Development of a new biosensor for glucose, fructose and sucrose using an oxidoreductase of zymomonas mobilis

Abstract

A new biosensor for the determination of glucose, fructose and sucrose was developed using an oxidoreductase of Zymomonas mobilis, and this biosensor was employed to construct the flow injection analysis (FIA) system. In this study, cells of Z. mobilis were permeabilized with toluene in order to utilize thd enzymes of glucose-fructose oxidoreductase and gluconolactonase inside the intact cells. For glucose (or fructose) biosensor, biocatalytic membrane was prepared by immobilizing permeabilized cells on a gelatin mimbrane and was fixed to the pH electrode using an Oring. For sucrose biosensor, invertase and permeabilized cells were coimmobilized in the membrane using the same method. The hydrogen ion produced by the oxidoreductase reaction in the immobilized membrane was detected using the biosensor-connected microcomputer. Optimum conditions for biosensor response were pH 6.2 and temperature 35 - 40^℃. The biosensor was highly specific for glucose and fructose; which are substrates of the glucose-fructose oxidoreductase of Z. mobilis. The calibration curves for glucose, fructose and sucrose were linear up to 5g/L, 50g/L, and 70g/L, respectively. The biosensor was stable for at least 7 day at 35℃ and the immobilized enzyme membrane was stable for more than 3 months when stored in buffer at 4℃. The results were well agreed with those obtained by a conventional analytical method. For the elimination of the interference effect on biosensor response, the steady-state method and the fructose-addition method in the FIA system were applied. The determination of sucrose in the presence of both glucose and fructose was possible by adding 0.5 M fructose to sampling line in FIA system. In FIA system, the peak height was linearly related to fructose concentration in the range of 2-80g/L when glucose concentration was fixed at 0.3 M. The calibration curve for glucose was linear up to 8g/L when fructose concentration was fixed at 0.5 M, while sucrose was linear up to 10g/...