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.
journal_name
J Biotechnoljournal_title
Journal of biotechnologyauthors
Hubrich F,Müller M,Andexer JNdoi
10.1016/j.jbiotec.2014.06.003subject
Has Abstractpub_date
2014-12-10 00:00:00pages
93-8eissn
0168-1656issn
1873-4863pii
S0168-1656(14)00284-3journal_volume
191pub_type
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