Purification and characterization of a novel thermostable 4‐α‐glucanotransferase of Thermotoga maritima cloned in Escherichia coli

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Abstract
Maltodextrin glycoslyltransferase (4‐α‐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‐α‐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°C up to 80°C.The proposed action modus is the transfer of 1,4‐α‐glucanosyl chains, thus resulting in the disproportionation of 1,4‐α‐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‐α‐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‐α‐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.