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Monolithic Bioreactors for Macromolecules 265
3.3.2. Biological Activity: DNase
The modified Kunitz hyperchromicity assay (10) was used to determine
DNase biological activity. The DNase activity is manifested as an increase in
absorbance at 260 nm.
1. Prepare 40 mM Tris–HCl buffer, pH 8, containing 1 mM MgCl 2 , 1 mM CaCl 2
(buffer A).
2. Prepare substrate of calf thymus DNA at concentration of 0.006–0.08 g/l in
buffer A.
3. Connect DNase enzyme reactor to the HPLC system.
4. Set the wavelength on HPLC detector at 260 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 of calf thymus DNA at 25ºC through the
enzyme reactor and change the residence time by altering the flow rate in the
range of 0.1–10 ml/min. When the substrate solution at a certain concentration
is pumped through the enzyme reactor at fixed flow rate, immobilized DNase
has been hydrolyzing DNA which results in an increase of the absorbance at the
column outlet.
8. Read the steady-state absorbance for each flow rate (see Subheading 3.2.1).
9. Draw a graph showing absorbance at 260 nm versus the residence time (see
Fig. 2A).
10. The DNase biological activity (v) is determined as the slope of the linear increase
in absorbance at low residence time, and specific biological activity is calculated
from biological activity divided by the amount of immobilized enzyme.
11. Changing the substrate concentration enables calculation of kinetics parameters
v max and K m using Michaelis–Menten equation (see Note 2) (see Fig. 3).
3.4. RNase Immobilization
The dynamic immobilization procedure (see Subheading 3.1.2.) was used
to immobilize RNase on CIM disk via epoxy and imidazole carbamate groups.
The efficiency of RNase immobilization was determined by hydrolysis of the
low molecular weight substrate cytidine-2,3-cyclic monophosphate as described
in Subheading 3.4.2. Immobilization was performed at different pH values of
immobilization buffer as indicated in Table 3 and described for optimal case
in Subheading 3.4.1.
Biological activity of immobilized RNase is presented in Table 3. RNase
immobilized on CDI-activated monolith at pH 9 was six-fold more active than
the one immobilized on epoxy-activated monolith (see Table 3). Furthermore,
there was almost no change in activity over 42 days (see Table 4). The