Clonagem e expressão do gene da quitinase de Anopheles gambiae em Pichia pastoris

Abstract

As part of class of glycosidic hydrolases, chitinases (EC 3.2.1.14) acts on cleavage of random sites of chitin molecule through hydrolysis, forming chains of chitooligosaccharides. Structurally has catalytic domains that helps in the binding to them insoluble substrate and rupture of crystalline conformation. Its enzymatic potential mainly comprises the biological control of fungal phyto-pathogens and insect vectors, organisms where chitin is a structural component of cell wall or exoskeletons, however its applicability extends to synthesis of artificial polysaccharides, isolation of fungal protoplasts and estimation of biomass in industrial processes. From the sequencing of the genome of Anopheles gambiae the gene assigned to chitinase expression was obtained, so that by chemical sinthesis the gene was cloned and Escherichia coli (DH5α line) and integrated to the Pichia pastoris genome in the vectors of expression pPIC9 and pPICPGK. The enzymatic activity of 19 clones was confirmed by the presence of colloidal chitin degradation halo, and after selection was performed the submerged production, recovery, and biochemical caracterization by central composite rotational design (CCRD). The cloning and generation of recombinant expression vectors was consolidated so that integration into the yeast genome shows a significant level of expression, in the enzymatic activity, factors like incubation period and temperature didn’t show significance, and the pH factor demonstrates a positive response in more acidics ranges.