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Monolithic Bioreactors for Macromolecules 263
Table 1
Effect of Temperature, Immobilization Time and pH on Deoxyribonuclease
(DNase) Immobilization.
Temperature
(°C)
Time
(h)
pH
value
Immobilization
method
Biological
activity
(dA 260 nm /
min)
Specific
Amount biological
of enzyme activity
(mg DNase/g (dA 260 nm /
support) min/mg)
37 3 5 Static 0.1 4.3 0.15
37 24 5 Static 1.9 9.4 1.26
37 3 7 Static 0.1 1.9 0.33
22 24 7 Static 1.2 3.4 2.21
22 0.5 7 Dynamic 0.9 2.9 1.94
22 2 7 Dynamic 1.2 3.5 1.96
37 24 7 Static 1.32 5.0 1.65
37 24 9 Static 0 5.6 0
DNase (2 g/l) in 50 mM Tris, pH 7 or 9, or 50 mM acetate buffer, pH 5, containing
1 mM CaCl 2 was immobilized on a CIM epoxy disk, median pore size 6 μm. The amount
of immobilized enzyme was determined as described in Subheading 3.2.2. Biological activity
was determined as described in Subheading 3.3.2. DNA concentration was 0.02 g/l in 40
mM Tris buffer, pH 8, 1 mM MgCl 2 , 1 mM CaCl 2 and detection wavelength 260 nm.
Specific biological activity is expressed as DNase activity per mg of DNase. Adapted from ref (9).
respectively, 0.28 g of DNA/1 and 16 dA 260 nm /min/mg of immobilized DNase
(see Fig. 3) (see Note 4). The long-term stability of enzyme reactor was determined
by measuring biological activity (see Subheading 3.2.3) immediately
after immobilization and repeatedly for up to 1 month.
The immobilization procedure to obtain the highest biological activity of the
immobilized enzyme and measurement of biological activity are presented below.
3.3.1. Immobilization Procedure
1. Prepare DNase solution by dissolving enzyme (2 g/l) in 50 mM acetate buffer,
pH 5, containing 1 mM CaCl 2 .
2. Apply static immobilization procedure described in Subheading 3.1.1 for 24 h
at 37ºC.
3. After immobilization is completed, wash the enzyme reactor first with 40 mM
Tris-HCl buffer, pH 8, containing 1 mM MgCl 2 , 1 mM CaCl 2 , 0.1 M NaCl,
followed by 40 mM Tris-HCl buffer, pH 8, containing 1 mM MgCl 2 , 1 mM CaCl 2
buffer.
4. Immobilized CIM disk should be stored in immobilization buffer to better preserve
biological activity (see Table 2).
5. Determine quantity of immobilized enzyme as described in Subheading 3.2.2 if
specific biological activity is of interest.