Article ID Journal Published Year Pages File Type
34281 Process Biochemistry 2015 11 Pages PDF
Abstract

•Amylomaltase from Thermus filiformis (TfAM) is the shortest Thermus amylomaltase.•pH and temperature optima for disproportionation and cyclization were different.•E27R-TfAM displayed a shift to higher pH and temperature optima in cyclization.•E27R-TfAM exerted a significant increase in alkaline and thermo-stability.•E27R-TfAM showed changes in CD spectrum at pH 9.0 and DSC profile at above 350 K.

Amylomaltase catalyzes the α-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 °C to produce linear oligosaccharides. However, highest cyclization activity was observed at pH 5.0 and 70 °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 °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 350 K were observed through the circular dichroism spectra and the thermal transition profiles, respectively.

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