Production and characterization of chimeric proteins containing chitinase catalytic domain and modules for chitin binding

Abstract

Chitin, a polymer formed by monomers of ²-1,4-N-acetylglucosamine and second most biopolymer occurring on the planet after cellulose, is degraded by enzymes called chitinases (EC 3.2.2.14) that cleave the chitin chain at sites in a random manner. These enzymes are produced by a wide variety of organisms including bacteria, fungi, insects, plants and animals and have different functions such as nutrition, morphogenesis and defense against pathogens. In addition to the important biological function, chitinases have been used in biocontrol (activities as fungicidal, bactericide, insecticide). In insects chitinases have been related to the chitin remodeling, present in the exoskeleton and in the peritrophic matrix. Insect chitinases have been classified into eight distinct groups based primarily on the architecture of domains such as catalytic and CBM ('carbohydrate-binding module'). As a result of studies conducted in our research group on chitin synthesis and considering the lack of information on chitinase ants, we propose, in this project, the production of different chimeric proteins (containing chitinase catalytic domain and modules for binding in chitin) and the study of its catalytic function against chitin and analysis of the products through HPLC and mass spectrometry. Analysis of sequences deposited in databases has already been started and two pairs of oligonucleotides have been acquired. RNA from A. sexdens will be used to obtain cDNA that will be used as a template molecule in PCR for DNA amplification. The proteins expression of will be done in P. pastoris, a well established system in our Laboratory. It is also the objective of the project to evaluate if the proteins present potential use in biocontrol. To develop these experiments the proteins will be tested against the fungi A. fumigatus and C. albicans, important infectious agents in humans and against Spodoptera frugiperda, the main pest of the corn crop in Brazil. To better understand the protein-substrate interaction studies will be performed using the Nuclear Magnetic Resonance (NMR) technique. With this project, an effective contribution to the construction of protein modules with interesting activities, such as insecticide and fungicide, is expected. (AU)