Evaluation of kinetic parameters of chitinases produced by Beauveria bassiana (Bals.) Vuill.

Abstract

Entomopathogenic fungus Beauveria bassiana is currently used as a biocontrol agent for agricultural pests. The infection process involves extracellular enzymes such as proteases and chitinases that degrade the cuticle of the insects. The objective of this work was to evaluate kinetic parameters of pH, temperature, ionic concentration and time of reaction on chitinases activity. The fungus B. bassiana CG432 was cultivated on coffee berry borer Hypothenemus hampei (Ferrari) and the conidia grown on insect were used to prepare the inoculum containing 108conídia/mL. These conidia were inoculated at 1% (v/v) in culture liquid medium containing D-glucose (10g), yeast extract (5g), NaNO3 (1,58g), Na2HPO4.7H2O (1,05g), KCl (1g), MgSO4.7H2O (0,6g) and KH2PO4 (0,36g) per liter. The cultivation was carried at 28°C and 180rpm during 5 days. Culture fluid was obtained by filtration and centrifugation at 8.000g, and the chitinases were isolated and concentrated by ultrafiltration using 10 and 100kDa cut off membranes under nitrogen pressure. Chitinase activity was detected and quantified using N-acetylglucosamine released by hydrolysis of colloidal chitin at 40 to 60ºC, at 50, 100 and 200 mM ionic concentrations of buffers sodium acetate (pH 4.0 to 6.0); sodium phosphate (pH 6.0 to 8.0); and Glycine-NaOH (pH 8.0 to 10.0) during 60 minutes. Maximum chitinase activity was at 45ºC and pH 5.5, and was also high at pH 6.0 and pH 8.5 using 50mM buffer. The chitinase activity increased and was stable during an hour at optimum conditions of the reaction, shown the stable nature of this enzyme.