A micro-plate format assay for real-time screening for new aldolases accepting aryl-substituted acceptor substrates
2015 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 16, no 18, 2595-2598 p.Article in journal (Refereed) Published
Aldolases are potentially important biocatalysts for asymmetric synthesis of polyhydroxylated compounds. Fructose-6-phosphate aldolase (FSA) is of particular interest by virtue of its unusually relaxed dependency on phosphorylated substrates. FSA has been presented as a promising catalyst of aldol addition involving aryl-substituted acceptors such as phenylacetaldehyde that can react with donor ketones such as hydroxyacetone. Improvement of the low intrinsic activity with these type of bulky acceptor substrates is of great interest but has been hampered by the lack of powerful screening protocols applicable in directed evolution strategies. Here, we present a new screen allowing for direct spectrophotometric recording of retro-aldol cleavage. The assay utilizes an in vitro evolved aldehyde reductase that reduces the aldehyde product formed after FSA-afforded cleavage of the aldol. The assay is suitable both for steady state enzyme kinetics and real-time activity screening in a 96-well format.
Place, publisher, year, edition, pages
2015. Vol. 16, no 18, 2595-2598 p.
aldolase, dehydrogenase, directed evolution, retro-aldol, screening
Biochemistry and Molecular Biology
Research subject Biochemistry
IdentifiersURN: urn:nbn:se:uu:diva-264195DOI: 10.1002/cbic.201500466ISI: 000367720300008PubMedID: 26449620OAI: oai:DiVA.org:uu-264195DiVA: diva2:859479
FunderSwedish Research Council, 621-2011-6055