P40a: Isolation and screening of bacterial strains producing of chitosan degrading enzymes

Oligosaccharides from chitosan hydrolysis have actually considerable interest in the pharmaceutical, chemical, and medical area, due to their biological properties. Chitosanolytic enzymes have been found in a variety of microorganisms and can be potentially used in the preparation of chitosan oligomers. This work concerns with isolation of chitosanolytic enzyme-producing microorganism and screening of the best strains. Soil samples were obtained from Parque das Dunas in Natal, Brazil. To isolate organisms, 1g of a soil sample was suspended in 10mL of sterile saline solution and aliquots of the resulting suspension were inoculated onto agar plates supplemented with yeast extract, chitosan, CaCl₂, MgSO₄.7H₂O, NH₄Cl, NaCl, KH₂PO₄ and Na₂HPO₄. After incubation for 3 days at 30ºC, colonies were examined for chitosan-degrading activity, and the colonies that showed clear halos were sub-cultured in liquid medium at 30°C with stirring at 120rpm for 24 hours. This culture was centrifuged and the supernatant was used for enzyme activity assays. Evaluation of enzyme activity was conducted by the incubation of enzyme extract with 1% chitosan solution (pH 6.0). The reaction tubes were incubated at 55°C for 30 min. The amount of reducing sugar released was measured by a dinitrosalicylic acid method (DNS) with glucosamine as standard. The results showed that was possible to isolate chitosanolytic enzyme-producing microorganism. From the amount isolated were morphologically identified six bacterial strains  and screened three strains that showed chitosanolytic activity ranging from 0.32 to 0.60 U/mL such activities are higher than some previously purified enzymes reported in literature.