Effect of Spacer Modification on Enzymatic Synthetic and Hydrolytic Activities of Immobilized Trypsin

Publication date: Available online 13 January 2016 Source:Journal of Molecular Catalysis B: Enzymatic Author(s): Julia Andre, David Saleh, Christoph Syldatk, Rudolf Hausmann 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 an 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...
Source: Journal of Molecular Catalysis B: Enzymatic - Category: Biochemistry Source Type: research