Otimização de ensaio de atividade quitinolítica em bibliotecas metagenômicas

Abstract

Bioprospecting is the search for new compounds from microbes with industrial applications, hence several projects are developed in Post-Graduate Program in Genomic Sciences and Biotechnology from the Catholic University of Brasília, Brazil, among them there is the search of bacterial hydrolytic enzymes in goats’ rumen, marine coral mucus, the Brazilian Cerrado and the Brazilian Amazon soil metagenomic libraries. Metagenome is the set of genomes from all organisms in a certain sample. Clones with amylolytic, proteolytic and lipolytic activities were identified using the functional analysis of metagenomes without the need to cultivate the microorganisms. Other activities, such as chitinase, were not studied and the assays for their detection were not yet optimized. These enzymes degrade chitin and have various industrial applications, such as in the control of plant and animal’s fungal pathogens, in agriculture as pest control (biopesticides) and also controlling the larvae growing of transmitting dengue and malaria’s mosquitoes. This study aims are to optimize the chitinolytic activity plate assay and identify clones with this activity in an Amazon soil metagenomic library. As a control organism, Vibrio alginolyticus was used due to its well-known chitinase activity. To standardize the screening, two types of colloidal chitin were tested. The colloidal chitin is obtained through the hydrochloric acid hydrolysis of commercial chitin. Part of colloidal chitin was stored at 4oC (humid chitin) and the remaining colloidal chitin was lyophilized. Different concentrations of colloidal chitin lyophilized and humid were used in a medium composed of salts to determine the best conditions for microorganism growth as well as for detection of enzyme activity on chitin by degradation and halo formation. The medium containing colloidal humid chitin in the concentration of 2.0% was chosen for the detection of chitinolytic activity in metagenomic libraries because it allows the bacterial growth and the expected halo formation from chitin degradation. To enhance the detection of halo of degradation, Congo Red dye was used, a methodology already used for other polysaccharides. When stained with Congo Red dye, the medium stains in light blue and the halos stains in dark blue. To screen the chitinolytic activity on the library of the Amazon Soil, it was plated on LB medium with the concentration and type of chitin previously standardized. The plates were incubated at 37°C for 16 hours and after fifteen days of growth at room temperature; the observation of the chitin-degradation halos was enhanced by the use of the Congo Red dye. The screening of 4032 clones using this methodology allowed the identification of four chitinolytic positive clones.