Cholesterol esterase and cholesterol oxidase immobilized onto arylamine glass beads

Cholesterol esterase (CEase) from bovine pancreas and cholesterol oxidase (COD) from Bravibacterium recombinant type have been immobilized individually and co-immobilized onto arylamine glass (pore diameter 55 nm) through the process of diazotization. CEase and COD retained 92.65% and 85.54% of the initial activity with conjugation yields of 7.2 mg/g and 8.3 mg/g support respectively when immobilized individually on arylamine glass beads, but retained 89.58% of the initial activity with a conjugation yield of 2.9 mg/g support when co-immobilized on the same support. The effects of pH, temperature, time of incubation, substrate concentration, serum inorganic salts & metabolites, thermal stability, storage stability in cold and reusability on the immobilized enzymes were studied and compared with those of free enzymes. The analytic use of both individually immobilized and co-immobilized enzymes in discrete analysis of total and free cholesterol in serum is demonstrated.