Enhanced Proteolytic Activity of Covalently Bound Enzymes in Photopolymerized Sol Gel

Abstract

Trypsin is covalently linked to a photopolymerized sol-gel monolith modified by incorporating poly(ethylene glycol) (PSG-PEG) for on-column digestion of N(alpha)-benzoyl-l-arginine ethyl ester (BAEE) and two peptides, neurotensin and insulin chain B. The coupling of the enzyme to the monolith is via room-temperature Schiff chemistry in which an alkoxysilane reagent (linker) with an aldehyde functional group links to an inactive amine on trypsin to form an imine bond. The proteolytic activity of the immobilized trypsin was measured by monitoring the formation of N alpha-benzoyl-L-arginine (BA), the digestion product of BAEE. The BA is separated from BAEE by capillary electrophoresis and detected downstream (18.5 cm from the microreactor) by absorption (254 nm). Using the Bradford assay, we determined that 97 ng of trypsin is bound to the 1-cm microreactor located at the entrance of capillary column. The bioactivity of the trypsin-PSG-PEG microreactor at 20 degrees C for the digestion of BAEE was found to be 2270 units/mg of immobilized trypsin. The bioactivity of trypsin bound to the capillary wall in the open segment upstream from the monolith was 332 units/mg of immobilized trypsin under the same conditions. In contrast, the activity of free trypsin could not be observed for the digestion of BAEE at 20 degrees C after 16 h of incubation time.

Capillary Monolithic Bioreactor of Immobilized Snake Venom Phosphodiesterase for Mass Spectrometry Based Oligodeoxynucleotide Sequencing

“…The amount of immobilized SVP in the bioreactor was determined using a Coomassie blue dye staining based Bradford assay. 27 Briefly, the bioreactor was treated by 0.1 N NaOH for 2 h at room temperature to release immobilized SVP completely. The released SVP solution (1.0 μL) was mixed with 100 μL of Bradford reagent (Bio-Rad, Richmond, CA.)…”

Efficient proteolysis using a regenerable metal‐ion chelate immobilized enzyme reactor supported on organic–inorganic hybrid silica monolith

“…The walls of bare capillaries and channels [7,8], micro-/nanoparticles or beads [9,10], organic polymer monoliths [11][12][13], and silica monoliths [14][15][16] have been exploited for enzyme immobilization, largely quicken the protein digestion process. Recently, we exploited another kind of material, organic-inorganic hybrid silica monolith, for trypsin immobilization via covalent bonding [17,18].…”

Simple and Rapid Proteomic Analysis by Protease-Immobilized Microreactors

“…It is known that the flow rate of substrate can affect the efficiency of the immobilized enzyme activity (Fan & Chen, 2007;Honda et al, 2005;Ma et al, 2008;Nel et al, 2008;Wu et al, 2004;Dulay et al, 2005). Therefore, we studied the effect of different delivery speeds of substrates on hydrolysis activities (Yamaguchi et al, 2009).…”

“…These results indicate that the stability of proteases was improved after formation of enzyme-polymerization. This enhancement in the efficiency of activity of the immobilized protease compared to free protease can be ascribed to minimization or elimination of the auto-digestion of proteases (Dulay et al, 2005;Shui et al, 2006) and possible stabilization of the structure of proteases by cross-linking, thus resulting www.intechopen.com in higher accessibility of the substrate to the catalytic-site of the enzyme (Honda et al, 2005;Honda et al, 2006;Dulay et al, 2005;Shui et al, 2006). The operational stability of the protease-immobilized microreactors was also tested based on the digestions of cytochrome c (Cyt-C) for TY-microreactor and β-casein for CT-microreactor (Yamaguchi et al, 2010a).…”