Abstract
Yeast alcohol dehydrogenase (ADH), diaphorase (DI) and NAD were co-immobilized on Sepharose that had been repeatedly modified with hexamethylenediamine and glutaraldehyde. The activity and re-usability of the gel were investigated with changing the immobilization conditions of the enzymes and the reaction conditions of the glutaraldehyde used in the modification. The results suggested that the immobilization temperature and the immobilization time of the enzymes mainly had an effect on the stability and activity, respectively. The degree of polymerization of the glutaraldehyde affected both the activity and the re-usability, and A235/A280 was used as an index of the degree of polymerization. The optimum conditions were as follows: a temperature and reaction time in the immobilization of the enzymes of 20°C and 7 hr, respectively, and a degree of polymerization of the glutaraldehyde used in the modification of A235/A280 = 20. The gel prepared under these optimum conditions was applied to a flow injection analytical system for ethanol. A good linear relationship between the concentration and the response was observed in the range of 20-80 HIM, suggesting that the gel would be applicable to ethanol analysis.
