Affordable Access

Publisher Website

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

Journal of Biotechnology
DOI: 10.1016/j.jbiotec.2014.06.003
  • Isochorismic Acid
  • Chorismatase
  • Chorismic Acid
  • Enzyme Cascade
  • Isochorismate Synthase
  • Biology


Abstract 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.

There are no comments yet on this publication. Be the first to share your thoughts.


Seen <100 times

More articles like this

Rapid purification of heart muscle enzymes using d...

on Biochemical Society Transactio... February 1996

High-yield production of dihydrogen from xylose by...

on Angewandte Chemie Internationa... Apr 22, 2013

Direct measurement of in vitro antibody production...

on Journal of Immunological Metho... Jan 01, 1983

Direct measurement of in vitro antibody production...

on Journal of Immunological Metho... Jul 29, 1983
More articles like this..