Investigation of Catalytic Properties of Immobilized Enzymes and Cells by Flow Microcalorimetry - Advances in Biochemical Engineering Biotechnology - page 93

These results indicate that flow microcalorimetry may be considered to be a
method for rapid evaluation of the amount and accessibility of immobilized
Con A [35].
The proposed method of IME screening assisted by the FMC has significant-
ly simplified research directed towards increasing the amount of Con A and
glycoenzyme associated with solid supports.A simple strategy that has recently
been developed by the authors involves building up layers of Con A and
glycoenzymes on bead cellulose [31, 47] and poly(glycidyl methacrylate)
(Eupergit C) [47] matrices precoupled with Con A.Bioaffinity-layered prepara-
tions of invertase exhibited - layer by layer - up to a tenfold increase in cataly-
tic activity,as measured by FMC [31,47]. A somewhat similar approach was later
used by Farooqui et al. [48] in the preparation of superactive immobilized
glucose oxidase, improving the sensitivity of glucose oxidase-based glucose
monitoring FET-biosensor devices. It is anticipated that the technique will be
applicable to all of the glycoenzymes investigated, namely glucose oxidase,
invertase, -galactosidase and amyloglucosidase [42] for both FMC and FET

biosensors. Tailor-made neoglycoenzymes [49], exhibiting a similar effect of
bioaffinity layering ,should be considered for FMC signal amplification,as well.

b

6
General Strategy for FMC Experiments

Current knowledge, summarized in this review, is sufficient to define a general
strategy for the study of immobilized biocatalyst properties by flow microcalori-
metry.The principal simplification of the investigation of the kinetics of immobi-
lized biocatalysts consists in using only a small amount of the IMB and working
with low substrate conversions. The FMC column can then be considered to be a
differential reactor and the mathematical description is reduced to the system of
algebraic instead of differential equations.Another important improvement is the
developed procedure of“autocalibration”of the FMC,leading to the value of para-
meter ,enabling the determination of reaction rates from thermometric data.In

this way,the thermometric data are converted to more conventional,mass-balance
data, which can be treated by the mathematical tools that are generally used for
calculating the rates of reactions catalyzed by immobilized biocatalysts.
The optimal organization of the kinetic FMC experiment is proposed in the
flowchart depicted in Fig. 14. The procedure can be divided into the following
principal points:

a

1. Experiments in the flow-through mode,in which the FMC operates in steady-
state mode.Here,first of all,the appropriate amount of IMB in the column to
be used is determined.Then,the steady state kinetic data - the dependence of
the thermometric signal on substrate concentration - is measured;
2. Experiments in the total recirculation mode, in which the FMC operates as
a batch system in non-steady-state. The dependence of the thermometric
signal on time for a chosen substrate concentration is measured;
3. The mathematical treatment of the data depends on whether the reaction is
diffusion limited or not.This part of work depends on the aim of the study.If