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Application of trypsin Fe3O4@SiO2 core/shell nanoparticles for protein digestion

Paper ID Volume ID Publish Year Pages File Format Full-Text
34201 45010 2015 11 PDF Available
Title
Application of trypsin Fe3O4@SiO2 core/shell nanoparticles for protein digestion
Abstract

•Hydrophilic Fe3O4@SiO2 core/shell nanoparticles decorated by amino groups were applied for covalent immobilization of trypsin with resulting activity 104.4 U/mg of nanoparticles.•Kinetic constant KM (0.399 mM) indicates improvement in affinity between the immobilized trypsin and substrate.•Qualitatively superior trypsin cleavage of α-casein by (MALDI)-Orbitrap MS in only 3 h.

Trypsin was covalently immobilized by attaching it to porous Fe3O4@SiO2 core/shell nanoparticles with hydrodynamic diameter of about 100–200 nm and specific surface area up to 58 m2/g. The particles were prepared by a modified sol–gel method from hydrophilic Fe3O4 nanoparticles. The surface of the Fe3O4@SiO2 particles was then decorated with amino groups by reaction with 3-aminopropyltriethoxysilane (APTES). These amino groups were connected with the carboxylic acid groups of trypsin using EDAC and sulfo-NHS methodology. Core/shell structure was confirmed by scanning electron microscopy, Fourier transform infrared spectroscopy and powder X-ray diffraction analysis. The specific enzyme activity of the trypsin Fe3O4@SiO2-NH2 nanoparticles was quantified and verified in its recoverability. Kinetic constants KM and vmax were measured and compared to those for soluble form of trypsin. Relatively lower value of the apparent Michaelis–Menten constant KM (0.399–0.658 mM) indicates improvement in affinity between the immobilized enzyme and substrate. The activity and specificity of trypsin Fe3O4@SiO2-NH2 nanoparticles were evaluated using α-casein proteolytic digestion (3 h) followed by high-resolution measurements on a matrix-assisted laser desorption/ionization (MALDI)-Orbitrap mass spectrometer. All measurements confirmed high proteolytic activity and specificity compared to trypsin SiMAG-Amine microparticles and/or to soluble enzyme with 3 h digestion.

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Keywords
Magnetite nanoparticles; Modification by amine; Trypsin; Enzyme immobilization; Michaelis–Menten constant; MALDI-Orbitrap mass spectrometry
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Application of trypsin Fe3O4@SiO2 core/shell nanoparticles for protein digestion
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 50, Issue 12, December 2015, Pages 2088–2098
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us