Extracellular tannase from Emericella nidulans showing hypertolerance to temperature and organic solvents

Abstract

Abstract The filamentous fungus Emericella nidulans (=Aspergillus nidulans) produced high levels of extracellular tannase when grown at 30 °C, under agitation (100 rpm), for 24 h in Khanna medium supplemented with tannic acid as carbon source. The enzyme was purified 61-fold, with 30% yield. The molecular mass of the native protein was estimated to be 302 kDa by gel filtration, with a carbohydrate content of 50%. Two protein bands (45.8 and 52 kDa) were observed after 12% SDS–PAGE, suggesting a glycoprotein constituted by three copies of each subunit. The extracellular tannase showed temperature and pH optima of 45 °C and 5.0, respectively, and was fully stable in the temperature range of 22–50 °C, with a half-life (t50) of about 72 h at 90 °C. The enzyme retained around 80% of control activity when maintained for 60 h at pH 4.0 or 5.0. Tannase activity was stimulated by Zn2+, Hg2+, Co2+, and the detergents SDS and Triton X-100. Organic solvents (about 50%, v/v) also increased enzyme activity, particularly isopropanol, acetonitrile, and ethanol. The Km and Vmax values were 14.01 mM and 2.63 U mg−1 protein in the presence of tannic acid; and 4.78 mM and 0.29 U mg−1 protein in the presence of methyl gallate. For propyl gallate, the Vmax was 0.05 U mg−1 protein, with Km of 7.69 mM; for pyrogallol, the Vmax was 7.40 U mg−1 protein and the Km was 16.94 mM.