Enzimas para desconstrução da quitina codificadas no genoma de Chromobacterium violaceum: validação experimental, avaliação do potencial quitinoclástico in silico

Abstract

Enzymes capable of degrading chitin, a recalcitrant polysaccharide abundant in nature, are present in many microorganisms and have the potential to be used in different applications in agriculture, medicine and industry. Therefore, the main objective of this study was to transform Escherichia coli BL21 (DE3) cells with plasmids coding for proteins from Chromobacterium violaceum ATCC 12472, and verify if these proteins are produced both on a small and large scale and if they have enzymatic activity in chitin degradation. In silico analyzes were carried out in order to asses if different chitinolytic enzymes would be produced in a soluble way when expressed in E. coli. After that, some proteins from the families AA10 (3 proteins), GH3 (2 proteins) and GH23 (3 proteins) were chosen. Plasmids carrying the DNA sequences encoding these proteins were then introduced into E. coli BL21 (DE3) cells and the expression of the recombinant molecules was carried out, in a small scale, at different temperatures (20 ° C, 25 ° C, 30 ° C and 35 ° C). An electrophoresis analysis (SDS-PAGE) of the lysed cells revealed the presence of bands with an apparent molecular mass that correspond to the expected values of the protein mass. After verifying the proteins production, tests for enzymatic activity were performed for all the studied proteins. Only two of them, both members of the GH3 family (CV0259 and CV2065), showed chitinolytic activity while none of the studied proteins tested positive for chitosan degradation. The protein with the best yield on previous tests (CV2065) was produced in a slightly large scale and purified by metal affinity chromatography using a cobalt resin. Finally, both proteins were analyzed using bioinformatic tools in order to understand their protein domains and generate a 3D structure of them by homology-modeling. Overall, the production, purification and analysis of chitinolytic proteins have been successful, which sets precedents for a large-scale production and purification of recombinant proteins with promising industrial applications.