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262 Benčina et al.
[A]
abs orbance
[S 1 ] > [S 2 ]; [E]
Φ 1 > Φ 2 > Φ 3 > Φ 4
[S 2 ]
A 8 ; Φ 4
A 7 ; Φ 3
[S 1 ]
5 1
A 1 ; Φ 1 A;Φ 2 2 A 3 ; Φ 3 A 4 ; Φ 4
[B]
abs orbance
A 2
A 1
dA/dt dA/dt
[S 2 ]
A 5
A 7
A 8
[S 1 ]
Φ 1 Φ 2 Φ 3 Φ 4
A 6 A 3
A 4
Flow rate ml/min
t 1 t 2 t 3 t 4
residence time (s)
Fig. 2. Schematic presentation of results obtained by on-line frontal analysis of
biological activity. (A) Absorbance of substrate passed through an enzyme reactor at
different flow rates. (B) Absorbance of substrate at calculated residence time. [S 1 ]
and [S 2 ], concentration of substrate; [E], amount of enzyme immobilized to CIM disk;
1−4 , flow rates; A 1−8 , absorbance calculated as difference between absorbance of
substrate passed through enzyme reactor and of substrate passed through CIM disk
monolithic column of the same chemistry (epoxy or CDI) but without enzyme.
3.2.2. Amount of Immobilized Enzyme Using BCA Kit
Quantity of enzyme immobilized on the CIM disk was determined from
a difference in enzyme concentration in the immobilization solution before
and after immobilization using BCA protein determination kit according to the
manufacturer’s instructions.
3.2.3. Stability of Enzyme Reactor
Stability of enzyme reactor was determined by monitoring biological activity
regularly for prolonged periods of time. For all measurements, experimental
conditions had to be identical.
3.3. DNase Immobilization
The static and dynamic immobilization methods were used to immobilize
DNase on CIM disk via epoxy groups (9). The efficiency of DNase immobilization
was determined by hydrolysis of DNA as substrate, as described in
Subheading 3.3.2. Different immobilization conditions like temperature, pH
and immobilization time were tested (conditions are described in Table 1).
Immobilized DNase activity is presented in Table 1 and long-term stability of
enzyme reactor in Table 2. The highest specific biological activity of immobilized
enzyme was detected for immobilization on epoxy groups at pH 7 and at
22°C (see Table 1). Immobilized CIM disk stored in buffer had better longterm
stability compared to the one stored in water (see Table 2). The apparent
(see Note 3) Michaelis–Menten constant, k app
m
, and turnover number, kapp 3 , were,