Avaliação de fontes de carbono e condições de indução na expressão de canacistatina em Escherichia coli BL21 (DE3)

Abstract

Canecystatin (CC) is a competitive and reversible protease inhibitor which blocks the proteolytic enzymes activities of insets, fungus e nematodes, prejudicing thus the growth, development and reproduction from these pathogenics organisms. CC is produced by sugarcane, but with limited production, difficulting extraction and purification for production of commercials products, so recombinant DNA technology is one alternative for increase of CC production. Nowadays, CC is produced in the Molecular Biology Laboratory of the Department of Genetic and Evolution of UFSCar (LBM-DGE/UFSCar) from E. coli BL21(DE3) in shaker, utilizing a high cost commercial culture medium (Circlegrow®). With the purpose of produce CC in higher scale, the aim of present work was the study the CC expression in E. coli BL21(DE3), evaluating different carbon source, kind of inductor and induction moment in shaker, as well as confirming the expression conditions in airlift bioreactor of 6 L working volume. In the experiments carried out in shaker were obtained similar cellular growth when utilized glycerol, glucose, fructose e fructose + glucose, and low cellular growth when utilized galactose. It was observed high expression when galactose was utilized as carbon source and IPTG 0,4 mM as inductor. When lactose at 4 e 40 mM was utilized as inductor, CC expression occurred only in the culture containing galactose as carbon source. Volumetric production (in mg/L) of CC up to 61% those from standard culture was obtained, with exception the culture that utilized galactose induced with 0,4 mM of IPTG and 4 mM of lactose, and specific production (mgCC/gcells) greater 75% from standard culture. With respect of cultures in airlift bioreactor, CC expression was superior to expression culture carried out in shaker utilizing glucose with carbon source, approximately 80% of CC concentration obtained in the standard culture after 1 hour of induction and achieving more than 90% in the second hour of induction. In terms of specific production, in this culture was obtained 79,9 mgCC/gcell, value approximately 25% superior to the standard culture.