Development of an Enzyme Sensor Using Multi-enzyme System Immobilized in β-Chitin Membrane

Abstract

A multi-enzyme system was immobilized in a β-chitin membrane. β-Chitin was obtained from common squid pens and glutamate pyruvate transaminase and pyruvate oxidase were immobilized simultaneously in the β-chitin membrane. The immobilization was achieved by filtration of the chitin suspension containing enzymes without any chemical reagent or pH treatment. An enzyme electrode for the measurement of L-alanine was assembled with the β-chitin membrane and an oxygenelectrode, and enzyme activities were evaluated. A control membrane was prepared using a cross-linking method with enzymes, bovine serum albumin and glutaraldehyde. The electrode response of the β-chitin membrane was obtained immediately after the sample injection and leveled off in 60 s. In contrast, the control membrane showed a slower response. The maximum response of the β-chitin embrane electrode was obtained at 38°C and pH 7.1. Linearity of the calibration curve was obtained up to 30mM in the batch system and 200mM in the flow system. These results suggest that β-chitin is a useful supporting material for enzyme immobilization and applicable to enzyme electrodes.