Characterization of amylomaltase from Thermus filiformis and the increase in alkaline and thermo-stability by E27R substitution

Amylomaltase catalyzes the alpha-1,4 glycosyl transfer between oligosaccharides. The amylomaltase gene from Thermus filiformis JCM11600 (TfAM) was cloned, expressed in Escherichia coli and purified to homogeneity. TfAM, a member of glycoside hydrolase family 77, encoded the polypeptide of 485 amino acid residues, the shortest among Thermus amylomaltases, with a calculated molecular mass of 55.47 kDa and pI of 5.11. Highest disproportionation activity occurred with maltotriose substrate at pH 6.5 and 60 degrees C to produce linear oligosaccharides. However, highest cyclization activity was observed at pH 5.0 and 70 degrees C, resulting in large-ring cyclodextrins with CD22 as the smallest and CD24-CD29 as principle products. TfAM lost 80% of its disproportionation activity after incubation for 2 h at pH 9.0 or 1 h at 90 degrees C. Meanwhile, E27R-TfAM mutant, forming an Arg cluster (R27-R30-R31-R34) on the enzyme surface, showed a significant increase in stability at these extreme pH and temperature and a shift toward higher pH and temperature optima in cyclization reaction. Conformational change of the mutated enzyme at pH 9.0 and temperature above 350K were observed through the circular dichroism spectra and the thermal transition profiles, respectively. (C) 2015 Elsevier Ltd. All rights reserved.