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Immobilized protein films for assessing surface proteolysis kinetics

Paper ID Volume ID Publish Year Pages File Format Full-Text
25141 43555 2007 6 PDF Available
Title
Immobilized protein films for assessing surface proteolysis kinetics
Abstract

Enzymatic cleavage of protein substrates at solid surfaces is important in the food and detergent industries, and in biomedical applications. Creation of a reproducible protein substrate to study surface proteolysis is difficult as protein monolayers may not necessarily provide complete coverage of the surface, and protein multilayer systems are often unstable and nonuniform. We present a method to form a reproducible, immobilized, multilayer protein substrate. A 100-nm ovalbumin protein film is spin-cast onto an amine-functionalized silicon wafer and chemically cross-linked using glutaraldehyde to create a multilayer film. This protein film is stable in the presence of non-protease components such as detergents, and can be tailored to include different proteins and their mixtures, and varying degrees of susceptibility to proteolysis. Ellipsometry was used to measure the protein-film thickness as the substrate is cleaved by the protease subtilisin Carlsberg. The decrease in film thickness over time was found to be linear, indicating the depth-homogeneity of the model substrates. Lateral-homogeneity of the substrates was corroborated by atomic force microscopy (AFM) and by the reproducibility of the ellipsometric film thickness measured across different spots on the sample substrates. AFM of the multilayer protein surface before and after exposure to enzyme suggests uniform areal surface cleavage by the protease.

Keywords
Immobilized protein; Cross-linking; Surface enzyme catalysis; Biological films; Detergency
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Immobilized protein films for assessing surface proteolysis kinetics
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 132, Issue 1, 15 October 2007, Pages 32–37
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