Development of effective cross-linking method for bioactive substance--enzyme immobilization using glutaraldehyde...

When a 10% aqueous solution of glutaraldehyde (GA) was alkalized to pH 8.5 in borate buffer solution and heated at 60 degrees C, the ultraviolet spectrum of GA solution showed two distinct absorption maxima. The one at 280 nm with a weak absorbance ascribable to the C = O bond in the aldehyde group shifted to near 300 nm after 50 min with a slight increase in its intensity. Another maximum at 235 nm with a strong absorbance was ascribable to the C = C bond of the alpha,beta-unsaturated aldehyde group which was formed by aldol condensation reaction of GA monomer, and its absorbance increased markedly with increasing reaction time. The high performance liquid chromatography (HPLC) analysis with detection at 235 nm indicated that several GA oligomers were formed by the alkali treatment and their concentrations increased. The cross-linking ability of these oligomers was examined by immobilizing enzymes (alcohol dehydrogenase (ADH), glutamate dehydrogenase (GLDH] to an aminated polymer gel matrix by reaction with the treated GA solution. The enzyme activities increased with increasing concentration of GA oligomers. Then, the GA oligomers were isolated and used as the cross-linking agent. The activities of ADH and GLDH were 4-fold and 13-fold higher, respectively, than those obtained by using untreated GA solution, while the total amounts of immobilized enzymes were almost unchanged. These results suggest that GA oligomers may act as cross-linkers in a manner different from the generally accepted Schiff base formation reaction; a possible mechanism may involve addition reaction of an amino group to the double bond in the aldol condensate of GA.(ABSTRACT TRUNCATED AT 250 WORDS)