Enzymatic microreactors in chemical analysis and kinetic studies

Table 2 (continued)
Enzyme Medium Application Refs.
Horseradish peroxidase, Microbeads Measurement of enzyme kinetics Seong et al., 2003
h-galactosidase
(fluorescence imaging)
Lipase Glass beads, alginate gel
Hydrolysis of triacetin,
Pijanowska et al., 2001
beads, nitrocellulose sheets tributyrin and triolein
Lipase SiO2-coated microcapillary Hydrolysis of umbelliferone acetate Nakamura et al., 2004
Lipases, proteases,
glucose oxidase,
horseradish peroxidase
Sol–gel arrays Screening of proteins (enzymes) Park and Clark, 2002
Peroxidase, glucose oxidase Silicon wafer Continuous glucose measurements Laurell and Rosengren, 1994
PikC hydroxylase Ni-NTA agarose beads Rapid hydroxylation of macrolides Srinivasan et al., 2004
Protease Silica monolith Transesterification (glycidol, n-butyrate) Kawakami et al., 2005
Trehalase Aminopropyl glass particles Quantification of trehalose Bachinski et al., 1997
Urease Polydimethylsiloxane High urea conversion in continuous flow Jones et al., 2004
B-Fructosidase Porous silicon Determination of sucrose Lendl et al., 1997
offers a great advantage by shortening the analysis time.
In batch reactions, completion of enzyme-catalysed
transesterification may take days for some supported
lipases (Kamal et al., 2002).
On account of reproducible distribution of the products
formed along the axes of the microreactor, the
enzymatic process can be visualized by fluorescence
microscopy in order to acquire data on the concentration
patterns inside the device. Such an approach was presented
by Seong et al. (2003), and fluorescence images
of the reaction zone together with scaled numerical
results from the cross-sections of input and output
streams are shown in Fig. 4. The method provided
high sensitivity for product detection and short response
time, and kinetic graphs of the reaction catalyzed by the
enzyme (horseradish peroxidase) were obtained. The
Lilly–Hornby model was used to characterize the kinet-
ics of biocatalysis in the packed microcolumn, and
results were compared with those for kinetics of the
enzyme in homogeneous solution. The Michaelis constants
were found to be similar to those obtained from
the Lineweaver–Burke model for the homogeneous catalysis.
In comparison with standard assays, the amount
of enzyme used was very small: Seong et al. (2003)
estimated that 200 pmol (3 10 9 molecules of enzyme)
were required for the analysis. The current trend in
biochemical analysis is to decrease the amount of biocatalyst
used. Recently, Moore et al. (2004) presented an
assay for 500 lipase molecules, capable of application to
single cells. Rondelez et al. (2005) described an assay
for monitoring reaction catalyzed by a single molecule
of h-galactosidase and horseradish peroxidase.
The kinetics model described by Lilly et al. (1966) is
appropriate for systems with continuous flow of the
Fig. 3. Schematic for continuous-flow reaction and monitoring of hydrolysis of esters using microreactor packed with lipase immobilized onto either
nitrocellulose sheets or glass beads coated with keratin. Reprinted from Pijanowska et al. (2001) with permission from Elsevier.