A gene encoding the thermostable alpha-amylase from hyperthermophilic archaeon Thermococcus hydrothermalis was cloned in Escherichia coli. In an effort to achieve the improved expression, four DNA primers (three reverse and one forward) were designed to yield three recombinant variants of alpha-amylase. The additional aim of the re-cloning was to verify how the changes at the C-teminal end of the recombinant alpha-amylase introduced by the pET vector and 6-His tag may affect the resulting specific activity of the variants. Biochemical characteristics of the recombinant alpha-amylases, i.e. molecular weight, temperature optimum, and pH optimum, were confirmed to be the same as those of the original alpha-amylase: 53.6 kDa, 85 degrees C, and 5.5, respectively. Concerning the specific activities, the addition of two residues succeeded by six histidines (LEHHHHHH) had in fact no influence, whereas the insertion of a longer peptide between the original alpha-amylase C-terminus and the 6-His tag (DPNSSSVDKLAAALEHHHHHH) caused a substantial decrease (more than 40%) in comparison with the specific activity of the original protein. The recombinant alpha-amylases were preliminary tested for their ability to hydrolyze 1% and 10% corn starch suspensions. Two types of starch were used: Meritena 100 (amylose starch) and Meritena 300 (amylopectin starch). Concerning the a-amylase concentration sufficient to reach the industrial requirements, i.e. dextrose equivalent 6-12% in 3 h, one unit of enzyme activity at defined conditions per mg starch can be considered as optimal concentration with 10% starch suspension. It was found that even a simple and partial purification (boiling the enzyme supernatant for 5 min) is enough for improving the course of corn starch hydrolysis in order to accomplish the technological parameters in a reasonable time. (c) 2006 Elsevier Inc. All rights reserved.