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Neutralización enzimática del agente de guerra química VX: evolución de la fosfotriesterasa para la hidrólisis del
Andrew N Bigley1, Chengfu Xu, Terry J Henderson
1Department of Chemistry, P.O. Box 30012, Texas A&M University, College Station, Texas 77842, United States.
Journal of the American Chemical Society
|June 25, 2013
Resumen
Los investigadores diseñaron variantes de la enzima fosfotriesterasa (PTE) para mejorar la actividad catalítica contra los agentes nerviosos tipo V como VX. El mejor mutante mostró una mejora de 230 veces en la hidrólisis de VX, ofreciendo una prometedora estrategia de descontaminación.
Área de la Ciencia:
- La bioquímica es la bioquímica.
- Enzimología Enzimología.
- Ingeniería Química Ingeniería Química.
Sus antecedentes:
- Los agentes nerviosos tipo V (VX y VR) son altamente tóxicos y persistentes, lo que requiere métodos efectivos de descontaminación.
- La fosfotriesterasa de tipo silvestre (PTE) hidroliza VX pero con baja eficiencia en comparación con otros sustratos.
- Los residuos del sitio activo de PTE pueden ser manipulados a través de la evolución dirigida para mejorar la actividad catalítica.
Objetivo del estudio:
- Diseñar nuevas variantes de PTE con una actividad catalítica mejorada contra los agentes nerviosos tipo V.
- Investigar la eficacia de la evolución dirigida y la PCR propensa a errores para la mejora enzimática.
- Desarrollar catalizadores mejorados para la descontaminación de VX.
Principales métodos:
- Se crearon bibliotecas mutacionales secuenciales de dos sitios que se dirigieron a 12 residuos de PTE en el sitio activo.
- Las bibliotecas fueron examinadas por actividad catalítica contra un análogo de VX, DEVX.
- Para una mayor optimización se empleó la mutagenesis por PCR dirigida propensa a errores del bucle 7.
Principales resultados:
- Las variantes evolucionadas de PTE mostraron una mejora de 26 veces en la hidrólisis de DEVX en comparación con el tipo salvaje.
- La mutagenesis adicional produjo variantes con un aumento de hasta 78 veces en la velocidad de hidrólisis de DEVX.
- El mejor mutante exhibió una mejora de 230 veces en la hidrólisis de VX (kcat/Km = 7 × 10(4) M(-1) s(-1)) y un aumento de 152 veces en el número de facturación.
Conclusiones:
- Las variantes modificadas de PTE demuestran una actividad catalítica significativamente mejorada contra el agente nervioso VX.
- Estos mutantes mejorados de PTE representan los catalizadores más efectivos reportados para la hidrólisis de VX.
- El estudio valida las estrategias de evolución dirigida para el desarrollo de potentes agentes de descontaminación basados en enzimas.
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