Expresión y purificación de una enzima PETasa obtenida mediante reconstrucción ancestral

Abstract

In the world, there are a wide variety of organic polymers that have come to represent a significant problem of environmental contamination. Currently, the scientific field began to design appropriate and efficient alternatives to facilitate the recycling of plastic materials ecologically. Biodegradation by enzymes has become highly relevant in recent years due to its good capacity for plastic degradation. The present investigation aimed to express and purify an PETase enzyme obtained from an ancestral reconstruction (ASR); the ancestral enzyme was expected to have superior characteristics to I. sakaiensis PETase respect to its activity against PET and other organic polymers. The ancestral PETase N1 enzyme was expressed in E.coli Ta cells under stable conditions of 1,0 mM IPTG at room temperature, over 16 hours. The enzyme purification was carried out through affinity chromatography (FPLC) complemented with an SDS-PAGE electrophoresis to verify the presence of the ancestral PETase N1 enzyme according to its molecular weight. Over more, the purification used an extra serine protease inhibitor (PMSF) together with EDTA metalloprotease inhibitor. The activity of the ancestral PETase N1 enzyme was evaluated through degradation assays in six high-value polymers by SEM. The images obtained showed degradation features in the HDPE polymer, while in the LDPE, PS, and PVC polymers there was low activity. On the other hand, PP and PET polymers experienced a null activity of the enzyme that could be influenced by various factors.