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 carbo di-imide can be performed by using numerous variations of the protocol. Thi s 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. Th e following results were found.(1) The activity of the bioconjugate was cri tically dependent on the physical state of the polymer and the pH of the co upling reaction. For example, in the case of wheatgerm acid phosphatase, th e 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 th e corresponding values were 57 % and 23 % and with beta-glucosidase they we re 57% and 52% respectively. (2) In some cases, such as beta-glucosidase an d 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 t he cases investigated, a significant amount of the enzyme (more than 90%) c ould be bound to the matrix merely by adsorption. (4) More importantly, aft er the carbodi-imide coupling procedure, a sufficient fraction of the bound enzyme could be eluted off the matrix, indicating that this was merely ads orbed and not covalently coupled.