Description of Instrumentation and Procedures - Advances in Biochemical Engineering Biotechnology - page 27

[57]. The effects were most pronounced during the transformation of bac-
terial cells to spheroplasts. It was shown that the lower production rate of
molecular hydrogen by spheroplastic cells was due not only to a suggested
decrease in mixed-acid fermentation, but to a reduction in hydrogen lyase
activity as well.
The production of molecular hydrogen was measured in the effluent gas of
seven fermentations [58]. The aim of this primary investigation was to study the
use of a H 2 -sensitive metal-oxide-semiconductor structure in physiological
studies of Escherichia coli . In order to yield more information, the metabolic
heat was measured with a flow microcalorimeter in parallel with the determina-
tion of molecular hydrogen.

3.5
Miscellaneous Applications

The characterization of immobilized invertase was carried out, and the techni-
que was successfully coupled to the catalytic activity determination of immobil-
ized cells [59]. Similarly, the results of this technique were useful in the selection
of Trigonopsis variabilis strains for high cephalosporin-transforming activity
[60]. Also, the cephalosporin-transforming activity of D -amino acid oxidase
isolated from yeast was identified in a similar manner. The thermometric signal
was proportional to the number of cells as well as the amount of D -amino acid
oxidase immobilized in the ET microcolumn. The ET was also coupled to a
thermometric ELISA procedure (TELISA) for the determination of hormones,
antibodies and other biomolecules generated during the fermentation process
[61]. Genetically engineered enzyme conjugates, e.g. human proinsulin-alkaline
phosphatase conjugate,were used for the determination of insulin or proinsulin.
Alkaline phosphatase was used predominantly as the enzyme label for such an
assay [62]. In another instance TELISA was employed for monitoring insulin
separation [63]. The expense of the conjugate for such automated procedures
was found to be negligible compared to the higher costs of the non-automated
procedures. In addition these techniques are easily set-up in an industrial envi-
ronment and have already been tested in several instances, e.g. monitoring of
fermentation.
Enzyme thermistors have also found applications in more research-related
topics, such as the direct estimation of the intrinsic kinetics of immobilized bio-
catalysts [64]. Here, the enzyme thermistor offered a rapid and direct method
for the determination of kinetic constants (K i ,K m and V m ) for immobilized
enzymes. For the system being investigated, saccharose and immobilized inver-
tase, the results obtained with the enzyme thermistor and with an independent
differential reactor system were in very good correlation, within a flow-rate
range of 1 to 1.5 ml/min.
Determination of ADP and ATP by multiple enzymes in recycling systems:
pyruvate kinase and hexokinase co-immobilized on aminopropyl CPG, was
demonstrated by Kirstein et al. [25]. In addition, a second reactor,with L -lactate
dehydrogenase, lactate oxidase, and catalase, was used to increase the sensitivity
from 6

10 -5 M with no recycling, to 2 10 -6 M in the kinase bienzyme reactor,

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