Robust trypsin coating on electrospun polymer nanofibers in rigorous conditions and its uses for protein digestion.
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.
Ahn
Hye-Kyung
HK
Korea Research Institute of Chemical Technology (KRICT), Daejeon, Republic of Korea.
Kim
Byoung Chan
BC
Jun
Seung-Hyun
SH
Chang
Mun Seock
MS
Lopez-Ferrer
Daniel
D
Smith
Richard D
RD
Gu
Man Bock
MB
Lee
Sang-Won
SW
Kim
Beom Soo
BS
Kim
Jungbae
J
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*
D004800:Enzymes, Immobilized; D011108:Polymers; D014357:Trypsin