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 Fridjonsson, O.
Right arrow Articles by Mattes, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fridjonsson, O.
Right arrow Articles by Mattes, R.
Agricola
Right arrow Articles by Fridjonsson, O.
Right arrow Articles by Mattes, R.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, September 1999, p. 3955-3963, Vol. 65, No. 9
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Cloning of the Gene Encoding a Novel Thermostable alpha -Galactosidase from Thermus brockianus ITI360

Olafur Fridjonsson,* Hildegard Watzlawick, Axel Gehweiler, Thilo Rohrhirsch, and Ralf Mattes

Institut für Industrielle Genetik, Universität Stuttgart, 70569 Stuttgart, Germany

Received 25 March 1999/Accepted 29 June 1999

An alpha -galactosidase gene from Thermus brockianus ITI360 was cloned, sequenced, and expressed in Escherichia coli, and the recombinant protein was purified. The gene, designated agaT, codes for a 476-residue polypeptide with a calculated molecular mass of 53,810 Da. The native structure of the recombinant enzyme (AgaT) was estimated to be a tetramer. AgaT displays amino acid sequence similarity to the alpha -galactosidases of Thermotoga neapolitana and Thermotoga maritima and a low-level sequence similarity to alpha -galactosidases of family 36 in the classification of glycosyl hydrolases. The enzyme is thermostable, with a temperature optimum of activity at 93°C with para-nitrophenyl-alpha -galactopyranoside as a substrate. Half-lives of inactivation at 92 and 80°C are 100 min and 17 h, respectively. The pH optimum is between 5.5 and 6.5. The enzyme displayed high affinity for oligomeric substrates. The Kms for melibiose and raffinose at 80°C were determined as 4.1 and 11.0 mM, respectively. The alpha -galactosidase gene in T. brockianus ITI360 was inactivated by integrational mutagenesis. Consequently, no alpha -galactosidase activity was detectable in crude extracts of the mutant strain, and it was unable to use melibiose or raffinose as a single carbohydrate source.

* Corresponding author. Mailing address: Institut für Industrial Genetik, Universität Stuttgart, 70569 Stuttgart, Germany. Phone: 49 (0) 711 685 6982. Fax: 49 (0) 711 685 6973. E-mail: olaf{at}genius.biologie.uni-stuttgart.de.

Applied and Environmental Microbiology, September 1999, p. 3955-3963, Vol. 65, No. 9
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Saunders, L. P., Ouellette, A., Bandle, R., Chang, W. C., Zhou, H., Misra, R. N., De La Cruz, E. M., Braddock, D. T. (2008). Identification of small-molecule inhibitors of autotaxin that inhibit melanoma cell migration and invasion. Molecular Cancer Therapeutics 7: 3352-3362 [Abstract] [Full Text]  
  • Brouns, S. J. J., Smits, N., Wu, H., Snijders, A. P. L., Wright, P. C., de Vos, W. M., van der Oost, J. (2006). Identification of a Novel {alpha}-Galactosidase from the Hyperthermophilic Archaeon Sulfolobus solfataricus.. J. Bacteriol. 188: 2392-2399 [Abstract] [Full Text]  
  • Fridjonsson, O., Watzlawick, H., Mattes, R. (2002). Thermoadaptation of {alpha}-Galactosidase AgaB1 in Thermus thermophilus. J. Bacteriol. 184: 3385-3391 [Abstract] [Full Text]  
  • Jindou, S., Karita, S., Fujino, E., Fujino, T., Hayashi, H., Kimura, T., Sakka, K., Ohmiya, K. (2002). {alpha}-Galactosidase Aga27A, an Enzymatic Component of the Clostridium josui Cellulosome. J. Bacteriol. 184: 600-604 [Abstract] [Full Text]  
  • Coombs, J., Brenchley, J. E. (2001). Characterization of Two New Glycosyl Hydrolases from the Lactic Acid Bacterium Carnobacterium piscicola Strain BA. Appl. Environ. Microbiol. 67: 5094-5099 [Abstract] [Full Text]  
  • Fridjonsson, O., Mattes, R. (2001). Production of Recombinant {alpha}-Galactosidases in Thermus thermophilus. Appl. Environ. Microbiol. 67: 4192-4198 [Abstract] [Full Text]  
  • Ishiguro, M., Kaneko, S., Kuno, A., Koyama, Y., Yoshida, S., Park, G.-G., Sakakibara, Y., Kusakabe, I., Kobayashi, H. (2001). Purification and Characterization of the Recombinant Thermus sp. Strain T2 {alpha}-Galactosidase Expressed in Escherichia coli. Appl. Environ. Microbiol. 67: 1601-1606 [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»