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.
Nota bibliográficaFunding Information:
The financial support of Swedish Agency for Research Development Cooperation (SIDA-SAREC) and the Swedish Foundation for Strategic Environmental Research (Mistra) is gratefully acknowledged.