Investigation of Catalytic Properties of Immobilized Enzymes and Cells by Flow Microcalorimetry - Advances in Biochemical Engineering Biotechnology - page 67

Investigation of Catalytic Properties of Immobilized
Enzymes and Cells by Flow Microcalorimetry

Vladimír ˇ tefuca 1 · Peter Gemeiner 2

1 Department of Chemical and Biochemical Engineering, Slovak University of Technology,
Radlinského 9,SK-812 37 Bratislava,Slovak Republic. E-mail: [e-mail]
2 Institute of Chemistry,Slovak Academy of Sciences,Dúbravská cesta 9,SK-842 38 Bratislava,
Slovak Republic. E-mail: [e-mail]

The investigation of catalytic properties of immobilized biocatalysts (IMB) is a time-con-
suming and not-always-simple procedure, requiring a simple and accurate method of enzy-
me-activity measurement. In comparison with generally-used techniques, flow microcalori-
metry (FMC) has proven to be a very practical and versatile technique for direct monitoring
of the course of enzyme reactions.The principal advantage of FMC is integration of the enzy-
me reaction and its monitoring in one step. This review summarizes the information needed
for the complete kinetic or catalytic characterization of the IMB by FMC,without the require-
ment of any independent analytical method. The optimal experimental procedure is pro-
posed.Examples of experimental studies on immobilized biocatalysts using the FMC are pro-
vided.The method is applicable to purified enzymes as well as to enzymes fixed in cells.

Keywords: Flow microcalorimetry, Immobilized biocatalysts, Reaction rate monitoring, In-
vestigation of kinetic properties,Characterization of bioaffinity systems.

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

2 Equipment and Procedure . . . . . . . . . . . . . . . . . . . . . . 72

3 Heat and Material Balance . . . . . . . . . . . . . . . . . . . . . . 73

3.1 Mass Balance in Immobilized Enzyme Particle . . . . . . . . . . 75

3.2 Low Substrate Conversion in the Column . . . . . . . . . . . . . . 76

4 Transformation of Thermometric Data to Reaction Rates . . . . 77

5Applications . . . . . . . . . . . . . . . . . . . . . 79

5.1 Investigation ofEnzyme Kinetics . . . . . . . . . . . . . . . . . . 79
5.1.1 Conversion of Thermometric Data . . . . . . . . . . . . . . . . . 80
5.1.2 Reaction Systems Without Limitation by Particle Diffusion . . . 82
5.1.2.1 Low Enzyme Activity . . . . . . . . . . . . . . . . . . . . . . . . . 83
5.1.2.2 High Enzyme Activity . . . . . . . . . . . . . . . . . . . . . . . . 84
5.1.3

of the Method

Enzyme Systems Influenced by Particle Diffusion Limitation . . . 85

5.2 pH-Activity Profiles . . . . . . . . . . . . . . . . . . . . . . . . . . 89

5.3 Biocatalyst Stability . . . . . . . . . . . . . . . . . . . . . . . . . . 89

5.4 Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

5.5 Screening of Immobilized Biocatalysts . . . . . . . . . . . . . . . 92

5.6 Monitoring of Enzymes Immobilized on Affinity Sorbents . . . . 92