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Volume 65, Issue 3 (Jun 2010)


Tyrosine 39 of GH13 α-amylase from Thermococcus hydrothermalis contributes to its thermostability

Andrej Godány
  • Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, SK-84551, Bratislava, Slovakia
  • Department of Biotechnology, Faculty of Natural Sciences, University of SS. Cyril and Methodius, Nám. J. Herdu 2, SK-91701, Trnava, Slovakia
  • Email:
/ Katarína Majzlová
  • Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, SK-84551, Bratislava, Slovakia
  • Email:
/ Viera Horváthová
  • Department of Biotechnology, Faculty of Natural Sciences, University of SS. Cyril and Methodius, Nám. J. Herdu 2, SK-91701, Trnava, Slovakia
  • Email:
/ Barbora Vidová
  • Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, SK-84551, Bratislava, Slovakia
  • Email:
/ Štefan Janeček
  • Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, SK-84551, Bratislava, Slovakia
  • Department of Biotechnology, Faculty of Natural Sciences, University of SS. Cyril and Methodius, Nám. J. Herdu 2, SK-91701, Trnava, Slovakia
  • Email:
Published Online: 2010-04-24 | DOI: https://doi.org/10.2478/s11756-010-0030-x


The presented work is focused on the naturally thermostable α-amylase from the archaebacterium Thermococcus hydrothermalis. From the evolutionary point of view, the archaeal α-amylases are most closely related to plant α-amylases. In a wider sense, especially when the evolutionary trees are based on the less conserved part of their amino acid sequences (e.g. domain C succeeding the catalytic TIM-barrel), also the representatives of bacterial liquefying (Bacillus licheniformis) and saccharifying (Bacillus subtilis) α-amylases as well as the one from Thermotoga maritima should be included into the relatedness with the archaeal and plant α-amylases. Based on the bioinformatics analysis of the α-amylase from T. hydrothermalis, the position of tyrosine 39 (Y16 if the putative 23-residue long signal peptide is considered) was mutated to isoleucine (present in the α-amylase from T. maritima) by the in vitro mutagenesis. The biochemical characterization of the wild-type α-amylase and its Y39I mutant revealed that: (i) the specific activity of both enzymes was approximately equivalent (0.55 ± 0.13 U/mg for the wild-type and 0.52 ± 0.15 U/mg for the Y39I); (ii) the mutant exhibited decreased temperature optimum (from 85°C for the wild-type to 80°C for the Y39I); and (iii) the pH optimum remained the same (pH 5.5 for both enzymes). The remaining activity of the α-amylases was also tested by one-hour incubation at 80°C, 85°C, 90°C and 100°C. Since the wild-type α-amylase lost only 13% of its activity after one-hour incubation at the highest tested temperature (100°C), whereas 27% decrease was seen for the mutant Y39I under the same conditions, it is possible to conclude that the position of tyrosine 39 could contribute to the thermostability of the α-amylase from T. hydrothermalis.

Keywords: α-amylase; Thermococcus hydrothermalis; glycoside hydrolase family 13; site-directed mutagenesis; protein thermostability

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About the article

Published Online: 2010-04-24

Published in Print: 2010-06-01

Citation Information: Biologia, ISSN (Online) 1336-9563, ISSN (Print) 0006-3088, DOI: https://doi.org/10.2478/s11756-010-0030-x. Export Citation

© 2010 Slovak Academy of Sciences. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. (CC BY-NC-ND 3.0)

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