The action mode of Thermus aquaticus YT-1 4-α-glucanotransferase and its chimeric enzymes introduced with starch-binding domain on amylose and amylopectin

A thermostable 4-alpha-glucanotransferase (TA alpha GT) gene isolated from Thermus aquaticus YT-1 had an open reading frame of 1503 nucleotides, which encoded 500 amino acid residues for a 57,969 dalton protein. The maximum activity of the TA alpha GT was observed at pH 7.5 and 70 degrees C. The enzyme catalyzed intermolecular transglycosylation of maltooligosaccharides (disproportionation) to produce linear alpha-1,4-glucans of various sizes. The starch-binding domains (SBD) of Bacillus stearothermophilus ET1 CGTase (E and DE) were introduced into the C-terminus of TA alpha GT to enhance the starch utilizing activity. The chimeric enzymes, TA alpha GT-E and TA alpha GT-DE, showed no difference in temperature optimum, transglycosylation activity, and amylolytic degradation pattern compared to TA alpha GT wild-type. However, TA alpha GT-DE exhibited the highest molar specific activity toward amylose. TA alpha GT-DE modified amylopectin molecules by its disproportionating activities to produce modified amylopectin clusters (M-w 10(5)-10(6)). Also, it demonstrated the ability to produce cyclo-amyloses with DP of 19 through 35 from amylose molecules. (c) 2006 Elsevier Ltd. All rights reserved.