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270 Benčina et al.
Table 6
Long-Term Stability of Trypsin Enzyme Reactor Stored in Water at 4°C
Days
Biological
activity
(mAU/min)
% initial activity
epoxy-CIM
1 7111 100
99 7101 100
190 6972 98
220 8114 114
687 6615 93
CDI-CIM
1 9994 100
99 10960 100
190 9388 94
220 10676 107
687 10540 105
Biological activity was determined as described in Subheading 3.5.2. Digestion of Nbenzoyal-arginine
ethyl ester at concentration of 3×10 −4 M in 20 mM Tris-HCl buffer pH 8 at
wavelength of 254 nm is monitored.
5. Immobilized CIM disk should be stored at 4°C in distilled water to preserve
biological activity.
6. Determine quantity of immobilized enzyme as described in Subheading 3.2.2 if
specific biological activity is of interest.
3.5.2. Biological Activity: Trypsin
The biological activity of the immobilized trypsin is determined by on-line
frontal analysis using low molecular substrates BAEE (7,8).
1. Prepare 20 mM Tris-HCl buffer, pH 8 (buffer A).
2. Prepare the substrate solution of BAEE at concentration of 3 × 10 −4 Min
buffer A.
3. Connect trypsin enzyme reactor to the HPLC system.
4. Set the wavelength on HPLC detector at 254 nm for monitoring substrate
conversion.
5. Equilibrate enzyme reactor by washing with at least 10 column volumes of
buffer A.
6. Set to zero HPLC detector to compensate background absorbance of buffer A.