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Free Trypsin (free + trypsin)
Selected AbstractsFunctionalization Strategies for Protease Immobilization on Magnetic NanoparticlesADVANCED FUNCTIONAL MATERIALS, Issue 11 2010Dan Li Abstract A comprehensive study on the general functionalization strategies for magnetic nanoparticles (MNPs) is presented in this work. Using well-established techniques as well as modified protocols, the wide range of functional moieties grafted on ,-Fe2O3 (maghemite) nanosurfaces include those of amine, aldehyde, carboxylic, epoxy, mercapto, and maleimide ends. Among the modified protocols are the one-step water-catalyzed silanization with mercaptopropyltrimethoxysilane, resulting in dense distal thiols, and the direct functionalization with a heterogeneous bifunctional linker N -[p-maleimidophenyl]isocynanate (PMPI). The former results in a protective Stöber type coating while simultaneously reducing the iron oxide core to magnetite (Fe3O4). The conjugation of trypsin, hereby chosen as model biomolecule, onto the differently functionalized MNPs is further demonstrated and assessed based on its activity, kinetics, and thermo-/long-term stability as well as reusability. Besides aqueous stability and ease in recovery by magnetic separation, the immobilized trypsin on MNPs offers superior protease durability and reusability, without compromising the substrate specificity and sequence coverage of free trypsin. The MNP-based proteases can be used as valuable carriers in proteomics and miniaturized total analysis devices. The applicability of the functional surfaces devised in the current study is also relevant for the conjugation of other biomolecules beyond trypsin. [source] Preparation of trypsin-immobilised chitosan beads and their application to the purification of soybean trypsin inhibitorJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 13 2008Li Zhang Abstract BACKGROUND: Trypsin inhibitors are among the most important antinutritional factors in legumes. Recent research has shown that soybean trypsin inhibitor (SBTI) exhibits multiple bioactivities, but very few studies on the purification of SBTI are available. Enzymes are commonly used as biospecific ligands in affinity purification of their substrates or inhibitors. The aim of the present study was to prepare trypsin (EC 3.4.21.4)-immobilised chitosan beads and use them to purify trypsin inhibitor from soybean whey. RESULTS: Compared with free trypsin, the immobilised trypsin had higher thermal and pH stability. The adsorption ratio of SBTI from crude SBTI aqueous solution by trypsin-immobilised chitosan beads was 33.3%. The purified SBTI obtained by affinity chromatography was characterised by sodium dodecyl sulfate polyacrylamide gel electrophoresis as a single polypeptide band with an Mr of 8.3 kDa belonging to the Bowman,Birk family. CONCLUSION: Trypsin-immobilised chitosan beads were effectively used in the affinity separation of trypsin inhibitor from soybean seeds, thus indicating that immobilised trypsin may have practical application in the soybean-processing industry. The results of this study provide a background for further investigation of potential applications of soybean bioactive constituents in the areas of agriculture and food. Copyright © 2008 Society of Chemical Industry [source] The Properties of Covalently Immobilized Trypsin on Soap-Free P(MMA-EA-AA) Latex ParticlesMACROMOLECULAR BIOSCIENCE, Issue 4 2005Kai Kang Abstract Summary: The covalent immobilization of trypsin onto poly[(methyl methacrylate)- co -(ethyl acrylate)- co -(acrylic acid)] latex particles, produced by a soap-free emulsion polymerization technique, was carried out using the carbodiimide method. The catalytic properties and kinetic parameters, as well as the stability of the immobilized enzyme were compared to those of the free enzyme. Results showed that the optimum temperature and pH for the immobilized trypsin in the hydrolysis of casein were 55,°C and 8.5, both of which were higher than that of the free form. It was found that Km (Michaelis constant) was 45.7 mg,·,ml,1 and Vmax (maximal reaction rate) was 793.0 ,g,·,min,1 for immobilized trypsin, compared to a Km of 30.0 mg,·,ml,1 and a Vmax of 5,467.5 ,g,·,min,1 for free trypsin. The immobilized trypsin exhibited much better thermal and chemical stabilities than its free counterpart and maintained over 63% of its initial activity after reusing ten times. TEM photograph of latex particles after trypsin immobilization. [source] Efficient on-chip proteolysis system based on functionalized magnetic silica microspheresPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 14 2007Yan Li Abstract An easily replaceable enzymatic microreactor has been fabricated based on the glass microchip with trypsin-immobilized magnetic silica microspheres (MS microspheres). Magnetic microspheres with small size (,300,nm in diameter) and high magnetic responsivity to magnetic field (68.2,emu/g) were synthesized and modified with tetraethyl orthosilicate (TEOS). Aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA) were then introduced to functionalize the MS microspheres for enzyme immobilization. Trypsin was stably immobilized onto the MS microspheres through the reaction of primary amines of the proteins with aldehyde groups on the MS microspheres. The trypsin-immobilized MS microspheres were then locally packed into the microchannel by the application of a strong field magnet to form an on-chip enzymatic microreactor. The digestion efficiency and reproducibility of the microreactor were demonstrated by using cytochrome c (Cyt-C) as a model protein. When compared with an incubation time of 12,h by free trypsin in the conventional digestion approach, proteins can be digested by the on-chip microreactor in several minutes. This microreactor was also successfully applied to the analysis of an RPLC fraction of the rat liver extract. This opens a route for its further application in top-down proteomic analysis. [source] | |||||||||||||