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

Abstract

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 47Ϫ70% 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 A-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 β-cyclodextrinϪhydrolyzing 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.Keywords : Bacillus stearothermophilus; maltogenic amylase ; acarbose ; transglycosylation; site-directed mutagenesis.Many types of amylases with unique properties have been megaterium [7], and A-amylase of Thermoactinomyces vulgaris [8] have been reported to hydrolyze the A-1,4 linkages of pulluisolated and characterized for various applications in the starch industry [1,2]. These proteins share many structural and mecha-lan to produce panose. Amylolytic enzymes, such as cyclodextrin glucanotransferases (CGTase) and CDase exhibit their nistic characteristics. However, amylases can be divided into several groups according to substrate specificities, patterns of highest levels of activity on cyclomaltodextrins [9Ϫ11]. Some starch cleavage, transglycosylation or cyclization activities, and amylolytic enzymes, including debranching enzymes and structural features. Classical A-amylases (e.g. 1,4-A-D-glucan CGTase, catalyze transglycosylation by forming A-1,4 or A-1,6 glucanohydrolase) catalyze hydrolysis of A-1,4-glucosidic link-linkages. ages in starch, and different amylases give rise to oligosacchaJespersen et al.[12] used sequence alignments and structurerides with specific lengths of glucose as major product [2]. De-prediction models to predict the presence of A-amylase-type branching enzymes are capable of hydrolyzing A-1,6-glucosidic (β/A) 8 -barrel domains and the positions of the β-strands and Alinkages in starch and/or pullulan [1, 3Ϫ5] to produce maltotri-helices found in 47 amy...

Extracellular Synthesis, Specific Recognition, and Intracellular Degradation of Cyclomaltodextrins by the Hyperthermophilic Archaeon Thermococcus sp. Strain B1001

“…2). A search with the BLAST program (43) revealed that the protein encoded by the ORF is homologous to neopullulanases (19,26), CDases (22,35), maltogenic amylase (6), and other amylolytic enzymes. It was suggested that the ORF encoded a kind of amylolytic enzyme.…”

Engineering Thermus Maltogenic Amylase with Improved Thermostability: Probing the Role of the Conserved Calcium Binding Site in Cyclodextrin-degrading Enzymes

“…Although the Ca2 site is far from the active site, the presence or absence of calcium ions in the Ca2 site seems to affect the catalytic properties of the enzyme. The hydrolysis activities of MAases belonging to the type II group were increased by the addition of EDTA and decreased by calcium ions 28,31,42,43) although we still do not have a clear explanation. However, it seems obvious that the binding of calcium ions at the type II Ca2 site improves the catalytic activity of MAases (Fig.…”

Enterococcus faecalis Uses a Phosphotransferase System Permease and a Host Colonization-Related ABC Transporter for Maltodextrin Uptake

“…In isopanose, the ␣-1,6 linkage is located between the two glucose residues at the reducing end (25). The E. faecalis strain V583 has been shown to grow on panose (26), and several other bacteria were reported to utilize both panose and isopanose (27)(28)(29). We found that maltotetraose-positive E. faecalis JH2-2 can also grow on both maltotriose isomers (Fig.…”