Produção de cutinases por Escherichia coli recombinante e potencial para aplicação ambiental

Abstract

Cutinase (EC 3.1.1.74) are enzymes that catalyze the hydrolysis of cutin, an insoluble biopolyester that compound the cuticle of plants. These enzymes have potential in the synthesis of triglycerides, polymers, surfactants in chemical, pharmaceutical and agrochemical industries. Cutinases have been applied to the surface modification of polymers, facilitating the degradation of these compounds. The aim of this work was to produce and characterize cutinases by Escherichia coli genetically engineered for use in the treatment of polyethylene terephthalate (PET). E. coli CUT and E. coli CUT-N1 were grown in Lysogeny broth (LB) in the presence of 100 μg/ml ampicillin and isopropyl β-D-1-tiogalactopiranosídeo (IPTG) as inducer. Cutinases activities were determined in the presence of p-nitrophenyl butyrate (p- NPB). The maximum cutinase activity was 1.4 U/mL, determined in the cell-free metabolicliquid, produced by E. coli CUT-N1. The metabolic liquid with activity of cutinase produced by E. coli CUT and E. coli CUT-N1 were concentrated two times by ultrafiltration and formulated with microbial preservatives and stabilizing substances of protein structures. Cutinases had optimum pH equal to 7.0 and thermal stability at 30 - 50 °C. The addition of (NH4)2SO4 at concentrations less than 10% stabilized cutinase activity for 60 days at 28 °C. Cutinases produced by cultures of E. coli degraded the plastic polyethylene terephthalate (PET) with a weight loss of 0.90%. Recombinant microbial cutinases are an alternative for application in biological treatment of plastics.