Description of Instrumentation and Procedures - Advances in Biochemical Engineering Biotechnology - page 25

response as it restored half the oxygen consumed in the GOX reaction. It also
eliminated the H 2 O 2 formed in the GOX reaction. Linearity up to 0.7-1.0 mM
could be achieved by this technique. The limit of detection was about 1 M.

Glucose in blood plasma and serum were determined by this method [3]. The
use of this system has also been extended to the detection of glucose in hydroly-
sates of cellobiose, lactose and maltose. This approach was advantageous, as low
enthalpy changes made it difficult to monitor the hydrolysis directly due to low
enthalpy changes [44-46].
Employing L -ascorbate oxidase, vitamin C (ascrobic acid) was determined in
food samples between 0.05-0.6 mM [44]. In order to measure ethanol in bever-
ages and blood samples, alcohol oxidase from Candida boidinii had been
employed. Linearity was obtained between 0.01 and 1 mM. These measurements
were also found to be useful in monitoring fermentation [47, 48].
L -lactate and oxalate were also tested with lactate-2-monoxygenase and oxalate
decarboxylase and excellent results were obtained. CPG columns were employed
in both instances. Good linearity was obtained between 0.005-1 mM for L -lac-
tate [3] and between 0.1-3 mM for oxalate [24]. Similarly, urea was measured
with a precision better than 1% in the linearity range 0.01-200 mM using Jack
bean urease. The reaction of urea with ethanol to produce ethylcarbamate is of
interest in fermentation monitoring.
Lipids such as triglycerides were determined with lipoprotein lipase and
phospholipids with phospholipase D. In the case of triglycerides, good linearity
between 0.05-10 mM (tributyrin) and 0.1-5 mM (triolein) was obtained, whe-
reas for phospholipids linearity was obtained between 0.03-0.19 mM [41].

m

3.4.3
Environmental

Two different concepts were employed for this purpose: substance-specific ana-
lysis using enzymes (substrate or inhibition) and more general measurements
applying whole cells.ET was successfully applied [49] to the monitoring of heavy
metal (Hg 2+ ,Cu +2 and Ag + ) toxicity in the environment by measuring the in-
hibition of urease activity down to ppb levels of the metal ions. Restoration of
activity was also tested upon chelation of the metal ions with strong chelating
agents. In the recent past, a study of Cu(II) determination was carried out using
acid urease [50]. In addition, Satoh et al. [51, 52] described flow injection micro-
determinations using enzyme thermistors with different immobilized enzymes
for the detection of heavy metal ions. The heavy metal ions were detected due to
their reactivating effect on apoenzymes.
In another configuration [53] two different approaches for pesticide analysis
were employed. A crude enzyme solution capable of hydrolyzing organophos-
phate insecticides was prepared. The enzyme was coupled to controlled-pore
glass with glutaraldehyde. The insecticides, e.g., parathion, cyanophos, and dia-
zinon, were dissolved in a perfusion buffer (Tris pH 8.9, 1% Triton X-100) and
injected as a 10 min pulse into an ET in split-flow mode. The instrument mea-
sured the heat output due to insecticide hydrolysis and consecutive buffer pro-
tonization. For parathion, the detection limit was approximately 10 ppm.