Laccase mediator system for activation of agarose gel: Application for immobilization of proteins

Cross-linked Sepharose beads were treated with laccase–TEMPO system for oxidation of the primary alcohol groups on the sugar moieties. Optimal activation conditions using Trametes versicolor laccase were at pH 5 and 22 °C, giving an aldehyde content of 55 μmol g−1 Sepharose with 28 units g−1 of laccase and 12.5 mM TEMPO. The activated Sepharose was used for immobilization of trypsin as model protein. Highest degree of immobilization was obtained at pH 10.5 but the activity yield was only 31% of that loaded on the gel. The yield of gel bound trypsin activity was increased to 76% (corresponding to about 43 U g−1 Sepharose) when the immobilization was performed in the presence of trypsin inhibitor, benzamidine. The immobilization yields were comparable to that obtained on the matrix activated using sodium periodate (containing 72 μmol aldehyde per g Sepharose). Recycling and storage of the immobilized trypsin preparations showed high stability of the enzyme bound to laccase–TEMPO activated gel.

Cross-linked Sepharose matrix was activated using laccase-mediator system to contain aldehyde groups. The activated gel was used to immobilize trypsin. Immobilization yield was comparable to that using a chemically activated gel.Figure optionsDownload as PowerPoint slideResearch highlights▶ Laccase-mediator system was used for activation of cross-linked agarose, Sepharose CL-6B, giving 55 μmol aldehyde per g Sepharose. ▶ The activated gel was used as a matrix for immobilization of trypsin. ▶ Immobilized trypsin activity, 43 U g−1 Sepharose, was comparable to that obtained with the periodate activated gel. ▶ Immobilized trypsin exhibited high stability during recycling and storage.