Molecular and enzymatic characterization of a maltogenic amylase that hydrolyzes and transglycosylates acarbose

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<jats:p>A gene encoding a maltogenic amylase of <jats:italic>Bacillus stearothermophilus</jats:italic> ET1 was cloned and expressed in <jats:italic>Escherichia coli</jats:italic>. DNA sequence analysis indicated that the gene could encode a 69 627‐Da protein containing 590 amino acids. The predicted amino acid sequence of the enzyme shared 47−70 % identity with the sequences of maltogenic amylase from <jats:italic>Bacillus licheniformis</jats:italic>, neopullulanase from <jats:italic>B. stearothermophilus</jats:italic>, and cyclodextrin hydrolase (CDase) I‐5 from an alkalophilic <jats:italic>Bacillus</jats:italic> I‐5 strain. In addition to starch, pullulan and cyclodextrin, <jats:italic>B. stearothermophilus</jats:italic> could hydrolyze isopanose, but not panose, to glucose and maltose. Maltogenic amylase hydrolyzed acarbose, a competitive inhibitor of amylases, to glucose and a trisaccharide. When acarbose was incubated with 10 % glucose, isoacarbose, containing an α‐1,6‐glucosidic linkage was produced as an acceptor reaction product. <jats:italic>B. stearothermophilus</jats:italic> maltogenic amylase shared four highly similar regions of amino acids with several amylolytic enzymes. The β‐cyclodextrin−hydrolyzing activity of maltogenic amylase was enhanced to a level equivalent to the activity of CDase when its amino acid sequence between the third and the fourth conserved regions was made more hydrophobic by site‐directed mutagenesis. Enhanced transglycosylation activity was observed in most of the mutants. This result suggested that the members of a subfamily of amylolytic enzymes, including maltogenic amylase and CDase, could share similar substrate specificities, enzymatic mechanisms and structure/function relationships.</jats:p>

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