Pichia stipitis OYE 2.6 variants with improved catalytic efficiencies from site-saturation mutagenesis libraries.
Bioorg Med Chem
; 22(20): 5628-32, 2014 Oct 15.
Article
em En
| MEDLINE
| ID: mdl-25087048
ABSTRACT
An earlier directed evolution project using alkene reductase OYE 2.6 from Pichia stipitis yielded 13 active site variants with improved properties toward three homologous Baylis-Hillman adducts. Here, we probed the generality of these improvements by testing the wild-type and all 13 variants against a panel of 16 structurally-diverse electron-deficient alkenes. Several substrates were sterically demanding, and as hoped, creating additional active site volume yielded better conversions for these alkenes. The most impressive improvement was found for 2-butylidenecyclohexanone. The wild-type provided less than 20% conversion after 24h; a triple mutant afforded more than 60% conversion in the same time period. Moreover, even wild-type OYE 2.6 can reduce cyclohexenones with very bulky 4-substituents efficiently.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Oxirredutases
/
Pichia
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Mutagênese Sítio-Dirigida
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Biocatálise
Idioma:
En
Revista:
Bioorg Med Chem
Assunto da revista:
BIOQUIMICA
/
QUIMICA
Ano de publicação:
2014
Tipo de documento:
Article
País de afiliação:
Estados Unidos