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Monolithic Bioreactors for Macromolecules 269
Table 5
Effect of Immobilization Procedure on Biological Activity of Trypsin Enzyme
Reactor
Static method
Immobilization time c (min)
Trypsin a Trypsin b 5 30 60 120 1440
Monolith (mAU/min) (mAU/min) (mAU/min)
Epoxy 1005 7066 115 436 1937 1981 5883
CDI 11530 11222 8430 5312 6329 7987 8369
a Immobilization performed without benzamidine hydrochloride.
b Immobilization performed with benzamidine hydrochloride.
c Immobilization of trypsin under dynamic conditions.
Trypsin (2 g/l) in 0.1 M borate buffer pH 8 with or without 50 mM benzamidine
hydrochloride was immobilized on CIM epoxy or CDI CIM disk, median pore size 1.5 μm.
Biological activity was determined as described in Subheading 3.5.2. Hydrolysis of N-benzoyl-
L-arginig ethyl ester at concentration of 3 × 10 −4 M in 20 mM Tris-HCl buffer pH 8 at wavelength
of 254 nm was monitored. Adapted from ref (7).
The efficiency of trypsin immobilization could be determined by hydrolysis of
high molecular weight substrates (12) or low molecular weight substrates, for
example, BAEE (7,8), as described in Subheading 3.5.2.
Biological activity of immobilized trypsin at different conditions is presented
in Table 5. Immobilization efficiency via imidazole carbamate groups is 10
times higher then those obtained for epoxy groups if the immobilization
procedure was performed without addition of benzamidine hydrochloride
(see Table 5). Dynamic immobilization method was completed in 120 min
while static immobilization method lasted 24 h (see Table 5). Furthermore,
biological activity of trypsin enzyme reactor was not changed over 2 years
(see Table 6).
The immobilization procedure to obtain the highest biological activity and
measurement of biological activity are presented below.
3.5.1. Immobilization Procedure
1. Prepare 0.1 M borate buffer, pH 8, containing 50 mM benzamidine hydrochloride
(immobilization buffer).
2. Prepare trypsin solution 2 g/l by dissolving the trypsin in immobilization buffer.
3. Apply dynamic immobilization procedure described in Subheading 3.1.2 at room
temperature for 5 min.
4. After immobilization is completed, the residual protein is removed by washing
the enzyme reactor with 10 column volumes of immobilization buffer and finally
with deionized water.