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

Piriya Kaewpathomsri, Yui Takahashi, Shigeyoshi Nakamura, Jarunee Kaulpiboon, Shun Ichi Kidokoro, Shuichiro Murakami, Kuakarun Krusong, Piamsook Pongsawasdi

Research output: Contribution to journalArticlepeer-review

15 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1814-1824
Number of pages11
JournalProcess Biochemistry
Volume50
Issue number11
DOIs
Publication statusPublished - Nov 2015

Keywords

  • Alkaline stability
  • Amylomaltase
  • Cyclization
  • Large-ring cyclodextrin
  • Thermostability
  • Thermus filiformis

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