Expression of α-Amylase in a Tropical Strain Penicillium rubrum with Bread as Growth Substrate

Background: Penicillium rubrum is an Ascomycete known to produce the toxic metabolites rubratoxins A and B associated with jaundice and convulsions in infected patients. The fungus is known to contaminate grains and grain products in the tropics. Materials and methods: In this current investigation, sterile fresh bread was inoculated with spore suspensions of approximately 6 x 10 5 spores of isolate per ml of a tropical strain Penicillium rubrum . Incubation was at 25 o C. Extracellular proteins produced were subjected to partial purification by ammonium sulphate precipitation and dialysis. Further purification using gel permeation and ion-exchange chromatography was employed. Results: The proteins produced by Penicillium rubrum in the inoculated medium exhibited α-amylase activity. The molecular weights of the α-amylase fractions obtained and estimated by gel filtration using Sephadex G-100 were approximately 47, 316 Daltons and 15, 849 Daltons. The apparent Michalis-Menten constant (K m ) values for the hydrolysis of starch by the purified α-amylase fractions were approximately 10 mg/ml, 5 mg/ml, 25 mg/ml and 16 mg/ml. Optimum activities were at 35 o C for all the fractions and were at pH 6.0. The activities of the α-amylase fractions produced by the fungus were stimulated at varying degrees by NaCl, KCl, CaCl 2 and MgCl 2 but inhibited by ethylene diamine tetraacetic acid (EDTA), mercuric chloride (HgCl 2 ) and 2,4-dinitrophenol (DNP). The α-amylase fractions were sensitive to heat, losing all their activities within twenty minutes of heating at 80 o C. Conclusion: Apart from the toxic metabolites rubratoxins produced by Penillium rubrum , the fungus produces α-amylases in contaminated bread at 25 o C in the tropics. These α-amylases are stable at 35 o C and at pH 6.0.