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.
|Award date||2 Jun 1995|
|Place of Publication||Espoo|
|Publication status||Published - 1995|
|MoE publication type||G5 Doctoral dissertation (article)|