Cloning and characterization of a maltotriose-producing alpha-amylase gene from Thermobifida fusca.

The gene (tfa), encoding a maltotriose-producing alpha-amylase from Thermobifida fusca NTU22, was cloned, sequenced and expressed in Escherichia coli. The gene consists of 1,815 base pairs and encodes a protein of 605 amino acids. The base composition of the tfa coding sequence is 69% G+C and the protein has a predicted pI value of 5.5. The deduced amino acid sequence of the tfa amylase exhibited a high degree of similarity with amylases from Thermomonospora curvata and Streptomyces amylases. The purified amylase could be detected as a single band of about 65 kDa by SDS-polyacrylamide gel electrophoresis and this agrees with the predicted size based on the nucleotide sequence. The optimal pH and temperature of the purified amylase were 7.0 and 60 degrees C, respectively. The properties of purified amylase from the E. coli transformant are similar to that of an amylase purified from the original T. fusca NTU22.