Increasing activity of a monoamine oxidase by random mutation / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 109-118, 2014.
Artigo
em Chinês
| WPRIM
| ID: wpr-242407
ABSTRACT
The monoamine oxidase mutant A-1 (F210V/L213C) from Aspergillus niger showed some catalytic activity on mexiletine. To futher improve its activity, the mutant was subjected to directed evolution with MegaWHOP PCR (Megaprimer PCR of Whole Plasmid) and selection employing a high-throughput agar plate-based colorimetric screen. This approach led to the identification of a mutant ep-1, which specific activity was 189% of that for A-1. The ep-1 also showed significantly improved enantioselectivity, with the E value increased from 101 to 282; its kinetic k(cat)/K(m) value increased from 0.001 51 mmol/(L x s) to 0.002 89 mmol/(L x s), suggesting that catalytic efficiency of ep-1 had been improved. The mutant showed obviously higher specific activities on 7 of all tested 11 amines substrates, and the others were comparable. Sequence analysis revealed that there was a new mutation T162A on ep-1. The molecular dynamics simulation indicated that T162A may affect the secondary structure of the substrate channel and expand the binding pocket.
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Aspergillus niger
/
Especificidade por Substrato
/
Cinética
/
Engenharia de Proteínas
/
Catálise
/
Reação em Cadeia da Polimerase
/
Estrutura Secundária de Proteína
/
Genética
/
Metabolismo
/
Monoaminoxidase
Tipo de estudo:
Ensaio Clínico Controlado
Idioma:
Chinês
Revista:
Chinese Journal of Biotechnology
Ano de publicação:
2014
Tipo de documento:
Artigo
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