MOLECULAR AND ENZYMATIC CHARACTERIZATION OF A MALTOGENIC AMYLASE THATHYDROLYZES AND TRANSGLYCOSYLATES ACARBOSE

A gene encoding a maltogenic amylase of Bacillus stearothermophilus ET 1 was cloned and expressed in Escherichia coli. DNA sequence analysis indicated that the gene could encode a 69627-Da protein containing 590 amino acids. The predicted amino acid sequence of the enzyme shared 4 7-70% identity with the sequences of maltogenic amylase from Bacillus licheniformis, neopullulanase from B. stearothermophilus, and cyclodex trin hydrolase (CDase) I-5 from an alkalophilic Bacillus I-5 strain. I n addition to starch: pullulan and cyclodextrin, B. stearothermophilus could hydrolyze isopanose, but not panose, to glucose and maltose. Ma ltogenic amylase hydrolyzed acarbose, a competitive inhibitor of amyla ses, to glucose and a trisaccharide. When acarbose was incubated with 10% glucose, isoacarbose, containing an alpha-1,6-glucosidic linkage w as produced as an acceptor reaction product. B. stearothermophilus mal togenic amylase shared four highly similar regions of amino acids with several amylolytic enzymes. The beta-cyclodextrin-hydrolyzing activit y of maltogenic amylase was enhanced to a level equivalent to the acti vity of CDase when its amino acid sequence between the third and the f ourth conserved regions was made more hydrophobic by site-directed mut agenesis. 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 sha re similar substrate specificities, enzymatic mechanisms and structure /function relationships.