In vivo screening of haloalkane dehalogenase mutants.
Bioorg Med Chem
; 7(10): 2175-81, 1999 Oct.
Article
en En
| MEDLINE
| ID: mdl-10579523
ABSTRACT
Haloalkane dehalogenase (Dh1A) from Xanthobacter autotrophicus GJ10 catalyzes the dehalogenation of short chain primary alkyl halides. Due to the high Km and low turnover, wild type Dh1A is not optimal for applications in bioremediation. We have developed an in vivo screen, based on a colorimetric pH indicator, to identify Dh1A mutant with improved catalytic activity. After screening 50,000 colonies, we identified a Dh1A mutant with a lower pH optimum. Sequence analysis of the mutant revealed a single substitution, alanine 149 to threonine, which is located close to the active site of Dh1A. Replacement of alanine 149 via site-directed mutagenesis with threonine, serine or cysteine retained the mutant phenotype. Other substitutions at position 149 show little or no activity.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Ingeniería de Proteínas
/
Hidrolasas
/
Mutación
Tipo de estudio:
Diagnostic_studies
/
Prognostic_studies
/
Screening_studies
Idioma:
En
Revista:
Bioorg Med Chem
Asunto de la revista:
BIOQUIMICA
/
QUIMICA
Año:
1999
Tipo del documento:
Article
País de afiliación:
Estados Unidos