Effect of spacer modification on enzymatic synthetic and hydrolytic activities of immobilized trypsin

• Comprehensive comparison of spacer-mediated immobilization of trypsin.
• Improved synthetic activity after immobilization via spacer.
• Slightly lowered stability of spacer-bound immobilized trypsin within several reuse cycles.

A thorough comparison of spacer-mediated covalent and non-covalent immobilization of trypsin on micro-magnetic particles was accomplished in the present study. Trypsin was coupled via diaminoalkanes, aminoalkanoic acids, bovine serum albumin (BSA), and biotin-derivate spacers onto magnetic particles. A comparison of resulting synthetic and hydrolytic activities after immobilization was performed. Whereas hydrolytic trypsin activity was measured employing N-α-benzoyl-dl-arginine 4-nitroanilide (BAPNA) assay, synthetic trypsin activity was measured employing a dipeptide synthesis assay. Within spacer-mediated trypsin immobilization, diaminoalkanes, aminoalkanoic acids and biotin spacers showed up to 40% increased synthetic specific activity of trypsin compared to the spacer-free method. Within the hydrolytic reaction type, coupling of trypsin via diaminoalkanes and biotin spacers resulted in a specific activity increase of up to 30%. BSA-bound trypsin displayed only minor increasing effects on both activities of trypsin. Furthermore, protein loading-dependent specific synthetic and hydrolytic activities were evaluated for 8-aminooctanoic acid, 12-aminododecanoic acid and 1,12-diamonododecane as spacers and compared to the direct covalent binding method. The protein binding capacity of spacer-modified particles was lower compared to the direct binding method. Synthetic activity of 8-aminooctanoic acid-bound trypsin was higher than in the case of the spacer-free method over a broad protein loading range with an up to 10-fold increase. The hydrolytic activity was increased by 64% using 8-aminooctanoic acid as spacer within a lower protein loading range. Spacer-bound trypsin showed a slightly lowered reusability over ten sequential cycles compared to spacer-free covalent binding method.

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