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Efficient protein digestion using highly-stable and reproducible trypsin coatings on magnetic nanofibers

Cited 12 time in Web of Science Cited 13 time in Scopus
Authors
Lee, Byoungsoo; Kim, Byoung Chan; Chang, Mun Seock; Kim, Han Sol; Na, Hyon Bin; Park, Yong Il; Lee, Jinwoo; Hyeon, Taeghwan; Lee, Hookeun; Lee, Sang-Won; Kim, Jungbae
Issue Date
2016-03
Citation
Chemical Engineering Journal, Vol.288, pp.770-777
Keywords
Trypsin digestionNanobiocatalysis
Abstract
Protein digestion, using an enzyme called trypsin (TR), is one of the key steps in proteomic analysis. The current technology of protein digestion in proteomic analysis is time-consuming, tedious and not automated due to the poor stability and autolysis of trypsin. To improve the protein digestion process, trypsin was immobilized and stabilized on polymer nanofibers entrapping superparamagnetic nanoparticles (magnetic nanofibers, NP-NFs). By electrospinning the homogeneous mixture of superparamagnetic nanoparticles (NPs) and polystyrene-poly(styrene-co-maleic anhydride), NPs could be effectively entrapped within polymer nanofibers, generating magnetically-separable nanofibers with high surface area for trypsin immobilization via the approach of enzyme coatings. Trypsin coatings on magnetic nanofibers (EC-TR/NP-NFs; EC-TR), fabricated via simple attachment of crosslinked trypsin molecules onto NP-NFs, were highly stable and could be recycled via facile magnetic separation. EC-TR showed negligible loss of trypsin activity even after incubation in an aqueous buffer under rigorous shaking (200 rpm) for 80 days, while the control samples of covalently-attached trypsin on NP-NFs (CA-TR/NP-NFs; CA-TR) and free trypsin lost more than 90% of their initial activities within 11 and 6 days, respectively. When highly-stable and magnetically-separable EC-TR was employed for the repetitive digestions of enolase under recycled uses for the duration of 50 days and even after treatment with another protease (chymotrypsin) for 32 h, the performance of enolase digestion was successfully maintained. The use of EC-TR for the enolase digestion in the ultra-sonication system resulted in fast (similar to 10 min) and efficient digestions with reproducible performance under recycled uses.
ISSN
1385-8947
URI
http://hdl.handle.net/10371/165960
DOI
https://doi.org/10.1016/j.cej.2015.12.003
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College of Engineering/Engineering Practice School (공과대학/대학원)Dept. of Chemical and Biological Engineering (화학생물공학부)Journal Papers (저널논문_화학생물공학부)
College of Engineering/Engineering Practice School (공과대학/대학원)Dept. of Chemical and Biological Engineering (화학생물공학부)Chemical Convergence for Energy and Environment (에너지환경 화학융합기술전공)Journal Papers (저널논문_에너지환경 화학융합기술전공)
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