Modes of Action of Acarbose Hydrolysis and Transglycosylation Catalyzed by a Thermostable Maltogenic Amylase, the Gene for Which Was Cloned from a Thermus Strain

Abstract

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°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 β-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 tomethyl-α-d-glucopyranoside by forming an α-(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.

Enzymatic characterization of a maltogenic amylase fromLactobacillus gasseriATCC 33323 expressed inEscherichia coli

“…The difference in molecular masses was due to the presence of the six-histidine tag and extra two amino acids (methionine and glutamic acid) attached to the N-terminus of the recombinant protein. The molecular mass of LGMA is much lower than that of other extracellular a-amylases found in LAB, such as L. amlylovorus (140 kDa), L. plantarum (230 kDa) and L. amlylophilus (100 kDa), but is similar to other MAases from Bacillus and Thermus strains [3,[10][11][12]. Gel permeation chromatography revealed that LGMA is present at a molecular mass of 211 kDa, implying that LGMA may exist in a functional tetrameric form in solution (Fig.…”

“…lactis, 44% with the MAase of Bacillus subtilis (BBMA) [15] and 44% with the MAase of Thermus sp. (ThMA) [3].…”

“…E. coli transformants were grown in LB containing ampicillin (100 lg/ml) at 37°C. Plasmid p6xHis119 was used as the cloning and expression vector [3].…”

“…Finally, LGMA was purified to apparent homogeneity and the purity was confirmed by SDS-PAGE analysis. The molecular mass of the purified enzyme was determined by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) [3].…”

“…This type of enzyme is distinguishable from typical a-amylases by its extensive transglycosylation activity in addition to its hydrolysis activity. It transfers a donor molecule released by the hydrolyzing activity of the enzyme to an acceptor molecule by mainly forming an a-(1,6)-glycosidic linkage be-tween the donor and acceptor molecules [2,3]. The combined transglycosylation and hydrolysis activities of MAase have been used to produce branched oligosaccharides from liquefied starch [4].…”