title: Purification and characterization of a novel thermostable 4-alpha-glucanotransferase of Thermotoga maritima cloned in Escherichia coli.
abstract: Maltodextrin glycosyltransferase (4-alpha-glucanotransferase) of the extremely thermophilic ancestral bacterium Thermotoga maritima has been purified from an Escherichia coli clone expressing the corresponding T. maritima MSB8 chromosomal gene. T. maritima 4-alpha-glucanotransferase, an approximately 53-kDa monomeric enzyme, is the most thermophilic glycosyltransferase described to date. It retained more than 90% of its maximum activity at temperatures from 55 degrees C up to 80 degrees C. The proposed action modus is the transfer of 1,4-alpha-glucanosyl chains, thus resulting in the disproportionation of 1,4-alpha-glucans. It converted soluble starch, amylopectin, and amylose, thereby changing the iodine staining properties of these substrates. The addition of low-molecular-mass malto-oligosaccharides, which act as glucanosyl acceptor molecules, enhanced the reaction and resulted in the formation of a series of linear maltohomologues from two to more than nine glucose units in size. Use of either of the malto-oligosaccharides maltotetraose, maltopentaose, maltohexaose, or maltoheptaose as sole substrate also yielded linear maltohomologues. On the other hand, maltose and maltotriose were not disproportionated by 4-alpha-glucanotransferase, although both were good acceptors for glucanosyl transfer. Glucose did not function as an acceptor in transfer reactions. Glucose also never appeared as a reaction product. The chain length of glucanosyl segments transferred ranged from two to probably far more than six glucose residues. Comparison of the N-terminal amino acid sequence of 4-alpha-glucanotransferase with other published protein sequences revealed significant similarity to sequences near the N-termini of various eucaryotic maltases and bacterial cyclodextrin glycosyltransferases, suggesting its relatedness on the molecular level with other starch- and maltodextrin-converting enzymes.
authors.affiliation: Institute for Microbiology, Technical University Munich, Federal Republic of Germany.
authors.forename: W
authors.initials: W
authors.lastname: Liebl
authors.forename: R
authors.initials: R
authors.lastname: Feil
authors.forename: J
authors.initials: J
authors.lastname: Gabelsberger
authors.forename: J
authors.initials: J
authors.lastname: Kellermann
authors.forename: K H
authors.initials: KH
authors.lastname: Schleifer
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002241:Carbohydrates / Q000032:analysis; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D006004:Glycogen Debranching Enzyme System / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D002241:Carbohydrates; D006004:Glycogen Debranching Enzyme System; D009844:Oligosaccharides; D011994:Recombinant Proteins; C020569:4 alpha-glucanotransferase