Abstract
In this work an enzyme electrode was constructed for
amperometric determination of xylose and glucose. The
electrode was based on the pyrroloquinoline quinone
(PQQ)-dependent membrane-bound aldose dehydrogenase
(ALDH) from Gluconobacter oxydans. PQQ-dependent enzymes
are particularly suitable for amperometric detection of
analytes, since they are oxygen insensitive and the
redox-cofactor PQQ remains tightly bound to the enzyme.
In order to construct the biosensor based on ALDH, a
simple, large-scale applicable purification procedure for
the enzyme was developed consisting of extraction of the
crude cell homogenate and its clarification with cell
debris remover combined with chromatographic
purification. The activity recovery from membrane extract
of Gluconobacter oxydans was 33%, with 130-fold
purification. The purified enzyme was most stable in the
pH range between 3.5 and 6.5. The pH optimum for xylose
oxidation was in the range between 7.5 and 8.0 for the
solubilized enzyme.
The preliminary experiments on amperometric aldose
detection with ALDH were performed in a batch system,
with ALDH covalently immobilized on a graphite
electrode. Immobilized dimethylferrocene, soluble
ferrocene carboxylic acid and phenazine methosulphate
could act as electron transfer mediators. With
immobilized dimethylferrocene as the mediator xylose
could be measured up to about 100 mM, whereas for glucose
measurement the measurement range was generally about one
tenth of that for xylose.
Another immobilization method used in this study was the
adsorption of ALDH on carbon paste electrodes. Carbon
paste can easily be modified with various compounds and
several different mediators for ALDH were investigated.
The lowest working potential with highest catalytic
current was obtained with dimethylferrocene as a
mediator.
Different flow injection configurations based on
ALDH-modified solid graphite or carbon paste electrodes
or on controlled pore glass immobilized ALDH were
investigated during this work. The best stability was
achieved with a flow injection system based on controlled
pore glass immobilized ALDH. Both the storage and the
operational stability of the carbon paste ALDH electrodes
could be increased by attaching a poly(ester sulphonic
acid) cation exchanger membrane on the electrode.
Polymer-bound ferrocene derivatives were also studied as
electron transfer mediators between the coenzyme of ALDH,
PQQ, and the carbon paste electrode. The operational
stability of the polymer-bound-ferrocene modified
electrodes was better than that of corresponding
dimethylferrocene-modified electrodes.
The controlled pore glass immobilized ALDH and carbon
paste electrodes were successfully used in the
measurement of real fermentation samples. The measurement
of samples containing only xylose as a carbon source
correlated very well with liquid chromatography
measurement. Furthermore, samples containing both xylose
and glucose could be measured satisfactorily when the
measurement with ALDH was combined with separate glucose
measurement.
Original language | English |
---|---|
Qualification | Doctor Degree |
Awarding Institution |
|
Award date | 2 Jun 1995 |
Place of Publication | Espoo |
Publisher | |
Print ISBNs | 951-38-4767-5 |
Publication status | Published - 1995 |
MoE publication type | G5 Doctoral dissertation (article) |
Keywords
- enzymes
- dehydrogenases
- aldoses
- detection
- amperometric
- electrochemistry
- thesis
- purification
- biosensors
- electrodes
- xylose
- glucose