Activity and stability of glucose oxidase in molecular films assembled alternately with polyions

Anionic glucose oxidase (GOD) was assembled alternately with polycations, namely, poly(ethylenimine) (PEI) and poly(dimethyldiallyl-ammonium chloride) (PDDA), in the preparation of molecular films. Enzymatic activity of the films was investigated by sequential redox reaction with glucose, peroxidase (POD) and DA67 dye. The apparent activity was not influenced by substrate diffusion at up to 5 microg of immobilized GOD (at the area of 5 x 5 mm(2) x 2 faces). This is ascribed to the less dense packing of the alternate molecular film compared with Langmuir-Blodgett (LB) films. Immobilized GOD could be released into solution, and its activity was about 80% of native GOD, indicating that the immobilization did not cause significant denaturation. The enzyme activity of the GOD film was maintained for 14 weeks when stored in buffer and in air at 4 degrees C. Activity measurement after incubation at elevated temperatures showed that significant deactivation was not observed up to 50 degrees C. This shows that GOD in the film has higher thermostability than native GOD. The pH profile of the GOD activity in the film became broad and shifted towards higher pH than that of native GOD. The GOD film was also prepared by the premixing method, in which a GOD-polyion complex was assembled alternately with another oppositely-charged polyion. The enzyme activity of the alternate film obtained by premixing was much higher (maximal enhancement, 67-fold) than that of the conventionally assembled films. Better dispersion of GOD in the premixed film appears to enhance the enzyme activity.