Extracellular tannase from Emericella nidulans showing hypertolerance to temperature and organic solvents
2011
Gonçalves, Heloísa Bressan | Riul, Alana Jacomini | Terenzi, Héctor Francisco | Jorge, João Atílio | Guimarães, Luis Henrique Souza
The filamentous fungus Emericella nidulans (=Aspergillus nidulans) produced high levels of extracellular tannase when grown at 30°C, under agitation (100rpm), for 24h 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 302kDa by gel filtration, with a carbohydrate content of 50%. Two protein bands (45.8 and 52kDa) 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 72h at 90°C. The enzyme retained around 80% of control activity when maintained for 60h 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 14.01mM and 2.63Umg⁻¹ protein in the presence of tannic acid; and 4.78mM and 0.29Umg⁻¹ protein in the presence of methyl gallate. For propyl gallate, the Vₘₐₓ was 0.05Umg⁻¹ protein, with Kₘ of 7.69mM; for pyrogallol, the Vₘₐₓ was 7.40Umg⁻¹ protein and the Kₘ was 16.94mM.
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