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

Abstract

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.

Expression of Thermobifida fusca thermostable raw starch digesting alpha-amylase in Pichia pastoris and its application in raw sago starch hydrolysis

“…The plasmid propagation in the expression work was accomplished with E. coli Top 10 (Invitrogen, San Diego, CA). The plasmid pAMY13H8 was constructed with pUC118 and a 3.0-kb BamHI-HindIII inserted fragment, which possessed an amylase gene tfa [10].…”

“…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 [10].…”

Heterologous expression of Thermobifida fusca thermostable alpha-amylase in Yarrowia lipolytica and its application in boiling stable resistant sago starch preparation

“…The base composition of the tfa coding sequence is 69% G ? C and the protein has a predicted pI value of 5.5 [6].…”

Characterization and gene cloning of a maltotriose-forming exo-amylase from Kitasatospora sp. MK-1785

“…Maltotriose-forming amylases have been reported for S. griseus (Nakakuki et al 1984;Kashiwagi et al 2014), Streptomyces avermitilis (Kashiwagi et al 2014), Bacillus subtilis (Takasaki 1985), Microbacterium imperiale (Takasaki et al 1991;Wu et al 2014), Natronococcus sp. Ah-36 (Kobayashi et al 1992), Thermobifida fusca (Yang and Liu 2004;Yang and Liu 2007), and Microbulbifer thermotolerans (Lee et al 2014), although few maltotrioseforming amylase with an exo-pattern has been found.…”