Investigation of Catalytic Properties of Immobilized Enzymes and Cells by Flow Microcalorimetry - Advances in Biochemical Engineering Biotechnology - page 69

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Introduction
The immobilized biocatalyst (IMB) is a key component of biotransformation
systems that are used to transform substrates to desired products.The improve-
ment of biocatalyst properties has a direct influence on the overall effectiveness
of the process based on the biotransformation. The basic catalytic characteri-
stics of biocatalyst that are followed include kinetic properties, pH optima,
stability,and inhibition.The investigation of catalytic properties of immobilized
enzymes is still a time consuming procedure and is not always simple. In the
1980s,a major effort was made to standardize the rules by which IMB is charac-
terized. The Working Party of EFB on immobilized biocatalysts has formulat-
ed principles of individual methods, among them the requirement of kinetic
characterization [1].It was recommended to use a packed-bed reactor,equipped
with temperature control and with infinite flow circulation. The system should
be equipped with a post-column unit to measure the time-dependence of the
product or substrate concentration [2, 3], the most commonly used analytical
methods being spectrophotometry, chemiluminiscence, automatic titration,
bioluminiscence, chromatography, polarimetry, and biosensors based on the
oxygen electrode. There are two main drawbacks to the application of these
methods:
1. The need to vary the analytical principles, depending on the chemical and
physical-chemical properties of analytes;
2. In some cases, mainly in the study of hydrolytic enzymes, the natural sub-
strate must be replaced by an artificial one,that is chromolytic,chromogenic,
chemiluminiscent,bioluminiscent,or fluorescent.
Therefore, in the same period, there was great interest in the development of a
standard measurement technique, that would join the advantages of immobi-
lized enzymes with a universal detection principle,that lead to a variety of devi-
ces for flow enthalpimetry [4].The first such effort considered the measurement
of glucose concentration using immobilized glucose oxidase [5].An important
improvement of this measurement system was the integration of thermistors
with immobilized enzyme columns [6] and further technical modifications
[7-13].The research on enthalpimetric methods was motivated by the potential
use of enthalpimeters in bioanalytical chemistry [14]. It was impelled by the
need to replace complicated and expensive commercial equipment by a system,
that would be simpler, cheaper, and have a shorter response time [15]. These
requirements were best fulfilled by the concept of an “enzyme thermistor”,
developed by Danielsson [16].The basic design and further development of this
equipment was aimed at providing a system of flow-injection analysis of the
concentration of metabolites, inhibitors [17-19], lipids [20], antigens [21], and
of on-line monitoring of fermentation processes and biotransformations [22].
In addition to the analytical applications, there was sporadic work on the
employment of flow calorimetry for the investigation of enzyme kinetics
[23, 24]. In 1985 Owusu et al. [25] published the first report on the use of flow
microcalorimetry for the study of immobilized enzyme kinetics approaching