Production and characterization of a thermostable antifungal chitinase secreted by the filamentous fungus Aspergillus niveus under submerged fermentation

Abstract

The filamentous fungus Aspergillus niveus produced extracellular antifungal chitinase when cultured under submerged fermentation (SbmF) using crab shells as the carbon source. Maximal chitinase production was achieved at 192 h of cultivation using minimal medium containing 1% chitin. The enzyme was purified 1.97-fold with 40% recovery by ammonium sulfate precipitation and Sephadex G-100 gel filtration. The molecular mass was estimated to be 44 kDa by both 12% SDS-PAGE and Sepharose CL-6B gel filtration. Maximal A. niveus chitinase activity was obtained at 65 degrees C and pH 5.0. The enzyme was fully stable at 60 degrees C for up to 120 min and the enzymatic activity was increased by Mn2+. In the presence of reducing and denaturing compounds, the enzyme activity was not drastically affected. The chitinase was able to hydrolyze colloidal chitin, azure chitin, and 4-nitrophenyl N-acetyl-beta-D glucosaminide; for the latter, the K-0.5 and maximal velocity (V-max) were 3.51 mM and 9.68 U/mg of protein, respectively. The A. niveus chitinase presented antifungal activity against Aspergillus niger (MIC = 84 mu g/mL), A. fumigatus (MIC = 21 mu g/mL), A. flavus (MIC = 24 mu g/mL), A. phoenicis (MIC = 24 mu g/mL), and Paecilomyces variotii (MIC = 21 mu g/mL). The fungus A. niveus was able to produce a thermostable and denaturation-resistant chitinase able to inhibit fungal development, signaling its biotechnological potential.