Expressão e purificação de uma esterase (LIPG7) isolada a partir de uma biblioteca metagenômica de sedimentos de manguezal

Abstract

The microorganisms hold the richest repertoire of chemical processes and genetic diversity found in nature. Their biomolecules can be employed in many sectors of modern industry for the production of various commercial compounds. Lipolytic enzymes such as esterases are used in pharmaceuticals, cosmetics and food industries. New tools, including metagenomics, have been helping researchers to prospect novel enzymes from diverse environmental samples with improved features. Mangroves are considered as potential ecosystems for the prospection of new enzymes due to their extreme environmental conditions, high biological activity and the low number of studies on the microbiome associated to these environments. In this context, the present work aimed the screening of metagenomic clones constructed from mangrove sediments for production of lipolytic enzymes as well as expression and purification of the esterase LipG7, which belongs to the VIII family of carboxylesterases. For that, clones were tested for lipolytic activity in Luria Bertani Agar (LB) containing 1% tributyrin. Positive activities were confirmed by the formation of halos around the colonies after 72 h of incubation at 37 ° C. From a screening of 1152 plasmidial clones (3 kb DNA), 3 positives clones for esterases were detected. The analysis of insert in clone LipG7 showed to be an esterase belonging to the VIII family of true esterases. This gene was expressed in three commercial E. coli strains, Rosetta-gami, ArcticExpress and BL21, but as insoluble aggregates. After treatment with guanidine hydrochloride and subsequent purification on affinity chromatography using nickel column, LipG7 was obtained in soluble form and showed an activity of 216.3 ± 16.4 U/mg against the synthetic substrate 4-nitrophenyl butyrate at 30 °C that was increased by addition of the bivalent Mg2+ ion.