Skip Navigation

This Article
Right arrow Full Text Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (17)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Schmitt, J.
Right arrow Articles by Pleiss, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schmitt, J.
Right arrow Articles by Pleiss, J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Protein Engineering, Vol. 15, No. 7, 595-601, July 2002
© 2002 Oxford University Press

Blocking the tunnel: engineering of Candida rugosa lipase mutants with short chain length specificity

Jutta Schmitt1, Stefania Brocca2, Rolf D. Schmid1 and Jürgen Pleiss1,3

1 Institute of Technical Biology, University of Stuttgart, Allmandring 31,D-70569 Stuttgart, Germany and 2 Department of Biotechnology and Biosciences, University of Milano-Bicocca, I-20126 Milano, Italy

The molecular basis of chain length specificity of Candida rugosa lipase 1 was investigated by molecular modeling and site-directed mutagenesis. The synthetic lip1 gene and the lipase mutants were expressed in Pichia pastoris and assayed for their chain length specificity in single substrate assays using triglycerides as well as in a competitive substrate assay using a randomized oil. Mutation of amino acids at different locations inside the tunnel (P246F, L413F, L410W, L410F/S300E, L410F/S365L) resulted in mutants with a different chain length specificity. Mutants P246F and L413F have a strong preference for short chain lengths whereas substrates longer than C10 are hardly hydrolyzed. Increasing the bulkiness of the amino acid at position 410 led to mutants that show a strong discrimination of chain lengths longer than C14. The results obtained can be explained by a simple mechanical model: the activity for a fatty acid sharply decreases as it becomes long enough to reach the mutated site. In contrast, a mutation at the entrance of the tunnel (L304F) has a strong impact on C4 and C6 substrates. This mutant is nevertheless capable of hydrolyzing chain lengths longer than C8.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Nucleic Acids ResHome page
A. Pavelka, E. Chovancova, and J. Damborsky
HotSpot Wizard: a web server for identification of hot spots in protein engineering
Nucleic Acids Res., July 1, 2009; 37(suppl_2): W376 - W383.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. Athenstaedt and G. Daum
Tgl4p and Tgl5p, Two Triacylglycerol Lipases of the Yeast Saccharomyces cerevisiae Are Localized to Lipid Particles
J. Biol. Chem., November 11, 2005; 280(45): 37301 - 37309.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. Chaloupkova, J. Sykorova, Z. Prokop, A. Jesenska, M. Monincova, M. Pavlova, M. Tsuda, Y. Nagata, and J. Damborsky
Modification of Activity and Specificity of Haloalkane Dehalogenase from Sphingomonas paucimobilis UT26 by Engineering of Its Entrance Tunnel
J. Biol. Chem., December 26, 2003; 278(52): 52622 - 52628.
[Abstract] [Full Text] [PDF]


Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.