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Amylomaltase of Pyrobaculum aerophilum IM2 produces thermoreversible starch gels

Author: Kaper, T. · Talik, B. · Ettema, T.J. · Bos, H. · Maarel, M.J.E.C. van der · Dijkhuizen, L.
Type:article
Date:2005
Institution: TNO Kwaliteit van Leven
Source:Applied and Environmental Microbiology, 9, 71, 5098-5106
Identifier: 238663
doi: doi:10.1128/AEM.71.9.5098-5106.2005
Keywords: Nutrition · Food technology · Escherichia coli · Gels · Glucose · Mathematical models · pH effects · Starch · Achaeal amylomaltase · Amylomaltase · Binding mode · Pyrobaculum aerophilum IM2 · Enzyme kinetics · 4alpha glucanotransferase · acarbose · cysteine · disulfide · dithiothreitol · gelatin · gene product · glucan · glucose · glycosidase · hydroxyl group · oligosaccharide · potato starch · starch · enzyme · Archean · article · chemical reaction kinetics · enzyme active site · enzyme activity · enzyme inhibition · enzyme substrate complex · Escherichia coli · food industry · gene expression · heat sensitivity · molecular cloning · nonhuman · Pyrobaculum · pyrobaculum aerophilum · thermostability · Binding Sites · Cloning, Molecular · Enzyme Stability · Escherichia coli · Food Industry · Gels · Glycogen Debranching Enzyme System · Heat · Kinetics · Pyrobaculum · Starch · Archaea · Escherichia coli · Pyrobaculum aerophilum · Solanum tuberosum

Abstract

Amylomaltases are 4-α-glucanotransferases (EC 2.4.1.25) of glycoside hydrolase family 77 that transfer α-1,4-linked glucans to another acceptor, which can be the 4-OH group of an α-1,4-linked glucan or glucose. The amylomaltase-encoding gene (PAE1209) from the hyperthermophilic archaeon Pyrobaculum aerophilum IM2 was cloned and expressed in Escherichia coli, and the gene product (PyAMase) was characterized. PyAMase displays optimal activity at pH 6.7 and 95°C and is the most thermostable amylomaltase described to date. The thermostability of PyAMase was reduced in the presence of 2 mM dithiothreitol, which agreed with the identification of two possible cysteine disulfide bridges in a three-dimensional model of PyAMase. The kinetics for the disproportionation of malto-oligosaccharides, inhibition by acarbose, and binding mode of the substrates in the active site were determined. Acting on gelatinized food-grade potato starch, PyAMase produced a thermoreversible starch product with gelatin-like properties. This thermoreversible gel has potential applications in the food industry. This is the first report on an archaeal amylomaltase. Copyright © 2005, American Society for Microbiology. All Rights Reserved.