Alpha-amylase gene of thermophilic Streptomyces sp TO1:: nucleotide sequence, transcriptional and amino acid sequence analysis

The nucleotide sequence of a 1860-bp region encoding a thermostable cc-amylase of Streptomyces sp. T01 was determined. Frame analysis revealed the presence of a 1359-bp long open reading Frame (amy T01) encoding a 453 amino acid protein with a deduced M-r of 49 kDa. Northern blot analysis revealed that amy T01 gene was expressed as approximately 1.5-kbp monocistronic transcript in both SL1326/pLM1 and Streptomyces sp. T01 strains. Primer extension experiments indicated that the transcriptional start site lies 30 bp upstream of the ATG start codon, and allowed the identification of -35 (TTGCTG) and -10 (TACGCG) eubacterial-like promoter sequences. Amy T01 exhibits strong amino acid identities with those from other Streptomyces species with a maximum of 78% with S. thermoviolaceus alpha-amylase. Nevertheless, subtle amino acid changes such as the substitution of four conserved residues found at similar positions in other Streptomyces alpha-amylases by proline residues, and the substitution of three conserved hydrophilic amino acids by hydrophobic ones in Amy T01 might account for the thermostable properties of Amy T01. (C) 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.