This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, Y.-W.
Right arrow Articles by Park, K.-H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, Y.-W.
Right arrow Articles by Park, K.-H.
Agricola
Right arrow Articles by Kim, Y.-W.
Right arrow Articles by Park, K.-H.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, August 2003, p. 4866-4874, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.4866-4874.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Directed Evolution of Thermus Maltogenic Amylase toward Enhanced Thermal Resistance

Young-Wan Kim,1 Ji-Hye Choi,1 Jung-Wan Kim,2 Cheonseok Park,3 Jung-Woo Kim,1 Hyunju Cha,1 Soo-Bok Lee,4 Byoung-Ha Oh,5 Tae-Wha Moon,1 and Kwan-Hwa Park1*

National Laboratory for Functional Food Carbohydrates, Center for Agricultural Biomaterials, and Department of Food Science and Technology, Seoul National University, Suwon 441-744,1 Department of Biology, University of Incheon, Incheon 402-749,2 Department of Food Science and Technology, Kyunghee University, Yongin 449-701,3 Department of Food and Nutrition, Yonsei University, Seoul 120-749,4 Department of Life Science, Pohang University of Science and Technology, Pohang 790-784, Korea5

Received 31 October 2002/ Accepted 21 May 2003

The thermostability of maltogenic amylase from Thermus sp. strain IM6501 (ThMA) was improved greatly by random mutagenesis using DNA shuffling. Four rounds of DNA shuffling and subsequent recombination of the mutations produced the highly thermostable mutant enzyme ThMA-DM, which had a total of seven individual mutations. The seven amino acid substitutions in ThMA-DM were identified as R26Q, S169N, I333V, M375T, A398V, Q411L, and P453L. The optimal reaction temperature of the recombinant enzyme was 75°C, which was 15°C higher than that of wild-type ThMA, and the melting temperature, as determined by differential scanning calorimetry, was increased by 10.9°C. The half-life of ThMA-DM was 172 min at 80°C, a temperature at which wild-type ThMA was completely inactivated in less than 1 min. Six mutations that were generated during the evolutionary process did not significantly affect the specific activity of the enzyme, while the M375T mutation decreased activity to 23% of the wild-type level. The molecular interactions of the seven mutant residues that contributed to the increased thermostability of the mutant enzyme with other adjacent residues were examined by comparing the modeled tertiary structure of ThMA-DM with those of wild-type ThMA and related enzymes. The A398V and Q411L substitutions appeared to stabilize the enzyme by enhancing the interdomain hydrophobic interactions. The R26Q and P453L substitutions led potentially to the formation of genuine hydrogen bonds. M375T, which was located near the active site of ThMA, probably caused a conformational or dynamic change that enhanced thermostability but reduced the specific activity of the enzyme.

* Corresponding author. Mailing address: Department of Food Science and Technology, Seoul National University, 103 Seodun Dong, Kwensun Gu, Suwon 441-744, Korea. Phone: 82-31-290-2582. Fax: 82-31-294-1336. E-mail: parkkh{at}plaza.snu.ac.kr.

Applied and Environmental Microbiology, August 2003, p. 4866-4874, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.4866-4874.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Lee, H., Wang, P., Hoshino, H., Ito, Y., Kobayashi, M., Nakayama, J., Seeberger, P. H, Fukuda, M. (2008). {alpha}1,4GlcNAc-capped mucin-type O-glycan inhibits cholesterol {alpha}-glucosyltransferase from Helicobacter pylori and suppresses H. pylori growth. Glycobiology 18: 549-558 [Abstract] [Full Text]  
  • Ni, J., Takehara, M., Miyazawa, M., Watanabe, H. (2007). Random exchanges of non-conserved amino acid residues among four parental termite cellulases by family shuffling improved thermostability. Protein Eng Des Sel 20: 535-542 [Abstract] [Full Text]  
  • Lee, J.-K., Ang, E.-L., Zhao, H. (2006). Probing the Substrate Specificity of Aminopyrrolnitrin Oxygenase (PrnD) by Mutational Analysis.. J. Bacteriol. 188: 6179-6183 [Abstract] [Full Text]  
  • Lee, J., Simurdiak, M., Zhao, H. (2005). Reconstitution and Characterization of Aminopyrrolnitrin Oxygenase, a Rieske N-Oxygenase That Catalyzes Unusual Arylamine Oxidation. J. Biol. Chem. 280: 36719-36727 [Abstract] [Full Text]  
  • Yuan, L., Kurek, I., English, J., Keenan, R. (2005). Laboratory-Directed Protein Evolution. Microbiol. Mol. Biol. Rev. 69: 373-392 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»