Investigation of Catalytic Properties of Immobilized Enzymes and Cells by Flow Microcalorimetry - Advances in Biochemical Engineering Biotechnology - page 76

independent analytical technique still complicates the measurement and makes
it more time consuming.In addition,in many reactions substrate conversion in
the FMC column is too low to be measured with sufficient precision by conven-
tional analytical techniques. Any enhancement of the substrate conversion by
decreasing the volumetric flow rate increases external mass transfer limitations
and heat losses.This complicates the mathematical description necessary for the
estimation of kinetic parameters.Therefore,it was proposed that the activity of
the immobilized biocatalyst can be determined more conveniently by carrying
out an additional reaction rate measurement outside the FMC column [28].
The disadvantages of the previous techniques provided the motivation to
develop another approach: an autocalibration technique.This method does not
require either an independent analytical method for calibration, post-column
analysis,or separate rate determination.
The novel feature of the method is the modification of experimental setup
to apply total recirculation of the reaction mixture, as depicted in Fig. 1. By
switching the valve from waste to the circulation loop, the system is closed and
the steady state - cannot be reached, because of a continuous consumption of
substrate in the enzyme reaction.In order to simplify the mathematical descrip-
tion,the following experimental assumptions were made:
1. perfect mixing in the stirred reservoir,
2. sufficiently high volumetric flow rate in the system in relation to the reaction
rate in the column so that the concentration difference between the reservoir
and the column can be neglected.
Conditions (1) and (2) imply that, from the point of view of the mathematical
description, the circulating system will behave as a stirred batch reactor. Then,
the initial rate of reaction,v 0 ,in the reactor is

dc S V C

6

v 0 = = v obs (22)

62

dt t=0 V T e

where V C and V T are the volume of the packed bed in the column and the total
volume of the circulation system, respectively. According to condition (2), the
substrate concentration measured in the stirred reservoir,c S ,corresponds to the
concentration in the column.At the beginning of the experiment, FMC is oper-
ated in the open flow mode.After achieving the steady state (the signal remains
stable), at time = 0 min, the column output is shunted into the stirred reservoir
and the thermometric signal is registered continuously.An example of the time-
variation of the measured signal is depicted in Fig. 4B.
Considering the differential packed bed and the slow dynamics of the reac-
tion system, a pseudo-steady-state in the column can be assumed for a short
period of time, in spite of the non-steady-state in the whole system. This
assumption is valid when the total volume of liquid in the circulation system is
sufficiently large compared to the volume of the column and the flow rate is so
high that in the time interval equal to the column residence time the signal
decrease is less than 1%.This means that each thermometric signal corresponds
to one value of the substrate concentration, the concentration that would