Modes of action of acarbose hydrolysis and transglycosylation catalyzed by a thermostable maltogenic amylase, the gene for which was cloned from a Thermus strain.
A maltogenic amylase gene was cloned in Escherichia coli from a gram-negative thermophilic bacterium, Thermus strain IM6501. The gene encoded an enzyme (ThMA) with a molecular mass of 68 kDa which was expressed by the expression vector p6xHis119. The optimal temperature of ThMA was 60 degrees C, which was higher than those of other maltogenic amylases reported so far. Thermal inactivation kinetic analysis of ThMA indicated that it was stabilized in the presence of 10 mM EDTA. ThMA harbored both hydrolysis and transglycosylation activities. It hydrolyzed beta-cyclodextrin and starch mainly to maltose and pullulan to panose. ThMA not only hydrolyzed acarbose, an amylase inhibitor, to glucose and pseudotrisaccharide (PTS) but also transferred PTS to 17 sugar acceptors, including glucose, fructose, maltose, cellobiose, etc. Structural analysis of acarbose transfer products by using methylation, thin-layer chromatography, high-performance ion chromatography, and nuclear magnetic resonance indicated that PTS was transferred primarily to the C-6 of the acceptors and at lower degrees to the C-3 and/or C-4. The transglycosylation of sugar to methyl-alpha-D-glucopyranoside by forming an alpha-(1,3)-glycosidic linkage was demonstrated for the first time by using acarbose and ThMA. Kinetic analysis of the acarbose transfer products showed that the C-4 transfer product formed most rapidly but readily hydrolyzed, while the C-6 transfer product was stable and accumulated in the reaction mixture as the main product.
Kim
T J
TJ
Department of Food Science and Technology and Research Center for New Bio-Materials in Agriculture, Seoul National University, Suwon 441-744, Korea.
Kim
M J
MJ
Kim
B C
BC
Kim
J C
JC
Cheong
T K
TK
Kim
J W
JW
Park
K H
KH
D020909:Acarbose; D000681:Amylases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006868:Hydrolysis; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D013816:Thermodynamics; D013824:Thermus / Q000201:enzymology / Q000235:genetics*; D014312:Trisaccharides / Q000378:metabolism*
D014312:Trisaccharides; D000681:Amylases; D020909:Acarbose