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Monolithic Bioreactors for Macromolecules 267
Table 4
Long-Term Stability of Ribonuclease Enzyme Reactor Stored in 20% Ethanol
at 4°C.
Days
Biological
activity
(dA 288 nm /min)
% initial activity
epoxy-CIM
0 75 100
7 57 76
28 58 77
CDI-CIM
0 349 100
14 342 98
42 343 98
Biological activity was determined as described in Subheading 3.4.2. Cytidin-2,3-cyclic
monophosphate concentration was at 0.57 mM in 10 mM Tris, pH 7.5, 2 mM EDTA, 0.1 M
NaCl buffer and detection wavelength 288 nm.
3.4.2. Biological Activity-RNase
The biological activity of immobilized RNase (11) was determined by online
frontal analysis using cytidine-2,3-cyclic monophosphate as substrate. The
initial velocity was calculated as the slope of linear increase in absorbance at
288 nm ( 288 nm = 1308/M/cm) of cytidine-2,3-cyclic monophosphate at low
residence time.
1. Prepare 10 mM Tris–HCl, pH 7.5, 2 mM EDTA, 0.1 M NaCl buffer (buffer A).
2. Prepare substrate cytidine-2,3-cyclic monophosphate at concentration of
0.39–0.68 mM in buffer A.
3. Connect RNase enzyme reactor in to the HPLC system.
4. Set the wavelength on HPLC detector at 288 nm for monitoring the substrate
conversion.
5. Equilibrate enzyme reactor by washing it with at least 10 column volumes of
buffer A.
6. Set to zero HPLC detector to compensate background absorbance of buffer A.
7. Pump different substrate solutions through the enzyme reactor and change the
residence time by altering the flow rate in the range of 0.1–10 ml/min at 25°C.
When the substrate solution at a certain concentration is pumped through the
enzyme reactor at fixed flow rate, immobilized RNase has been hydrolyzing
cytidine-2,3-cyclic monophosphate which results in an increase of the absorbance
at the column outlet.