Directed Evolution of the Epoxide Hydrolase from Aspergillus niger

An efficient and genetically stable expression system for the directed evolution of epoxide hydrolase from Aspergillus niger (ANEH) has been constructed. Error prone polymerase chain reaction (PCR) with defined mutation rates was used to create biodiversity in two libraries of mutants. Screening for...

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Bibliographic Details
Published in:Biocatalysis and biotransformation 2003-12, Vol.21 (6), p.357-364
Main Authors: Cedrone, Frederic, Niel, Sebastien, Roca, Sanja, Bhatnagar, Tej, Ait-abdelkader, Nadra, Torre, Claudia, Krumm, Holger, Maichele, Andrea, T. Reetz, Manfred, C. Baratti, Jacques
Format: Article
Language:English
Subjects:
Aspergillus niger
Biological and medical sciences
Biotechnology
Catalytic efficiency
Directed evolution
Enantioselectivity
Enzyme engineering
Epoxide hydrolase
Expression system
Fundamental and applied biological sciences. Psychology
Methods. Procedures. Technologies
Protein engineering
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Summary:An efficient and genetically stable expression system for the directed evolution of epoxide hydrolase from Aspergillus niger (ANEH) has been constructed. Error prone polymerase chain reaction (PCR) with defined mutation rates was used to create biodiversity in two libraries of mutants. Screening for activity allowed the isolation of clones with improved properties. One of these clones shows an expression level 3.4 higher than the original wild type clone in E. coli SG13009 and a 3.3 fold increased catalytic efficiency on 4-(p-nitrophenoxy)-1,2-epoxybutane. In addition, a screening assay for determining the enantioselectivity in the kinetic resolution of styrene oxide has been established using mass spectrometry.
ISSN:1024-2422
1029-2446
DOI:10.1080/102420310001630137