Biophysical Properties of Thermostable Amidase Produced by Aspergillus fumigatus in Submerged Fermentation

Abstract

Amidases, also known as amidohydrolases (EC 3.5.1.4), are enzymes within the nitrilase superfamily or amidase signature family. They hydrolyze amides and nitriles into their corresponding acids while releasing ammonia. These enzymes are widely used in biotechnology and industry. However, their production is limited to a few organisms, which cannot meet industrial demand. Therefore, exploring new amidase sources is crucial. This study focuses on purifying and characterizing amidase from Aspergillus fumigatus using cold acetone precipitation and column chromatography with Sephadex G-100. The effects of temperature, pH and metal ions, on the enzyme activity were examined for both crude and purified amidase. The thermal and pH stability of the enzyme alongside the enzyme kinetics were also assessed. The purified amidase exhibited optimal activity of 13.1 U/mL at 70°C, with 65% residual activity after 5 hours at this temperature. The pH studies showed optimal activity at pH 5.0 for the purified enzyme (7.8 U/mL) and at pH 4.0 for the crude enzyme (5.5 U/mL). Metal ion effects indicated that Zn²⁺, Mg²⁺, and EDTA enhanced, while Cu²⁺ inhibited, the activities of both crude and purified amidase. Kinetic analysis showed that the enzyme followed Michaelis-Menten kinetics, with Km and Vmax values of 0.104 mM and 1.884 U/mL for crude amidase, and 0.017 mM and 1.872 U/mL for purified amidase. These findings suggest that amidase from Aspergillus fumigatus holds potential for industrial applications where amidases are essential.