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Multimeric immobilization of alcohol oxidase on electrospun fibers for valid tests of alcoholic saliva

Paper ID Volume ID Publish Year Pages File Format Full-Text
23294 43430 2013 9 PDF Available
Title
Multimeric immobilization of alcohol oxidase on electrospun fibers for valid tests of alcoholic saliva
Abstract

•Multimeric AOX consisting of eight subunits was immobilized on electrospun fibers.•Branched PEI allows multipoint binding with AOX through glutaraldehyde activation.•Secondary and tertiary amino groups of PEI intensify interactions with AOX subunits.•Color strips were established on electrospun fibrous mats dyed with indigo Carmine.•Color fading ratio remained no significant change after repeat tests for 9 cycles.

An accurate quantitation of ethanol is of great importance in clinical and forensic analyses. In the current study, alcohol oxidase (AOX) from Pichia pastoris, a multimeric enzyme consisting of eight identical subunits, was immobilized on electrospun polystyrene-co-maleic anhydride (PSMA) fibers for valid tests of alcoholic saliva. Branched polyethyleneimine (PEI) was grafted on PSMA fibers with a density of 0.15 nmol/cm2 as tethers to allow multipoint covalent binding of enzyme molecules through glutaraldehyde activation, and the secondary and tertiary amino groups of PEI could intensify the interactions with AOX subunits to stabilize the quaternary structure. PSMA-PEI-AOX fibers were less sensitive than free AOX to the incubation temperature and pH, and indicated no detectable subunit release from the immobilized AOX after boiling in the presence of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol. Color strips were established on PSMA-PEI-AOX fibrous mats dyed with indigo Carmine after incubation into ethanol solutions of different concentrations. The color fading ratio remained no significant change after repeat tests for 9 cycles after immersion in 0.2 and 0.8 mg/mL of alcoholic saliva. It was indicated that multipoint immobilization of the multimeric enzyme was essential to improve the enzyme stability by stabilizing both the quaternary structure of the enzyme and the structure of each individual subunit.

Keywords
Electrospun fiber; Multimeric enzyme; Alcohol oxidase; Multipoint immobilization; Alcoholic saliva
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Multimeric immobilization of alcohol oxidase on electrospun fibers for valid tests of alcoholic saliva
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 168, Issue 1, 10 October 2013, Pages 46–54
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