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

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A gene encoding a maltogenic amylase of Bacillus stearothermophilus ET1 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 47-70% identity with the sequences of maltogenic amylase from Bacillus licheniformis, neopullulanase from B. stearothermophilus, and cyclodextrin hydrolase (CDase) I-5 from an alkalophilic Bacillus I-5 strain. In addition to starch: pullulan and cyclodextrin, B. stearothermophilus 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 alpha-1,6-glucosidic linkage was produced as an acceptor reaction product. B. stearothermophilus maltogenic amylase shared four highly similar regions of amino acids with several amylolytic enzymes. The beta-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.
Publisher
SPRINGER VERLAG
Issue Date
1998-04
Language
English
Article Type
Article
Keywords

ALPHA-AMYLASE; BACILLUS-STEAROTHERMOPHILUS; THERMOACTINOMYCES-VULGARIS; BRANCHED OLIGOSACCHARIDES; CATALYTIC PROPERTIES; STRUCTURAL FEATURES; NUCLEOTIDE-SEQUENCE; AMYLOLYTIC ENZYMES; ESCHERICHIA-COLI; GENE

Citation

EUROPEAN JOURNAL OF BIOCHEMISTRY, v.253, no.1, pp.251 - 262

ISSN
0014-2956
DOI
10.1046/j.1432-1327.1998.2530251.x
URI
http://hdl.handle.net/10203/77659
Appears in Collection
BS-Journal Papers(저널논문)
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