Carbodi-imide coupling of enzymes to the reversibly soluble insoluble polymer Eudragit S-100

The coupling of proteins and enzymes to soluble-insoluble polymers by carbodi-imide can be performed by using numerous variations of the protocol. This protocol has been investigated for the coupling of five different enzymes, namely wheatgerm acid phosphatase, beta-glucosidase, beta-galactosidase, trypsin and xylanase, to an enteric methacrylate polymer Eudragit S-100. The following results were found. (1) The activity of the bioconjugate was critically dependent on the physical state of the polymer and the pH of the coupling reaction. For example, in the case of wheatgerm acid phosphatase, the activity of the bioconjugate was 49% when coupling was performed at pH 7.2 and 67% when coupling was performed at pH 4. 5. With beta-galactosidase the corresponding values were 57% and 23% and with beta-glucosidase they were 57% and 52% respectively. (2) In some cases, such as beta-glucosidase and beta-galactosidase, it might be necessary to remove excess carbodi-imide before the addition of the enzyme to the activated matrix. (3) In most of the cases investigated, a sig-nificant amount of the enzyme (more than 90%) could be bound to the matrix merely by adsorption. (4) More importantly, after the carbodi-imide coupling procedure, a sufficient fraction of the bound enzyme could be eluted off the matrix, indicating that this was merely adsorbed and not covalently coupled.