Computer-aided design of bromelain and papain covalent immobilization

Titulo en espanol: Diseno asistido por computadora de la inmovilizacion covalente de bromelina y papaina. Titulo corto: Computer-aided design of bromelain and papain.  Abstract:  Enzymes as immobilized derivatives have been widely used in Food, Agrochemical, Pharmaceutical and Biotechnological industries. Protein immobilization is probably the most used technology to improve the operational stability of these molecules. Bromelain ( Ananas comosus ) and papain ( Carica papaya ) are cystein proteases extensively used as immobilized biocatalyst with several applications in therapeutics, racemic mixtures resolution, affinity chromatography and others industrial scenarios. The aim of this work was to optimize the covalent immobilization of bromelain and papain via rational design of immobilized derivatives strategy (RDID) and RDID 1.0 program. It was determined the maximum protein quantity to immobilize, the optimum immobilization pH (in terms of functional activity retention), and the most probable configuration of the immobilized derivative and the probabilities of multipoint covalent attachment was also predicted. As support material Glyoxyl-Sepharose CL 4B was used.  The accuracy of RDID 1.0 program´s prediction was demonstrated comparing with experimental results. Bromelain and papain immobilized derivatives showed desired characteristics for industrial biocatalysis, such as: elevate pH stability retaining 95% and 100% residual activity at pH 7.0 and 8.0, for bromelain and papain, respectively; high thermal stability at 30 °C retaining 90% residual activity for both immobilized enzymes; a catalytic configuration bonded by immobilization at optimal pH; and the ligand load achieved, ensures the minimization of diffusional restrictions. Key words: bromelain, covalent immobilization, immobilized derivatives, papain, rational design. Resumen:  Las enzimas inmovilizadas han sido ampliamente utilizadas en las industrias Alimentaria, Agroquimica, Farmaceutica y Biotecnologica. La inmovilizacion de proteinas es, probablemente, la tecnologia mas empleada para elevar la estabilidad operacional de estas moleculas. La bromelina ( Ananas comosus ) y la papaina ( Carica papaya ) son cistein proteasas extensamente usadas como biocatalizadores inmovilizados con disimiles aplicaciones en la terapeutica, resolucion de mezclas racemicas, cromatografia de afinidad, entre otros escenarios industriales. El objetivo del presente trabajo fue optimizar la inmovilizacion covalente de las enzimas bromelina y papaina a traves de la estrategia de diseno racional de derivados inmovilizados (RDID) y el programa RDID 1.0 . Se predijo la cantidad maxima de proteina a inmovilizar, el pH optimo de inmovilizacion (en terminos de retencion de la actividad funcional), la configuracion mas probable del derivado inmovilizado y la probabilidad de enlazamiento covalente multipuntual. Como soporte de inmovilizacion de empleo Glioxil-Sepharose CL 4B. La precision de las predicciones llevadas a cabo con el programa RDID 1.0 fue validada comparando con los resultados experimentales obtenidos. Los derivados inmovilizados de bromelina y papaina mostraron caracteristicas deseadas para la biocatalisis a nivel industrial, tales como: elevada estabilidad al pH reteniendo el 95% y 100% de actividad residual a pH 7.0 y 8.0, para la bromelina y la papaina, respectivamente; una elevada estabilidad termica con la retencion del 90% de actividad residual a  30 °C para ambas enzimas; al pH de inmovilizacion optimo la configuracion obtenida es cataliticamente competente; y la carga de ligando alcanzada asegura la disminucion de las restricciones difusionales. Palabras clave: bromelina, derivados inmovilizados, diseno racional, inmovilizacion covalente, papaina.

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