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In vitro production and purification of isochorismate using a two-enzyme cascade

Paper ID Volume ID Publish Year Pages File Format Full-Text
23003 43409 2014 6 PDF Available
Title
In vitro production and purification of isochorismate using a two-enzyme cascade
Abstract

•Pure isochorismate is obtained by an in vitro two-enzyme cascade.•First, isochorismate synthase generates an isochorismate/chorismate equlibrium.•Second, the chorismatase degrades exclusively chorismate.•Finally, isochorismate is purified using an automated FPLC system.

Combining the isochorismate synthase EntC and the chorismatase FkbO in a sequential enzyme cascade provides a useful system for the biocatalytic production and subsequent purification of isochorismate from an isochorismate/chorismate mixture. FkbO has a strict preference for chorismate – isochorismate is not accepted as a substrate – therefore the enzyme can be used to selectively hydrolyse chorismate, leading to the chiral building block 3,4-dihydroxycyclohexa-1,5-dienecarboxylate. This simplifies the final purification step, as isochorismate is much easier to separate from the chorismate hydrolysis products than from chorismate itself.The presented procedure starts with an optimised method for purifying chorismate from Escherichia coli culture supernatants, which is followed by conversion into isochorismate with the isochorismate synthase EntC, removal of the remaining chorismate by FkbO and a final purification step using an automated flash chromatography system. Isochorismate was isolated in up to 20% yield and >95% purity from chorismate, and has been characterised with respect to its degradation and suitability as a substrate in enzyme assays.

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Keywords
Isochorismic acid; Chorismatase; Chorismic acid; Enzyme cascade; Isochorismate synthase
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In vitro production and purification of isochorismate using a two-enzyme cascade
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 191, 10 December 2014, Pages 93–98
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