Optimizing protease production from an isolate of the nematophagous fungus Duddingtonia flagrans using response surface methodology and its larvicidal activity on horse cyathostomins

Abstract

Protease production from Duddingtonia flagrans (isolate AC001) was optimized and the larvicidal activity of the enzymatic extract was evaluated on infective horse cyathostomin larvae (L3). Duddingtonia flagrans was grown in liquid medium with eight different variables: glucose, casein, bibasic potassium phosphate (K 2 HPO 4 ), magnesium sulphate (MgSO 4 ), zinc sulphate (ZnSO 4 ), ferrous sulphate (FeSO 4 ), copper sulphate (CuSO 4 ) and temperature. The Plackett– Burman analysis showed a significant influence of MgSO 4 , CuSO 4 and casein (P , 0.05) on protease production by D. flagrans in liquid medium. Central composite design indicated that the highest proteolytic activity was 39.56 U/ml as a function of the concentrations of casein (18.409 g/l), MgSO 4 (0.10 g/l) and CuSO 4 (0.50 mg/l). A significant difference (P , 0.01) was found for the larval number between the treated and control groups at the end of the experiment. A reduction of 95.46% in the number of free-living larvae was found in the treated group compared with the control. The results of this study suggest that protease production by D. flagrans (AC001) in liquid medium was optimized by MgSO 4 , CuSO 4 and casein, showing that the optimized enzymatic extract exerted larvicidal activity on cyathostomins and therefore may contribute to large-scale industrial production.