SSF production, purification and characterization of chitin deacetylase from Aspergillus flavus

Abstract

The production of an extracellular chitin deacetylase (CDA) produced by Aspergillus flavus under solid-substrate fermentation (SSF) using wheat bran as substrate was optimized using statistical methods. The CDA production in SSF increased 1.79-fold in comparison to the unoptimized basal level medium. It was purified to a final purity of 3.94-fold by ammonium sulphate precipitation, ion-exchange chromatography, and gel-permeation chromatography (GPC) consecutively and further characterized. The molecular mass of the enzyme was estimated to be about 28 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and GPC analysis. The optimum pH and temperature of the purified enzyme were pH 8.0 and 50 °C, respectively. Additionally, the effect of some cations and other chemical compounds on the CDA activity was studied. A marginal increase in enzyme activity was observed with metal ions mainly Mn2+ and Zn2+. No inhibition of the enzyme was observed by the end product, that is, acetate up to 70 mM concentration. The Km and kcat values of the enzyme were determined to be 9.45 mg mL−1 and 26.72 s−1 respectively, using colloidal chitin as substrate. Among various substrates tested, glycol chitin and colloidal chitin were deacetylated.