially purified inulinase was immobilized on a molecular sieve in order to hydrolyze inulin sugars present in Jerusalem artichoke tubers to fructose. Materials Materials
and Methods
Molecular
sieve was obtained from
* Correspondence at: Thapar Centre, Patiala 147001, India
Corporate
R
&
the Fisher Scientific Company,
U.S.A.
The material
was supplied in the form of beads (812 mesh). and inulin (from chicory root, moLwt.=5000)
TiCls were
obtained from the Sigma Chemical Company (U.S.A.). All other chemicals used were of analytical grade. Support activation The molecular sieve was treated with 5% nitric acid at 80°C for about an hour. It was washed repeatedly with distilled water and dried at 100°C for 24 h.
Titanous
chloride was oxidized
by the dropwise addition of 70% perchloric
acid or
30% HsOs till the solution became colourless. Ten grams of molecular sieve was suspended in 20 ml of titanous chloride (TiCls or oxidized form) and left at 40°C overnight. Excess titanium ions were removed washing with distilled water
and the
molecular sieve was dried. Enzyme preparation and immobilization
by repeated
An
inulinase of KruyuGromyus marxianus was partially purified by tan& acid precipitation to 14 units/mg proteins)
Activated
molecular
sieve was suspended
in inulinase solution and left overnight at 4°C. The immobilized enzyme was washed with distilled water, 0.5 M NaCl, and distilled water successively. The activity was measured by the sugars released from inulin by dinitrosalicylic acid.‘)
Results
and Discussion
D
Titanium
chloride
was
used
to activate
[J. Ferment. Technol.,
BAJPAIand MARGARITIS
240
It was observed that storing molecular sieve in a humid atmosphere over a long period resulted in the non-reactivity of the This molecular sieve for binding enzymes. may be due to the masking of groups on the surface of molecular sieve by water molecules, because reactivation is possible by drying at 105°C for 3 to 4 days. Molecular sieve activated with titanium chloride can be stored in a dry state at a room temperature for a month. The optimum pH for binding of enzyme to the molecular sieve was studied in the presence of citrate phosphate buffer and the maximum activity was observed at pH 3.0. The activity decreased rather sharply when the pH was increased to 6.0. However, with acetate buffer, the activity was found to be more or less constant in the range of pH 3.5 to 5.5. The maximum value was almost the same as that without buffer. Hence in all further experiments, buffer was not used during immobilization. Optimum molecular sieve enzyme Inulinase was immobilized with ratio the molecular sieve in the presence of varying initial protein concentrations to establish the optimum ratio (Table 2). The bound inulinase activity increased as the protein loading was increased. However, increasing the amount of protein beyond 9 mg did not cause any significant increase in the retention of inulinase activity. The percent activity
Table 1. Effects of pretreatment on the retention of inulinase on molecular sieve.
acid.
Umts of mulmase retained/g of molecular sieve
Treatment Untreated molecular sieve
5.2
Perchloric acid treated molecular sieve
10.8
Titanium trichloride treated molecular sieve
18.0
Titanium trichloride oxidized with HsOs
35. 1
Titanium trichloride oxidized with perchloric acid
60.2
One gram of molecular sieve was suspended in a total volume of 6 ml of enzyme containing a total of 8 mg of protein. After 24 h of contact, the support was washed with distilled water, 0.5 M NaCl, and diitilled water successively.
molecular sieves as described by Emery et al.8) As acidic conditions were reported to induce loss in the crystalline structure of molecular sieves,91 we tried perchloric acid The results presented in Table 1 treatment. clearly indicated that untreated molecular sieve and perchloric acid treated molecular sieve retained only a limited amount of protein. However, titanium chloride increased the retention of inulinase activity. Activation of molecular sieve with Tic18 and perchloric acid increased the activity J-fold as compared to molecular sieve treated with TiCls. Hence in all further experiments Tic18 was oxidized with perchloric Table 2. Protein added*
Measurement of optimum molecular .sieve/inulinase ratio. Protein** removed
W/g of molecular sieve)
Theoretical immobilized activity O-mWg molecular sieve) ~__~~ (A) ~.
Actsvny of immobilized enzyme Wits/g molecular sieve) (B) __
Retention (%) B/Ax
100
_.
~~_
2.8
2. 1
30
22.1
74.6
6.02
5.6
80
48. 1
60.5
9.2
8.5
120
59. 1
49. 1
13. 1
11.2
158
60.2
38. 1
18.4
14. 1
199
61. 2
30.8
* Specific activity of inulinase was 14.1 units/mg protein. ** Protein removed is calculated by taking the difference between protein added and protein present in the supematant.
Vol. 65, 19871 retained loading
was
Immobilized Inulinase dependent
on
the
protein
Effects of pH Soluble inulinase had an optimum pH around 6.0 while the maximum activity for immobilized inulinase was at pH 5.0. The shift of the pH optimum of immobilized inulinase to the acidic side was also noted when DEAE cellulose and tygon tubing
100
and varied from 74 to 30%.
were used as solid supports.112)
80 g z
5
60
I
::
40 : 5 g
20
0
Effects of temperature With the immobilized inulinase, the highest activity for inulin hydrolysis was obtained at 55°C while with the free inulinase, maximum activity occurred at 50°C. The activation energies for the free and immobilized inulinase were estimated to be 7.8 kcal/mol and 3.62 kcal/mol respectively, from the activity at 30 to 65°C. Kim et d.1) found that the optimum temperature of aminoethyl-celluloseimmobilized inulinase was lower than that for the soluble inulinase. Effects of substrate concentration Molecular-sieve-bound and soluble inulinases were assayed at various initial inulin concentrations (2 to 32 mm). The michaelis constants calculated from Lineweaver-Burk plots were 6.66 mM for free enzyme and 13.3 mM for the bound enzyme. Kim et aZ.1) have reported K, values for soluble inulinase from Kluyveromyces fragilis as 6.7 mM and immobilized inulinase on tygon tubing and aminoethyl cellulose as 10 mM and 7.7 mM, respectively. Thermal stability Soluble and bound enzymes were exposed to temperatures from 30 to 65°C at pH 6 in 0.1 M acetate buffer for various lengths of time to 140 mm. Although no inulinase activity of the soluble enzyme was lost at 30 and 40°C for 140 min, the activity decreased rapidly over 50°C. At 60 and 65”C, about 75 and 90% of the activities were lost in 30 min, respectively (Fig. 1A). The activity of immobilized enzyme after 140 min remained fairly stable up to 55°C but decreased beyond that (Fig. 1B). At 60°C about 30% activity was lost in 140 min and at 65°C about 70% activity was lost in 30 min. Thus immobilization brings about an increase in the thermal
Fig. 1. Thermal stability of free enzyme (A) and immobilized enzyme (B) after preincubation at different temperatures: (0) 80%; (A)40°C; (0) 50°C; (0) 55°C; (A) 60%; (0) 65°C. stability. Storage stability Storage stability of molecular-sieve-bound inulinase was measured at room temperature and the activity losses were found to be only 24% for 40 days. Operational stability Operational stability of molecular-sieve-bound inulinase was measured by passing 5% inulin solution at a flow rate of 105 ml/h for 25 days through a column where 43 g of molecular sieve was placed in a water jacketed glass column (42 x 28 cm). Reducing sugar formed were measured at regular interval. The half life was found to be about 30 days. References 1) Kim, W. Y., Byun, S. M., Nahm, B. H.: J. Food Sci. Technol., 11, 238 (1979). 2) Kim, W. Y., Byun, S. M., Uhm, T. B.: Enz. ML-rob. Technol., 4, 239 (1982). 3) Guiraud, J. P., Dcmeulle, S., Galzy, P.: technol. Z.&t., 3, 683 (1981). 4) Bajpai, P., Margaritis, A.:
Em
Bio-
Microb. Tcchnol.,
BAJPAIand MAFCGARITIS
242 7, 373 (1985). 5) Bajpai, P., Margaritis,
8) Emery, A. N., Hough, J. S., Novais, J. M., Lyons, A.:
Microbial., 31, 297 (1985). 6) Bajpai, P., Margaritis, A.: tation, 11, p. Rickmansworth, 7) Miller,
G. L.:
J.
Gen.
&
A@.
Advances in Fermen-
103, Turret Wheatland England (1985). Anal. Chem., 31, 426 (1959).
Ltd.,
Chem. Eng., 258, 71 (1982). T. P.: 9) Lee, M. N. Y.: Recent Developments in Separation Science, Vol. 1,p. 75, CRC Press, Cleaveland (1972). (Received August 13, 1986)