Study on gene cloning, expression and enzymatic characterization of a high maltose-producing alpha-amylase from Thermobifida fusca

The alpha-amylase genes lacking signal peptide and containing signal peptide were amplified from genomic DNA of T.fusca by PCR and cloned into expression vecor pSE380, respectively.Then constructed recombined plasmids pSE380-tfa and pSE380-sptfa were expressed in host cell of E.coli JM109.The recombinant enzyme of recombinant strains E.coli JM109 containing pSE380-tfa was purified by nickel affinity chromatography.The moleculer weight of purified protein was 64kua by the SDS-PAGE analyzing.The Km of purified enzyme was 1.305mg/mL, its optimum temperature and pH were 60℃ and 7.0, respetively.Extracellular amylase activity was observed in the supernatant fluid of recombinant strains E.coli JM109 containing pSE380- sptfa, then the supernatant fluid was concentrated by ultrafiltration.The results of HPLC showed that the hydrolysis products of soluble starch using this α-amylase all contained glucose, maltose and maltotriose.So this amylase could apply to manufacture high maltose syrup.