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

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.