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

Anionic glucose oxidase (GOD) was assembled alternately with polycations, n amely, poly(ethylenimine) (PEI) and poly(dimethyldiallyl-ammonium chloride) (PDDA), in the preparation of molecular films. Enzymatic activity of the f ilms 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 mu g 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 molec ular him compared with Langmuir-Blodgett (LB) films. Immobilized GOD could be released into solution, and its activity was about 80% of native GOD, in dicating that the immobilization did not cause significant denaturation. Th e enzyme activity of the GOD him 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 observe d up to 50 degrees C. This shows that GOD in the him has higher thermostabi lity than native GOD. The pH profile of the GOD activity in the him became broad and shifted towards higher pH than that of native GOD. The GOD him wa s 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 him obtained by premixing was much higher (maxima l enhancement, 67-fold) than that of the conventionally assembled films. Be tter dispersion of GOD in the premixed film appears to enhance the enzyme a ctivity.