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Protease adsorption, diffusion and reaction on an immobilized substrate surface

Record Type:	Electronic resources : Monograph/item
Title/Author:	Protease adsorption, diffusion and reaction on an immobilized substrate surface
Author:	Roy, Shaunak.
Description:	112 p.
Notes:	Advisers: Channing R. Robertson; Alice P. Gast.
Notes:	Source: Dissertation Abstracts International, Volume: 65-04, Section: B, page: 1985.
Contained By:	Dissertation Abstracts International65-04B.
Subject:	Engineering, Chemical.
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3128468
ISBN:	049675730X

Protease adsorption, diffusion and reaction on an immobilized substrate surface
Roy, Shaunak.

Protease adsorption, diffusion and reaction on an immobilized substrate surface [electronic resource] - 112 p.

Advisers: Channing R. Robertson; Alice P. Gast.

Thesis (Ph.D.)--Stanford University, 2004.

Enzymatic reactions with surface bound substrates present an interesting problem in biomolecular surface science, as they require us to consider traditional enzyme kinetics in the context of protein adsorption. These reactions are important in such applications as detergent enzyme additives, food processing and contact lens cleaning. We study the interaction of a serine protease (subtilisin) with an immobilized substrate (bovine serum albumin) surface.

ISBN: 049675730XSubjects--Topical Terms:

226989
Engineering, Chemical.

Protease adsorption, diffusion and reaction on an immobilized substrate surface
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245 1 0 $a Protease adsorption, diffusion and reaction on an immobilized substrate surface $h [electronic resource]
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500 $a Advisers: Channing R. Robertson; Alice P. Gast.
500 $a Source: Dissertation Abstracts International, Volume: 65-04, Section: B, page: 1985.
502 $a Thesis (Ph.D.)--Stanford University, 2004.
520 # $a Enzymatic reactions with surface bound substrates present an interesting problem in biomolecular surface science, as they require us to consider traditional enzyme kinetics in the context of protein adsorption. These reactions are important in such applications as detergent enzyme additives, food processing and contact lens cleaning. We study the interaction of a serine protease (subtilisin) with an immobilized substrate (bovine serum albumin) surface.
520 # $a In the second portion of our study, we probe the notion that under certain conditions, the overall rate of reaction may be limited by the surface diffusion of the enzyme. We use microfluidic patterning to decorate a bovine serum albumin substrate surface with stripes of adsorbed enzyme in the absence of physical barriers. Any spreading of the enzyme from this initial region indicates surface diffusion, while removal of the substrate provides a measure of reactivity. Diffusion data is analyzed using a one dimensional diffusion model with a no-flux boundary condition. The technique is most applicable to systems where the surface reaction is slow enough to allow surface diffusion to be measurable. In this study, the reaction is retarded by glutaraldehyde treatment of the substrate to satisfy this condition. A surface reaction rate constant of 0.082 min-1 and a surface diffusion coefficient of 1.38 x 10-10 cm2/s was determined with excellent internal reproducibility. These results provide a baseline for the comparative assessment of the surface diffusion/surface reaction behavior of other protease mutants.
520 # $a We first employ a tandem surface plasmon resonance/surface plasmon enhanced fluorescence (SPR/SPEF) technique to simultaneously monitor the adsorption/reaction behavior of the subtilisin enzyme as it cleaves the surface bound substrate. By varying the ionic strength of the reaction environment and studying several single point mutations of subtilisin, we find the adsorption behavior of the enzyme is strongly influenced by its electrostatic interactions with the charged BSA surface. The surface reactivity of each of the mutants is coupled to its adsorption properties. Based on these findings, we propose a modified Michaelis-Menten enzyme surface adsorption and reaction model.
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