Robust Trypsin Coating on Electrospun Polymer Nanofibers in Rigorous Conditions and Its Uses for Protein Digestion

Journal Article · Wed Dec 15 04:00:00 EST 2010 · Biotechnology and Bioengineering, 107(6):917-923

DOI:https://doi.org/10.1002/bit.22895· OSTI ID:1001091

Ahn, Hye-Kyung; Kim, Byoung Chan; Jun, Seung-Hyun; Chang, Mun Seock; Lopez-Ferrer, Daniel; Smith, Richard D; Gu, Man Bock; Lee, Sang-Won; Kim, Beom S; Kim, Jungbae

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 °C 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 0.24 and 163.20 hours at 40 ºC, 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 one 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.

• Research Organization: Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
• Sponsoring Organization: USDOE
• DOE Contract Number: AC05-76RL01830
• OSTI ID: 1001091
• Report Number(s): PNNL-SA-72385; 32707; KP1601010
• Journal Information: Biotechnology and Bioengineering, 107(6):917-923, Journal Name: Biotechnology and Bioengineering, 107(6):917-923 Journal Issue: 6 Vol. 107; ISSN 0006-3592
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
99 GENERAL AND MISCELLANEOUS
AUTOLYSIS
COATINGS
DIGESTION
ENZYME STABILIZATION
ENZYMES
Environmental Molecular Sciences Laboratory
IDENTIFICATION
IMMOBILIZATION
INCREASE
LEACHING
NANOPARTICLES
PERFORMANCE
POLYMERS
PRINCIPLES
PROTEINS
PROTEOLYSIS
STABILITY
STABILIZATION
THERMOSTABILITY
TRYPSIN
TRYPTIC DIGESTION
electrospun nanofibers
enzyme coating
protein digestion
trypsin