Expression and characterization of α-Amylases from penicillium citrinum with bread as growth substrate

Abstract

In an attempt to enhance the industrial production of α-amylases in the tropics, sterile fresh bread was inoculated with spore suspensions of Penicillium citrinum at 25 ^oC. Extracellular α-amylases were produced and subjected to partial purification by ammonium sulphate precipitation and dialysis. Further purification by gel filtration and ion-exchange chromatography was engaged. The molecular weights of the α-amylase fractions obtained and estimated by gel filtration using Sephadex G-100 were approximately 56,234 Daltons, 53,089 Daltons and 11,885 Daltons. The apparent Michalis-Menten constant (K_m) values for the hydrolysis of starch by the purified α-amylase fractions were approximately 8.3 mg/ml, 10 mg/ml and 7.14 mg/ml respectively. Optimum activities were at 30 ^oC for one of the fractions and 35 ^oC for the other two fractions and were at pH 5.5 and pH 6.0. The activities of the α-amylase fractions produced by the fungus were stimulated at varying degrees by NaCl, KCl, CaCl₂ and MgCl₂ but inhibited by Ethylene Diamine Tetraacetic Acid (EDTA), mercuric chloride (HgCl₂) and 2,4-dinitrophenol (DNP). The α-amylase fractions were sensitive to heat, losing all their activities within twenty minutes of heating at 80 ^oC. The industrial production of α-amylases should be encouraged in the tropics using bread as a cheap source of substrate.

Keywords: α-Amylase, expression, bread, purification, characterization.