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266 Benčina et al.
Table 3
Effect of Buffer pH on Ribonuclease (RNase) Immobilization via Epoxy or
Carboxydiimidazole Groups
pH of
immobilization
buffer
RNase
biological
activity
(dA 288 nm /min)
Amount of
enzyme (mg
RNase/ disk)
Specific
biological
activity (dA 288 nm /
min/mg)
Epoxy
5 14 0.6 24
7 25 1.1 23
9 44 0.7 65
11 75 0.9 83
13 8 0.7 11
CDI
7 341 1.3 262
9 349 0.7 499
11 16 0.9 17
RNase (2g/l) in 50 mM Tris buffer, pH 7 and 9, or 50 mM acetate buffer, pH 5, or 50 mM
sodium carbonate buffer, pH 11, or 50 mM KCl NaOH buffer, pH 13, was immobilized on a
Connective Interaction Media (CIM) epoxy or CIM CDI 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 3.4.2. Cytidine-2,3-cyclic monophosphate concentration
was at 0.57 mM in 10 mM Tris-HCl pH 7.5, 2 mM EDTA, 0.1 M NaCl buffer and
detection wavelength 288 nm. Specific activity was expressed as RNase activity per mg of RNase.
Michaelis–Menten constant, K m , and turnover number, k 3 , for immobilized
RNase are 0.52 mM and 4.6 per second (see Fig. 4). The stability of RNase
enzyme reactor was determined as described in Subheading 3.2.3.
The immobilization procedure to obtain the highest biological activity and
measurement of biological activity are presented below.
3.4.1. Immobilization Procedure
1. Prepare RNase solution by dissolving enzyme (2 g/l) in 50 mM Tris buffer pH 9
(immobilization buffer).
2. Apply dynamic immobilization procedure described in Subheading 3.1.2 for2h
at room temperature.
3. After immobilization is completed, wash the enzyme reactor first with immobilization
buffer containing 0.1 M NaCl and with immobilization buffer afterwards.
4. Immobilized CIM disk should be stored in 20% ethanol solution to preserve
biological activity (see Table 4).
5. Determine quantity of immobilized enzyme as described in Subheading 3.2.3 if
specific biological activity is of interest.