title: Robust trypsin coating on electrospun polymer nanofibers in rigorous conditions and its uses for protein digestion.
abstract: An efficient protein digestion in proteomic analysis requires the stabilization of proteases such as trypsin. In the present work, trypsin was stabilized in the form of enzyme coating on electrospun polymer nanofibers (EC-TR), which crosslinks additional trypsin molecules onto covalently attached trypsin (CA-TR). EC-TR showed better stability than CA-TR in rigorous conditions, such as at high temperatures of 40 and 50°C, in the presence of organic co-solvents, and at various pH's. For example, the half-lives of CA-TR and EC-TR were 1.42 and 231 h at 40°C, respectively. The improved stability of EC-TR can be explained by covalent linkages on the surface of trypsin molecules, which effectively inhibits the denaturation, autolysis, and leaching of trypsin. The protein digestion was performed at 40°C by using both CA-TR and EC-TR in digesting a model protein, enolase. EC-TR showed better performance and stability than CA-TR by maintaining good performance of enolase digestion under recycled uses for a period of 1 week. In the same condition, CA-TR showed poor performance from the beginning and could not be used for digestion at all after a few usages. The enzyme coating approach is anticipated to be successfully employed not only for protein digestion in proteomic analysis but also for various other fields where the poor enzyme stability presently hampers the practical applications of enzymes.
authors.affiliation: Korea Research Institute of Chemical Technology (KRICT), Daejeon, Republic of Korea.
authors.forename: Hye-Kyung
authors.initials: HK
authors.lastname: Ahn
authors.forename: Byoung Chan
authors.initials: BC
authors.lastname: Kim
authors.forename: Seung-Hyun
authors.initials: SH
authors.lastname: Jun
authors.forename: Mun Seock
authors.initials: MS
authors.lastname: Chang
authors.forename: Daniel
authors.initials: D
authors.lastname: Lopez-Ferrer
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
authors.forename: Man Bock
authors.initials: MB
authors.lastname: Gu
authors.forename: Sang-Won
authors.initials: SW
authors.lastname: Lee
authors.forename: Beom Soo
authors.initials: BS
authors.lastname: Kim
authors.forename: Jungbae
authors.initials: J
authors.lastname: Kim
mesh_terms: D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D006207:Half-Life; D057139:Nanofibers / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry*; D011485:Protein Binding; D013696:Temperature; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004800:Enzymes, Immobilized; D011108:Polymers; D014357:Trypsin