Study on multiple-enzyme electrode for sucrose determination.

Invertase (INV), mutarotase (MUT), glucose oxidase (GOD) and BSA were coimmobilized via glutaraldehyde-bridged covalent bonding, and directly absorbed on the teflon membrane. This membrane was covered with a nylon mesh and placed over an oxygen electrode. An enzyme electrode for flow injection analysis system (EFIA) was adopted. The optimum enzyme composition (IU) for immobilization on the teflon membrane of INV-MUT-GOD was found to be in the ratio 72:48:2.4, with a recovery activity INV-MUT of more than 42.9%. pH 5.8-6.5 was the most suitable range of acidity for the sensor activity. The optimum temperature was 35-45 degrees C. The system exhibited good linearity in the range of 5 x 10(-4) approximately 10(-1) M sucrose (kinetic method) and 10(-5) approximately 2 x 10(-3) M sucrose (steady state method), in short response time (20 seconds for kinetic method, 2 minutes for steady state method), CV = 1.7% (kinetic method). The sensor had been used for determining sucrose concentration in fermentation broth, with an average recovery rate of 98%. The interference caused by the presence of glucose derived from decomposition of sucrose was eliminated by calibration with a GOD sensor. No significant loss of the enzyme electrode activity was observed after 120 hours of the continuous flow of fresh 1 mM sucrose. The multiple-enzyme membrane showed a relatively long lifetime (compared with 14 hours as reported previously) and good storage stability (30 days, stored in distilled water at 4 degrees C).