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 Tomschy, A.
Right arrow Articles by Wyss, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tomschy, A.
Right arrow Articles by Wyss, M.
Agricola
Right arrow Articles by Tomschy, A.
Right arrow Articles by Wyss, M.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, April 2002, p. 1907-1913, Vol. 68, No. 4
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.4.1907-1913.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Engineering of Phytase for Improved Activity at Low pH

Andrea Tomschy,1 Roland Brugger,1 Martin Lehmann,1 Allan Svendsen,2 Kurt Vogel,1 Dirk Kostrewa,1,{dagger} Søren F. Lassen,2 Dominique Burger,1 Alexandra Kronenberger,1 Adolphus P. G. M. van Loon,1,{ddagger} Luis Pasamontes,1 and Markus Wyss1*

Biotechnology Department, Roche Vitamins, Ltd., 4070 Basel, Switzerland,1 Novozymes A/S, DK-2880 Bagsværd, Denmark2

Received 12 November 2001/ Accepted 22 January 2002

For industrial applications in animal feed, a phytase of interest must be optimally active in the pH range prevalent in the digestive tract. Therefore, the present investigation describes approaches to rationally engineer the pH activity profiles of Aspergillus fumigatus and consensus phytases. Decreasing the negative surface charge of the A. fumigatus Q27L phytase mutant by glycinamidylation of the surface carboxy groups (of Asp and Glu residues) lowered the pH optimum by ca. 0.5 unit but also resulted in 70 to 75% inactivation of the enzyme. Alternatively, detailed inspection of amino acid sequence alignments and of experimentally determined or homology modeled three-dimensional structures led to the identification of active-site amino acids that were considered to correlate with the activity maxima at low pH of A. niger NRRL 3135 phytase, A. niger pH 2.5 acid phosphatase, and Peniophora lycii phytase. Site-directed mutagenesis confirmed that, in A. fumigatus wild-type phytase, replacement of Gly-277 and Tyr-282 with the corresponding residues of A. niger phytase (Lys and His, respectively) gives rise to a second pH optimum at 2.8 to 3.4. In addition, the K68A single mutation (in both A. fumigatus and consensus phytase backbones), as well as the S140Y D141G double mutation (in A. fumigatus phytase backbones), decreased the pH optima with phytic acid as substrate by 0.5 to 1.0 unit, with either no change or even a slight increase in maximum specific activity. These findings significantly extend our tools for rationally designing an optimal phytase for a given purpose.

* Corresponding author. Mailing address: Roche Vitamins, Ltd., Bldg. 203/113A, CH-4070 Basel, Switzerland. Phone: 41-61-688-2972. Fax: 41-61-687-1847. E-mail: markus.wyss{at}roche.com.

{dagger} Present address: Paul Scherrer-Institut, Life Sciences, OSRA/007, CH-5232 Villigen-PSI, Switzerland.

{ddagger} Present address: Waldhofstrasse 15, CH-4310 Rheinfelden, Switzerland.

Applied and Environmental Microbiology, April 2002, p. 1907-1913, Vol. 68, No. 4
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.4.1907-1913.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Kim, T., Mullaney, E. J., Porres, J. M., Roneker, K. R., Crowe, S., Rice, S., Ko, T., Ullah, A. H. J., Daly, C. B., Welch, R., Lei, X. G. (2006). Shifting the pH Profile of Aspergillus niger PhyA Phytase To Match the Stomach pH Enhances Its Effectiveness as an Animal Feed Additive.. Appl. Environ. Microbiol. 72: 4397-4403 [Abstract] [Full Text]  
  • Lee, S.-J., Lee, D.-W., Choe, E.-A., Hong, Y.-H., Kim, S.-B., Kim, B.-C., Pyun, Y.-R. (2005). Characterization of a Thermoacidophilic L-Arabinose Isomerase from Alicyclobacillus acidocaldarius: Role of Lys-269 in pH Optimum. Appl. Environ. Microbiol. 71: 7888-7896 [Abstract] [Full Text]  
  • Lehmann, M., Loch, C., Middendorf, A., Studer, D., Lassen, S. F., Pasamontes, L., van Loon, A. P.G.M., Wyss, M. (2002). The consensus concept for thermostability engineering of proteins: further proof of concept. Protein Eng Des Sel 15: 403-411 [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»