Carrier free immobilization and characterization of trypsin

Abstract

Pancreatic trypsin was immobilized by cross-linked enzyme aggregates (CLEA) which is a carrier free immobilization method. Ammonium sulfate was chosen for enzyme precipitation which was followed by cross linking of formed aggregates via glutaraldehyde. Concentrations of precipitant and cross linker were respectively optimized as 60% ammonium sulfate and 1% glutaraldehyde. Optimum pH and temperature for CLEA was increased compared to free enzyme. Furthermore, pH, thermal and storage stability were improved. Presence of additives had no effects on enzyme activity. Prepared cross-linked trypsin aggregates are convenient for in situ protein fragmentation and can be used for protein identification.

Bio-inspired trypsin-chitosan cross-linked enzyme aggregates: a versatile approach for stabilization through carrier-free immobilization

“…In addition, immobilized trypsin showed higher activity compared to the free trypsin enzyme, both at pH 6.0 and at alkaline pH (pH 9.0). These results are in accordance with previous results described in literature, where improved pH stability of enzymes upon immobilization was reported for trypsin (Menfaatli and Zihnioglu, 2015;Sun et al, 2017), lipase (Mohamed et al, 2011 and catalase (Abdel-Mageed et al, 2012) were reported.…”

“…In this study, ammonium sulfate was chosen for precipitation of trypsin as a mild precipitant and preferred over other organic solvents due to its minimal effect on enzyme activity (Velasco-Lozano et al, 2015). Precipitation using ammonium sulfate was previously reported by several authors (López-Serrano et al, 2002;Yu et al, 2006;Menfaatli and Zihnioglu, 2015). The second stage of CLEA preparation is the cross-linking step of the precipitated enzyme aggregates.…”

“…The reaction mixture was incubated at room temperature for 10 minutes. The release of yellow p-nitroaniline (p-NA) was determined spectrophotometrically at a wavelength of 405 nm (405A) (Daglioglu and Zihnioglu, 2012;Menfaatli and Zihnioglu, 2015). One activity unit (U) of trypsin is defined as the amount of enzyme hydrolyzing 1 μ mol of N-α-benzoyl-DL-arginine-p-nitroanilide per minute under standard assay conditions.…”

“…Operational stability of the trypsin CLEAs was examined according to the method described by Menfaatli and Zihnioglu (2015) in a reaction solution composed of 0.5 ml trypsin CLEA, 4 ml 100 mM sodium phosphate buffer (pH 7.5), 0.5 ml 0.9% NaCl and 2.5 ml 0.1% BAPNA (enzyme substrate). The reaction was kept at 37EC with continuous shaking for 6 h. Samples were collected at designated time intervals.…”