Purification and Characterization of Two Alkaline, Thermotolerant α-Amylases from Bacillus halodurans 38C-2-1 and Expression of the Cloned Gene in Escherichia coli

Abstract

A newly isolated strain, 38C-2-1, produced alkaline and thermotolerant α-amylases and was identified as Bacillus halodurans. The enzymes were purified to homogeneity and named α-amylase I and II. These showed molecular masses of 105 and 75 kDa respectively and showed maximal activities at 50–60 °C and pH 10–11, and 42 and 38% relative activities at 30 °C. These results indicate that the enzymes are thermotolerant. The enzyme activity was not inhibited by a surfactant or a bleaching reagent used in detergents. A gene encoding α-amylase I was cloned and named amyI. Production of AmyI with a signal peptide repressed the growth of an Escherichia coli transformant. When enzyme production was induced by the addition of isopropyl β-D(−)-thiogalactopyranoside in the late exponential growth phase, the highest enzyme yield was observed. It was 45-fold that of the parent strain 38C-2-1.